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Transcript
Biotic and abiotic factors influencing the development
of N2-fixing symbioses between rhizobia and
the woody legumes Acacia and Prosopis
LEENA A. RÄSÄNEN
Department of Applied Chemistry and Microbiology
Division of Microbiology
University of Helsinki
Finland
ACADEMIC DISSERTATION IN MICROBIOLOGY
To be presented, with the permission of the Faculty of Agriculture and Forestry
of the University of Helsinki, for public criticism in Auditorium 1041,
at Viikki Biocenter 2, (Viikinkaari 5, Helsinki)
on December 5th, 2002, at 12 o’clock noon.
Helsinki 2002
Supervisor:
Docent Kristina Lindström
Department of Applied Chemistry and Microbiology
University of Helsinki
Finland
Reviewers:
Prof. Ken Giller
Department of Plant Sciences
Wageningen University
The Netherlands
Dr. David Odee
Biotechnology Laboratory
Kenya Forestry Research Institute
Nairobi, Kenya
Opponent:
Docent Marjo Helander
Department of Biology
University of Turku
Finland
ISSN 1239-9469
ISBN 952-10-080-2 (paperback)
ISBN 952-10-0821-0 (PDF)
Electronic publication available at http://ethesis.helsinki.fi
Yliopistopaino, Helsinki 2002
Front cover: Root hairs of Acacia senegal stained with methylene blue.
Photo: Leena Räsänen.
Oi maailma,
elämä sä ihmeellinen!
Mi on sun tarkoitukses?
Mihin viimein?
Se arvoituspa kauan askaretta
on aatokseen tuonut.
Aleksis Kivi
To my parents
Contents
List of the original papers
The author’s contribution
Abbreviations
1. Introduction .............................................................................................................1
1.1. Background ......................................................................................................1
1.2. Characterisation of arid and semiarid regions.....................................................3
1.2.1. Dry tropical forests .................................................................................3
1.2.2. Soil types................................................................................................3
1.2.3. Saline soils ............................................................................................6
1.3. Role of N2-fixing trees in dry tropical ecosystems...............................................6
1.3.1. Trees improve soil fertility ......................................................................6
1.3.2. Significance of N2 fixation.......................................................................7
1.4. The family Leguminosae (or syn. Fabaceae)........................................................8
1.5. Acacia and Prosopis trees (Mimosoideae) ..........................................................9
1.5.1. Geographical distribution .......................................................................9
1.5.2. Biological and ecological properties .......................................................9
1.5.3. Capacity for N2 fixation ........................................................................12
1.5.4. Nodulation in the field .........................................................................13
1.5.5. Multipurpose Acacia and Prosopis trees ...............................................14
1.5.6. Problems .............................................................................................15
1.5.7. Systematics of Acacia spp. ....................................................................16
1.5.8. Systematics of Prosopis spp. .................................................................17
1.6. Rhizobia nodulating Acacia and Prosopis trees ................................................17
1.6.1. The most common species and genera among tree rhizobia .................18
1.6.2. Occurrence of rhizobia in deep soils ....................................................20
1.7. Development of symbioses between rhizobia and leguminous plants ..............21
1.7.1. Colonisation and attachment of rhizobia on the plant root....................21
1.7.2. Infection and nodulation ......................................................................23
1.7.3. A closer look at the infection process ..................................................24
1.7.4. The structure of nodules ......................................................................28
1.7.5. The symbiosome and the symbiosomal membrane ...............................30
1.7.6. N2 fixation ............................................................................................30
1.7.7. Altruistic behaviour of N2-fixing bacteroids...........................................31
1.8. Inhibition of N2-fixing symbiosis by abiotic factors...........................................32
1.8.1. Abiotic constraints of arid and semiarid regions....................................32
1.8.2. Movement of water in the plant and soil...............................................33
1.8.3. Drought stress and drought tolerance strategies of the plants ...............34
1.8.4. Dehydration postponement in Acacia and Prosopis ..............................34
1.8.5. Salt stress and salt tolerance mechanisms of the plants .........................35
1.8.6. Heat stress and heat tolerance mechanisms of the plants ......................36
1.8.7. Osmotic adjustment and compatible solutes in plants ..........................36
1.8.8. N2 fixation under stress .........................................................................37
1.8.9. Nodulation and infection under stress ..................................................38
1.9. Abiotic stresses and free-living rhizobia ................................................................40
1.9.1. Osmoadaptation in rhizobia .................................................................42
1.9.2. Role of trehalose during desiccation ....................................................45
1.10. Inhibition of N2-fixing symbioses by biotic factors..........................................46
2. Aims of the study ....................................................................................................48
3. Materials and methods............................................................................................49
3.1. Biological material...........................................................................................49
3.2. Parameters and stress conditions studied..........................................................49
3.3. Statistical analyses ...........................................................................................50
4. Results and discussion.............................................................................................53
4.1. Symbiotic properties of African sinorhizobia ...................................................53
4.1.1. Host specificity.....................................................................................53
4.1.2. Variation in the effectiveness of the symbiotic association ....................54
4.2. Development of the symbiosis between sinorhizobia and Acacia and
Prosopis trees ...............................................................................................55
4.2.1. Infection trough the root hairs...............................................................56
4.2.2. The structure of effective indeterminate nodules ...................................57
4.2.3. Ineffective nodules of A. holosericea and P. africana ............................57
4.3. Tolerance of sinorhizobia to abiotic stresses ....................................................58
4.3.1. Properties of two potential inoculant strains..........................................58
4.3.2. Tolerance of heat ..................................................................................58
4.3.3. Tolerance of salt ...................................................................................59
4.3.4. Roles of glycine betaine and trehalose for sinorhizobia ........................60
4.4. A. senegal-S. arboris symbiosis under heat and drought stress..........................61
4.4.1. Rhizobial population in soil..................................................................61
4.4.2. Adaptation of A. senegal seedlings to heat and drought ........................62
4.4.3. Infection and nodulation under stress ..................................................63
4.5. Role of compatible solutes on the A. senegal-Sinorhizobium symbiosis ...........64
4.5.1. Endogenous glycine betaine in A. senegal ............................................64
4.5.2. Effects of exogenous compatible solutes on A. senegal seedlings .........65
4.5.3. Effects of exogenous compatible solutes on rhizobia ............................66
4.6. Responses of S. arboris populations to changing environmental conditions......66
4.6.1. Alterations in EPS production................................................................67
4.6.2. Changes in cell activity and culturability ..............................................68
4.6.3. Moderately and highly stressed cultures................................................70
4.6.4. Changes in cell morphology .................................................................71
4.7. Is it possible to exploit symbiotic N2 fixation by inoculation of Acacia and
Prosopis seedlings? ..........................................................................................71
4.7.1. Evaluation of the role of inoculation in the field....................................72
4.7.2. Inoculation as a procedure ...................................................................73
5. Summary .............................................................................................................75
6. Tiivistelmä .............................................................................................................77
7. Acknowledgements ................................................................................................79
8. References ............................................................................................................80
List of the original papers
This thesis is based on the following papers, referred to in the text by their Roman numerals.
Additionally, some unpublished results are presented.
I
Räsänen, L. A., J. I. Sprent and K. Lindström. 2001. Symbiotic properties of sinorhizobia
isolated from Acacia and Prosopis nodules in Sudan and Senegal. Plant and Soil
235:193-210.
II
Räsänen, L. A. and K. Lindström. 1999. The effect of heat stress on the symbiotic
interaction between Sinorhizobium sp. and Acacia senegal. FEMS Microbiology Ecology
28:63-74.
III Räsänen, L. A., S. Saijets, K. Jokinen, and K. Lindström. Evaluation of the roles of two
compatible solutes, glycine betaine and trehalose, for the Acacia-Sinorhizobium symbiosis
exposed to drought stress. Submitted 2002.
IV Räsänen, L. A., A. M. Elväng, J. Jansson, and K. Lindström. 2001. Effect of heat stress on
cell activity and cell morphology of the tropical rhizobium, Sinorhizobium arboris. FEMS
Microbiology Ecology 34:267-278.
The author’s contribution
I and II Leena Räsänen planned and conducted the experiments, analysed and interpreted the
results and wrote the paper, under supervision of the project leader Kristina Lindström.
Janet Sprent acted as an expert on the symbioses between tropical leguminous trees
and rhizobia and assisted in writing of paper I.
III
Leena Räsänen planned and conducted the experiments analysed and interpreted the
results and wrote the paper, under supervision of the project leader Kristina Lindström.
Salla Saijets performed growth experiments on bacteria, foliar application of glycine
betaine and preliminary drought experiments on plants. Kari Jokinen acted as an expert
on the application of plant glycine betaine and assisted in writing.
IV
Leena Räsänen planned and conducted the experiments, analysed and interpreted the
results and wrote the paper, under supervision of the project leader Kristina Lindström.
Annelie Elväng introduced analyses and measurements of luc tagged bacteria. Janet
Jansson acted as an expert on the application of bacteria tagged with the marker genes,
was the supervisor of Annelie Elväng and assisted in writing.
Abbreviations
ABA
AM fungi
BD
bv.
cfu
CPS
DMSP
EPS
FAO
GFP
GUS
Hsps
LPS
NAGGN
NAS
PEG
SFDA
sHsps
subsp.
USDA
VBNC
YEM
abscisic acid
arbuscular mycorrhizal fungi
Brown and Dilworth medium
biovar
colony forming unit
capsular polysaccharides
b-dimethylsulfonepropionate
exopolysaccharides
Food and Agricultural Organizations of the United Nations
green fluorescent protein
ß-glucuronidase
heat shock proteins
lipopolysaccharides
N- acetylglutaminylglutamine amide
National Academy of Sciences
polyethylene glycol
fluorochromo 5- (and)-sulfofluorescein diacetate
small heat shock proteins
subspecies
United States Department of Agriculture
viable but nonculturable
yeast-mannitol
1. INTRODUCTION
1.1. BACKGROUND
Population growth together with recurrent drought periods have lead to
deforestation and degradation of many dry ecosystems in the tropics. Activities of the increased human population cause pressure on the environment,
resulting in reduction of biological productivity and diversity. The gathering
of fuelwood and production of charcoal, which have intensified near large
towns, overgrazing, shortening of forest fallow periods, cultivation of rangelands, and land clearance for agriculture and livestock husbandry are the
main factors that reduce vegetation cover. Moreover, forced migration due
to wars, population shifts or droughts have often pushed people to settle in
areas that are alien to them, and therefore agricultural and cropping practices
may be more destructive compared to those used by the local people (Kirmse
and Norton 1984, Bellefontaine et al. 2000).
Reduction of vegetation and, especially, replacement of perennial trees
by annual plants cause decreases in soil fertility, litter, and organic matter,
subsequently increasing erosion. Natural, periodic droughts enhance this
adverse development (Kirmse and Norton 1984). Land degradation does not
only disturb natural plant communities but causes disturbances in plantmicrobe symbioses, which are critical factors in helping further plant growth
in degraded ecosystems (Requena et al. 2001).
Planting of trees is one important practical step to interrupt or retard this
deleterious process. Agroforestry, in which crop plants are grown together
with perennial, N2-fixing trees, has traditionally belonged to the cultivation
systems of many tropical areas. These trees were rediscovered in late the
1970’s and 1980’s when the governments and foundations all over the world
funded the research of biological N2 fixation after the energy crisis 1973. The
term Multi-Purpose-Trees, including roughly 100 tree species, was invented
at that time. Besides being suitable for the production of fuelwood and
timber, rapid growth, production of human food and animal fodder, and the
ability to improve soils and crops nearby, were their typical features (Smith
and Smith 1992). In the beginning, new N2-fixing tree species were needed
for agroforestry purposes. This was important especially in Africa, where the
consumption of mineral fertilisers was, and still is low, 2.2-3.9% of the global
use (Dakora and Keya 1997). Nowadays, the need of trees with different
properties has exploded. Large areas have to be brought back under forest
cover in order to reverse the current trend of deforestation, to conserve biodiversity (Khurana and Singh 2001) and to prevent the development of the
global warming.
Acacia and Prosopis trees originating in arid and semiarid regions of the
tropics and subtropics, are good candidates for reforestation of dry, marginal
1
or degraded lands with low fertility and/or high salinity. As drought and salt
tolerant trees, they can grow in harsh conditions. Moreover, most Acacia
and Prosopis species are capable of forming root nodules in symbiosis with
root nodule bacteria called rhizobia. In the nodules, rhizobia fix atmospheric
nitrogen (N2) and transform it into a form that is available to the host plant.
Legume symbioses have been estimated to contribute 70 million metric tons
N per year out of the total global fixation of 175 million metric tons per year.
Approximately one half of N fixed by the legumes is derived from the tropics
and the other half from temperate zones (Brockwell et al. 1995).
In agriculture, crop legumes have been inoculated with appropriate,
effective rhizobial inoculants in order to assure formation of N2-fixing nodules and, subsequently to improve plant growth. However, whether the
exploitation of the N2 fixation capacity of leguminous trees, for example, by
using inoculation or inoculated seedlings or seeds when planting trees to
the field, is largely unknown. More basic knowledge about the symbiosis
and ecology of the associations between rhizobia and leguminous trees,
such as Acacia and Prosopis spp., is still needed. An understanding of this
relationship may facilitate growth of well N2-fixing plants in fields and may
help to develop appropriate field transplanting techniques. Consequently, the
amount of surviving tree seedlings in the field will increase, which improves
the chance to succeed in reforestation.
Table 1.1. Classification of tropical climates adapted by Giller and Wilson (1991).
Climatic
zone
Temperature Rainfall
month-1
year-1
Wet tropic
> 18oC
Dry
period
> 1 800 mm
Geographic
distribution
River basins of
Amazon and Congo,
lowland of South-East
Asia
Wet and dry > 18oC
tropics
300 1800 mm
A dry period
of at least
two months
Subhumid and
semiarid tropics; most
part of Asia, savannah
areas of South
America and Asia
Dry tropics
< 300 mm
Long dry
period of
several months
Africa North of 15oN,
Australia South
of 15oS
Cool tropics
-3oC - +18oC Variability
in rainfall
At high altitudes
2
1.2. CHARACTERISATION OF ARID AND SEMIARID REGIONS
The major climatic zones in the tropics are mainly distinguished by the
amount and distribution of the rainfall throughout the year (Table 1-1). Many
semiarid areas are characterised by large variations in rainfall. For example,
in a semiarid desert in the Sudan, where the average rainfall is 185.1 mm,
the rainfall ranged 1941-1980 between a minimum of 58.5 mm (1960) and
a maximum of 442.0 mm (1978) per year (Ahmed 1986).
Arid and semiarid zones cover approximately four-tenths of the earth’s
land area, and are inhabited by one tenth of the world’s population. They
are mainly located in two subtropical belts. The northern part includes the
Sahara and Sahel, Middle East, Southern Asia, and part of North America.
The other is in the south, including part of Southern Africa, Australia and the
dry zones of Peru, Chile, Argentina and Brazil (Olivares et al. 1988). According to FAO (Bellefontaine et al. 2000), 66% of the world’s dry and very dry
ecological zones lie within the African continent. Fifty-two percent of the
dry deciduous forests are found in Africa, 23% in the Asia-Pacific region and
25% in Latin America.
1.2.1. Dry tropical forests
FAO (Bellefontaine et al. 2000) has defined dry tropical forests as those areas,
where the annual rainfall over the past 10-15 years ranged between 300 and
1 200 mm, and where there were 5-10 dry months a year (rainfall < 30 mm
per month). The dry tropical forests include the following vegetation types:
dry deciduous forests, thickets, open woodlands, savannahs, and steppes
(wooded, tree and shrub). This classification is mainly based on the degree of
the crown cover and the height of the woody vegetation (Figure 1-1). Acacia
and Prosopis species are characteristic of areas where the trees grow more
separately (Table 1-2).
1.2.2. Soil types
Generally, in large areas of tropical and subtropical Africa and South America
continuous high temperatures with high rainfalls have caused extreme weathering and leaching of soils developed on old rocks. More fertile soils occur
in areas that have a relatively recent addition of volcanic ash or alluvium
containing minerals, or in climates in which the long dry season has retarded
the rate of weathering (Giller and Wilson 1991, Lal 2000).
Although the ability of soils to support plant growth mainly depends
on the surface soil horizons, the classification of soils gives background
information about the geographical distribution of different soil types, and
advances and problems occurring in them (Giller and Wilson 1991, Lal
3
Figure 1-1. Types of woody or partially woody stands of dry tropical areas according to
Letouzey (1982) and Bellefontaine et al. (2000).
4
Table 1-2. Acacia and Prosopis species found in dry tropical forests according to
FAO’s report (Bellefontaine et al. 2001). Dry areas with a Mediterranean climate
and dry zones of Australia and the Pacific was not included in the report.
Domain Acacia and
Prosopis species
Forest type
Occurrence
Dry deciduous forest
Africa: Sudanian domain
P. africana (in legume
forests)
Thickets
Africa
A. ataxacantha
(climbing acacia)
Open woodlands
“ Zambezian domain
Acacia spp.
Savannahs
“ Sudania and Sahelian
domains
Asia: central Indian region
A. seyal, A. senegal,
A. tortilis ssp. raddiana
Acacia spp.
Steppes
Africa: Sahelian domain
A. tortilis ssp. raddiana,
A. senegal,
A. ehrenbergiana
P. juliflora
Prosopis spp.,
Acacia spp.
Central America
Asia: north-west India
2000). According to the classification of USDA (Soil Survey Staff 1975), Ultisols, Oxisols, Alfisols, Aridisols and Vertisols are the major soil types in arid
and semiarid regions. Highly weathered Ultisols and Oxisols are found in
parts of West Africa and Australia, which used to be wetter but are now dry.
Alfisols, containing low activity clay similarly to Ultisols and Oxisols, are
widely distributed in the semiarid tropics (e.g. Sub-Sahara). Alfisols and Ultisols have low water retention capacity, and are susceptible to soil erosion
and compaction. Aridisols, affected by severe drought stress, contain high
concentrations of CaCO3 and CaSO4, and cover a large area of Africa (Giller
and Wilson 1991, Buresh and Tian 1998, Lal 2000). Vertisols with a high
proportion of clay (2:1), are found for example in acid savanna, and cover
large areas in India, Ethiopia and Sudan (Giller and Wilson 1991).
About 36% of the tropical soils are dominated by low nutrient reserves.
These, usually highly weathered soils, have limited capacity to supply P, K,
C, Mg and S (Sanchez and Logan 1992). Deficiency of N is common, especially in cultivated soils. In Africa, approximately 90% of the mineral N is
found in living plants Lack of micronutrients occurs in places. Extensively
weathered soils can have such a low pH that Al, and sometimes Mn, may
become soluble and, thus toxic to the plants. Vertisols can fix large amounts
of P (Giller and Wilson 1991, Dakora and Keya 1997).
5
1.2.3. Saline soils
Saline soils are formed under hot, arid and semiarid conditions due the accumulation of salts in the topsoil. They can form naturally or as the result of
poorly managed irrigation or clearing from trees (Giller and Wilson 1991).
Saline lands (e.g. salt desert) represent about 15% of the arid and semiarid
lands of the world, whereas salt-affected cultivated soils represent about
40% of the world’s irrigated lands (Zahran 1997). Saline soils predominantly
contain chlorides and sulfates of Na, Ca and Mg. Soils salinized with neutral,
soluble salts of Na are termed saline-sodic. There, toxic excess of neutral
salts prevents plants from normal growth (Gupta and Abrol 1990).
Many soils in arid and semiarid regions have CaCO3 in the soil profile.
If calcareous soils have an excess of exchangeable Na and high pH, caused
by the presence of NaHCO3 and NaCO3, the soils are termed alkali soils.
These soils have a poor physical structure, which together with Na toxicity
adversely affects plant growth. The elements commonly observed in toxic
concentrations include Na, Mo, Bo, and sometimes Se. Alkaline soils contain
adequately P and Zn but otherwise they may suffer from low fertility (Gupta
and Abrol 1990).
1.3. ROLE OF N2-FIXING TREES IN DRY TROPICAL ECOSYSTEMS
1.3.1. Trees improve soil fertility
Generally, savannah and desert soils have poor fertility. However, soil fertility
and growth of annual vegetation are frequently superior under tree crowns
compared to that of areas distant from the trees. The studies on African
tropical semiarid savannah and on warm desert ecosystems in the Sonoran
Desert, California, USA, showed that organic matter and N were concentrated in the soil surface, in the savannah 0-10 cm and in the desert 0-30 cm
(Barth and Klemmedson 1978, Bernhard-Reversat 1982, Virginia and Jarrel
1983, Belsky et al. 1989). Moreover, N and C concentrations were significantly higher in soils under tree or bush canopy than in open grassland or on
dunes. Soil Ca, K, P (Virginia and Jarrel 1983, Belsky et al. 1989) and Mg
(Virginia and Jarrel 1983) also were highest in surface soil beneath the trees.
The situation was similar with soil organic C, N, P and K in A. senegal plantations in Senegal. N and K contents increased in surface soils as the plantation aged (Deans et al. 1999). Five to 30-year-old P. juliflora plantations
founded in degraded alkaline soils in semiarid India also improved levels
of organic C, N, P, Ca and Mg. In addition, growth of the Prosopis stands
restored alkaline soils by decreasing soil pH, electrical conductivity (salinity)
and exchangeable Na levels (Bhojvaid and Timmer 1998). By reducing solar
radiation and soil temperatures and by improving the moisture status of the
6
soil the tree canopies created a more favourable microclimate compared to
that of open fields (Belsky et al. 1989, Belsky 1994, Bhojvaid and Timmer
1998). Microbial biomass was also higher in soils from the canopy than in
soils from the root or grassland zones (Belsky et al. 1989).
The origin of N and/or organic C accumulated under the tree canopy is
not well understood. For example, it is not known whether the N has resulted
from the symbiotic N2 fixation, from animal deposits or exclusively from
the root uptake of deeply located N reserves. It is not clear either, which factors actually are responsible for the increased herbaceous-layer productivity
under and near the tree (Bernhard-Reversat 1982, Belsky et al. 1989, Belsky
1994, Deans et al. 1999). However, it has been commonly agreed that the
greater fertility of canopy soils is linked to deep-rooted trees and shrubs,
which recycle nutrients from the depth to surface soils (Bernhard-Reversat
1982, Belsky et al. 1989, Buresh and Tian 1998). Buresh and Tian (1998)
summarised soil improvements by trees into three categories: i) increased
supply of nutrients through increased inputs and reduced outputs (reduced
leaching of nutrients); ii) increased availability of nutrients through enhanced
nutrient cycling and conversion of nutrients to more labile forms; and iii) a
more favourable environment for plant growth through improved soil chemical and physical properties.
1.3.2. Significance of N2 fixation
Methodological problems in the quantification of N2 fixation for adult trees
are major reasons for the uncertainty of the precise source of N accumulated beneath the woody legumes (Buresh and Tian 1998). Searching of
nodules from adult trees is also a very tedious work (Bernhard-Reversat
1982, Högberg 1986). An approach to estimate N2 fixation is the comparison
of the natural 15N (δ15N) abundance in tissue of presumed N2-fixing plants
with that of presumed non-N2-fixing plants growing on the same site, and
with that of soil N. 15N is usually more abundant in soils than it is in the
atmosphere. Consequently, the 15N abundance would be lower in N2-fixing
plants than in non-N2-fixing plants (Shearer et al. 1983, Högberg 1986).
The use of this technique has indicated that in some areas the N was
derived from N2 fixation. In the Sonoran Desert, N2 fixation of Prosopis was
concluded to be very important at six out of seven sites, the relative contribution of the N2 fixation being 43-61%. At the site where Prosopis were not N2fixers, N2 fixation was important for papilionoid legumes (70%; Shearer et
al. 1983). Along an aridity gradient in Namibia, the contribution of N2 fixation to the total N concentration of the mimosoid trees (e.g. several Acacia
species, Faidherbia albida, P. glandulosa) was estimated to be about 30%,
but the variation within and between species was large (Schulze et al. 1991).
The mulga is an arid or semiarid ecosystem in Australia, in which A. aneura
7
(mulga) and other acacias with similar properties comprise the dominant
arboreal element. The 15N abundance analysis showed that in the regular
mulga, acacias do not fix N2, apparently due to high amounts of soil mineral
N. Instead, in an exceptional, heavily leached sand plain mulga acacias and
two papilionoid legumes were nodulated and fixed N2 (Pate et al. 1998).
Prosopis spp. (P. alba, P. tamarugo) were able to grow in the rainless
region of the Atachma Desert, Chile. Lack of surface moisture inhibited leaf
decomposition and prevented N cycling. However, the trees were able to
extract water and nutrients from ground water through the deep roots, and
fixed N2 with nodules located in moist soil layers (Ehleringer et al. 1992).
Herbaceous legumes grown under tree canopies can also fix N2 (BernhardReversat 1982, Pate et al. 1998).
1.4. THE FAMILY LEGUMINOSAE (OR FABACEAE)
Of all plants used by man, only the grasses (Gramineae) are more important
than the legumes (Allen and Allen 1981). Leguminous plants are used as
forage for the cattle, and their cultivation alternated with cereals or other
plants improves soil fertility. Plant sources contribute about 70% of the
human protein need, and leguminous seeds account for 19% of this amount
(Peoples and Herridge 1990). With approximately 750 genera and nearly
20 000 species, Leguminosae is the third largest family of flowering plants
(Allen and Allen 1981). Its species are found throughout the world but the
greatest diversity is found in the tropics and subtropics. Whereas herbaceous
legumes are common in the temperate regions, most legumes in the tropics
are trees and bushes. All legumes bear pods, the characteristic by which they
most easily can be recognised (NAS 1979).
Based on mainly floral differences (Allen and Allen 1981), taxonomists
have traditionally divided the Leguminoseae family into three subfamilies.
The subfamily Caesalpinioideae contains approximately 2 500 - 2 800 species, which are mainly trees of the tropical savannahs and forests of Africa,
South America and Southeast Asia. Approximately 3 000 species of the subfamily Mimosoideae are mostly small trees and shrubs of tropical and subtropical regions of Africa, North and South America, Asia and Australia. The
subfamily Papilionoideae contains 12 000-13 500 species. They are mainly
herbs, distributed world-wide (NAS 1979, Doyle 1994, Sprent 2001). Nodulation is common among the subfamilies Mimosoideae and Papilionoideae
but within the Caesalpinioideae subfamily, only few species or genera can
nodulate (Sprent 2001).
8
1.5. ACACIA AND PROSOPIS TREES (MIMOSOIDEAE)
Due to the ecological and economical importance and morphological and
biological similarities, Acacia and Prosopis trees are very often studied
together (Bukhari 1997a). The name Acacia is derived from Greek and means
”point” or ”barb” in reference to thorns. Only Australian acacias are thornless. The Latin name Prosopis is an ancient Greek name for burdock (Allen
and Allen 1981). Like the Latin name reveals, Prosopis trees also have thorns,
though thornless cultivars occur. The name mesquite is used for several
Prosopis species in North America, whereas the name algarrobo is used
commonly in South America (NAS 1979).
1.5.1. Geographical distribution
The genus Acacia is pantropical having a wide distribution in the tropics.
However, in Australia, in which their main development has presumably
occurred (Brenan 1965), acacias have invaded temperate regions (Norris
1956). Out of the over 1200 Acacia species (Chappill and Maslin 1995)
about 850 are endemic to Australia, seven to South America and 135 to
Africa, with some species also occurring in Asia (Maslin and Stirton 1997).
Several Acacia species grow in the arid and semiarid regions of the tropics.
The genus Prosopis, a tree characteristic for the American continent, is
not particular tropical (Brenan 1965), with some species growing in warm
deserts of Texas, New Mexico, Arizona and California (Fisher 1977). The 40
species are distributed in arid and semiarid areas of North and South America, and three species are found in Northern Africa and Eastern Asia. Most
of the species (31 out of 44) are indigenous to South America. Only one species, P. africana, is indigenous to Africa (Burkart 1976). Like several tropical
or subtropical trees, many Acacia and Prosopis species have spread under
the influence of man from one continent to an other.
1.5.2. Biological and ecological properties
Information about the Acacia and Prosopis species used in this thesis is gathered in Table 1-3. In general, Acacia and Prosopis trees resemble each other
in appearance. They are small or medium-sized trees (5-15 m) or shrubs.
Only few species, e.g. A. sieberiana (Maydell 1990), can reach up to 25
m height. Leaves are typically small and bipinnate. In Australian acacias
they are reduced after the seedling stage to a leaf-like petiole termed a phyllodium. In dry areas, the trees are drought-deciduous, but otherwise they
tend to be evergreen (Roshetko 2001). Except Australian acacias, Acacia and
Prosopis trees bear thorns, which may disturb their cultivation if farmers are
not used to them. The flowers are fragrant, showy globular heads, spikes
9
Table 1-3. Geographical distribution, biological and ecological properties, and
utilisation of Acacia and Prosopis species used in this work.
Annual
Temperature
rainfall (mm) range °C
Tree species
Geographic origin
Tribe Acaciae
Acacia
A. angustissima
(Mill.) Kunze
- 6 varieties
Height
Central America
- in tropical and
subtropical areas
900-3000
5 - 30°C
Shrub 7m
A. holosericea G.
Don
Northern Australia
- hot, subhumid zones
200-1500
Mild frost 39°C
Shrub 8m
A. mellifera
(Vahl.) Benth.
Arabian Peninsula,
Egypt-South Africa
A. nilotica (L.) Del.
- 9 subspecies
Africa, Arabian
Peninsula-India-Burma
A. oerfota (Forssk.)
Schweinf.
Africa
A. senegal (L.) Willd.
- 4 varieties
Shrub
-9m
250-1500
-1 - +50°C
Shrub
- 20 m
Africa, Arabia, Iran
India, Pakistan
100-950
-4 - +48°C
2-6 m
A. seyal Del.
- 2 varieties
Semiarid zones
of Africa
250-1000
5 - 55°C
9-17 m
A. sieberiana DC.
Semiarid zones
of Africa
400-800
A. tortilis (Forsk.)
Hayne ”umbrella thorn”
- 4 subspecies
Arid and semiarid
zones of Africa, Arabia,
Near and Middle East
40-1200
Tribe Mimoseae
Prosopis
P. africana (Guill. &
Perr.) Taub.
Senegal-Ethiopia,
Egypt-Sudan-Lake Victoria
P. chilensis (Molina)
Stunz
Peru, Bolivia, Chile,
Argentina
350-400
-4 - +45°C
< 12 m
P. cineraria
(L. Druce)
India, Pakistan, Iran
Afghanistan, Arabia
75-880
-4 - +51°C
5-18 m
P. juliflora (Sweet)
DC
- several varieties
Central America,
Mexico, introduced in
Africa and Asia
150-750
-4 - +51°C
Shrub
- 15 m
P. pallida (Willd.)
Kunth.
Dry coastlines of Peru
and Ecuador
250-600
< 25 m
0 - 50°C
4-20 m
4-20 m
< 18 m
Other trees growing in arid and semiarid regions
Faidherbia
albida (Del.) A.
Chev. syn. A. albida Del.
”miracle tree” of the Sahel
P. glandulosa Torrey
- 2 subspecies
”honey mesquite”
Dry Africa, Near East
Mexico to southern
U.S.A.
300 -1800
-6 - +44°C
200 <
frost - 38°C
References: von Maydell 1990, Hocking 1993, Roshetko 2001
10
15-31 m
3-7 m
Uses
Special features
Potential for fodder
and agroforestry
Thornless, earlier medicinal
plant of Maya Indians
Windbreaks, erosion control, potential
for fuel and fodder (dried phyllodes)
Introduced recently in
Africa and South Asia
Fodder, fuel, fences, medicine
Baskets made from the
roots
Fodder, fuel, tannin, gum,
timber, medicine
Tolerates alkaline and
saline soils, evergreen
Synonym A. nubica Benth.
Gum arabic, fuel, fodder, honey,
food, soil stabiliser, medicines
Drought resistant, used
in agroforestry in Sudan
Fodder, fuel, gum talha,
medicines
Suitable tree for silvopastoral systems
Fodder (pods), fences, tools and
implements, honey, windbreaks
One of the biggest umbrellashaped acacias in Africa
Fodder, fuel, fences, timber,
honey, windbreaks, medicine,
food, dune stabiliser in India
Well-known Acacia species,
particular drought resistant,
a good silvo-pastoral tree
Fuel, fodder, medicines,
arts, craft
The only native Prosopis in
Africa, ”ironwood”
Fuel, timber, fodder (pods),
food, honey, windbreaks, shade
If ground water, growth
with 250 mm rainfall
Fodder, agroforestry, timber, fuel,
dune stabiliser, honey, medicines
Drought resistant, deep rooting, tolerates
moderate saline and alkaline soils
Fodder (pods), food, fuel, timber,
honey, windbreaks, shades,
live fence, dune stabiliser
Deep rooting, 35 m;
tolerates alkaline and saline
soils, easily weedy, evergreen
Dune stabiliser, fuel, fodder
(pods), syrup, honey
Tolerates saline soils
Agroforestry, fodder, tools and
implements, medicines, seeds
are eaten in times of famine
Deep rooting, retains leaves
through the dry season,
drought tolerant, fast growing
Food and beverage in Mexico,
fodder (pods), timber, fences
honey, medicines
Moderately salt and frost, tolerant,
drought resistant if the roots reach
the ground water
11
or short racemes, the colour varying from cream-white (Acacia) or greenish
white (Prosopis) to bright yellow (Acacia, Prosopis; Allen and Allen 1981,
Hocking 1993). Several species are good honey plants (Maydell 1990,
Roshetko 2001). The length of pods depends on the species (3-30 cm;
Hocking 1993, Roshetko 2001). Usually, the mature pods fall a short distance
from the tree. Both wild and domestic animals, particularly goats and sheep,
are effective long-distance dispersers of seeds (Ahmed 1986).
The temperatures on the growth sites of Acacia and Prosopis can vary
within a wide range. Both tree genera tolerate day temperatures of 38-51°C
but are usually sensitive to frost (e.g. A. nilotica, P. pallida), or tolerate only
mild frost. Especially the seedlings are sensitive to low temperatures (Hocking 1993, Roshetko 2001). Acacia and Prosopis trees are not very fire-resistant (Hocking 1993). However, the seeds of A. sieberiana and A. gerradii are
released from dormancy by heat shock generated by fire (Khurana and Singh
2001).
Many Acacia and Prosopis species are very drought resistant. However,
their survival in low rainfall conditions (e.g. 40-50 mm y-1 for A. tortilis and
75 mm y-1 for P. cineraria; Hocking 1993, Roshetko 2001) or during long
rainless periods is possible due to their long tap roots reaching the groundwater or moisture soil layers. The tap roots of several Prosopis species, A.
senegal and A. tortilis can penetrate 10-35 m of soil (NAS 1979, Hocking
1993, Deans et al. 1999, Roshetko 2001). The deepest plant roots (P. velutina) were recovered in Arizona from an open-pit copper mine at a depth of
53 m (NAS 1979).
Several Acacia and Prosopis species are capable of growing in saline
soils. P. articulata, P. pallida and P. tamarugo could grow and fix nitrogen
in 0.3 M NaCl (Felker et al. 1981). Highly salt tolerant Australian acacias
(A. auriculiformis, A. stenophylla, A. maconochieana) could survive even at
1.7-1.8 M NaCl and showed shoot growth at 1 M NaCl (Aswathappa et al.
1987).
1.5.3. Capacity for N2 fixation
In spite of the fact that several Acacia and Prosopis species form N2-fixing
nodules (Allen and Allen 1981, Sprent 2001), information about their
N2-fixing capacity is restricted due to methodological problems (Dakora and
Keya 1997). Available data (Table 1-4) suggest that Acacia and Prosopis trees
of arid and semiarid regions are not very efficient N2-fixers (6-34 kg N ha-1
y-1) compared to woody legumes originating from more humid climate (100
- 800 kg N ha-1 yr-1; Peoples and Herridge 1990, Dakora and Keya 1997).
Nevertheless, low N2-fixing capacity might be connected to their low growth
rate in dry regions.
12
Table 1-4. Estimation of the proportion and amount of plant N derived from N2
fixation for Acacia spp., Prosopis spp. and Faidherbia albida grown in arid and
semiarid regions of the tropics (ARA, acetylene reduction assay; 15N, 15N-isotopic
technique).
N derived
Annual from N2
kg N ha-1 fixation Method
Species
Location
A. holosericea
Australia
Australia
Senegal
12 ± 4
6
10-20
A. pennatula
Mexico
34
F. albida
Austria
P. glandulosa
Sonoran desert,
U.S.A.
P. juliflora
Senegal
25-30
Reference
ARA
ARA
15
N
Langkamp et al. 1979
Langkamp et al. 1982
Cornet et al. 1985
ARA
Peoples & Herridge 1990
20%
15
N, in
greenhouse
Sanginga et al. 1990
50%
N content
Rundel et al. 1982
with Kjeldahl,
field study
19%
30%
15
N, pots
20
Diagne and Baker 1994
1.5.4. Nodulation in the field
If mineral N is present in the soil, for example after fire, woody legumes
prefer soil N to N2 fixation, and the nodulation is poor (Monk et al. 1981,
Pate et al. 1998). The legumes develop nodules only if the soil is poor in N,
because development, maintenance, and function of nodules require extra
energy and other resources from the plant. Young Acacia and Prosopis trees
and seedlings are usually nodulated if the soil is deficient in N (Jenkins et al.
1988b), but it has been difficult to recover nodules from adult trees (Högberg
1986, Virginia et al. 1986). This has led to the question, whether these trees
actually fix N2 in the field. Johnson and Mayeux (1990) gave three explanations for the failure to find nodules under Prosopis canopies: i) nodules were
formed but decomposed too rapidly to be detected. ii) The density of nodules
on the root system was too low in order to detect them by the coring method
(ø 4.7-10 cm). iii) The nodules were fed on by insects.
A reason for the apparent lack of nodules might be that in areas with wet
and dry seasons, nodulation and N2 fixation are seasonal, nodule activity
being at its highest during the wet season (Monk et al. 1981, Langkamp et
al. 1982, Hansen and Pate 1987). In Australia, Acacia stands depended on
mineral N early and late in the growing season, whilst in the middle of the
growing season 68-75% of the plant N came from nodule activity (Monk et
al. 1981). It is generally believed that the tree nodules are perennial (Allen
and Allen 1981). However, survival of Australian Acacia nodules through
the dry period into the second rainy season was not common (Monk et al.
1981).
13
Another reason for the absence of nodule observations might be that the
deep-rooted woody legumes bear nodules in deep soil layers. Root nodules
of P. glandulosa were found in depths of 3-7 m in Sonoran Desert (Jenkins et
al. 1988a) and in depths of 0.01-2.5 m in Texas (Johnson and Mayeux 1990).
The nodules of P. alba and P. tamarugo grown in Atacama Desert, Chile,
were located in the moist soil layers, 0.5-1.5 m below the surface (Ehleringer
et al. 1992). In addition, nodules were not regularly distributed but occurred
in patches scattered both in the vertical and horizontal plane (Johnson and
Mayeux 1990). It is not known whether the nodulation near the soil surface is
inhibited by unfavourable conditions (e.g. low moisture content, high temperature) or due to the elevated N content.
1.5.5. Multipurpose Acacia and Prosopis trees
Mankind has utilised Acacia and Prosopis trees for thousands of years. In
Biblical times acacias (probably A. seyal, A. tortilis) were used to build the
Ark of the Covenant and the Table of Tabernacle (Allen and Allen 1981). Their
wood was also used by ancient Egyptians for pharaoh’s’ coffins (NAS 1979).
In historical times, mesquite (P. glandulosa) was a widespread and important
resource of the native people in south-western North America. It was utilised
for food, fuel, shelter, weapons, tools, fibre, and dye. The mesocarp of the
pods, being largely sucrose and prepared as flour and further as a gruel,
cakes and beverages, provided a major carbohydrate component in native
diets (NAS 1979). P. cineraria, which is an important tree for desert dwellers
in India, providing fuel and fodder, is still considered to be a religious tree
(Hocking 1993).
Nowadays, Acacia and Prosopis trees are still of great importance for the
inhabitants of the arid and semiarid regions. In Africa, the foliage and pods
of acacias are important fodders both for domestic and wild animals. The
digestible protein content is 8-15% for leaves and pods, respectively. They
also are rich in minerals. The seeds are high in crude phosphorus, an element usually scarce in grasslands. In Australia, cattle and sheep browse
harsh acacia phyllodes during the dry seasons, when other plants are scarce
(Roshetko 2001). In the case of American Prosopis species, only pods can
be used as fodder and food. Due to their sugar containing mesocarp, the
pods are sweet, maximum glucose content being 27-35% (Maydell 1990,
Roshetko 2001). In times of famine, powdered bark (e.g. P. cineraria) has
been mixed with flour. In some areas, dried seeds of A. senegal (Hocking
1993) or pods of A. nilotica (Maydell 1990) and P. cineraria (Roshetko 2001)
are included in the human diet. However, pods and leaves of some acacia
species contain cyanogenetic glycosides, which may yield free hydrocyanic
acid and alkaloids toxic to livestock (Allen and Allen 1981).
In Africa, Acacia and Prosopis are important sources of firewood (calo-
14
rific value 3000-5000 kcal/kg) and charcoal (Hocking 1993). The wood is
generally hard and dense, and therefore also used for house construction
and manufacturing of furniture, tools and implements (Maydell 1990, Hocking 1993). Leaves, bark, gum, roots, pods and seeds are used medicinally
against a wide variety of diseases, wounds and burns (Hocking 1993).
Leaves, pods and bark may contain tannin. A. nilotica is an important
source of tanning material in Sahel and India (Maydell 1990, Hocking 1993).
Acacia and Prosopis trees are commonly used for windbreaks, stabilisation
of sand dunes, shading, and ornamentals. Thorny species are used for fencing (Maydell 1990, Hocking 1993, Roshetko 2001).
Several Acacia and Prosopis trees produce gum after they have been
wounded. A. senegal is the source of gum arabic, which is a highly watersoluble gum of relatively low viscosity. It is safe as a food additive. Collection of gum, tapping, consists of slitting the bark and collecting walnut-sized
gum globules. Gum is formed at leaf-fall and is carried to roots even when
the tree has not been tapped. An average annual yield is 250 g of gum per
tree. A. seyal produces water-soluble gum thala, which is traditionally used
for non-food applications (textile and paper manufacture, explosives; Allen
and Allen 1981, Roshetko 2001).
1.5.6. Problems
Properties that help Acacia and Prosopis trees survive, propagate and spread
under harsh growth conditions may cause problems in more favourable environments. In South-Western United States at the turn of the 20th century,
after the big herds of cattle had grazed for 30-50 years, the population of
Prosopis plants (P. velutina, P. glandulosa) was so increased that they were
considered as an agricultural pest. At the same time, their growth form
changed from single or few-stemmed tall trees to shrubby thickets (Fisher
1977). A similar phenomenon has occurred in Argentina, Paraguay (Fisher
1977), and South Africa, in which Prosopis spp. were introduced at the turn
of 20th century (Zimmermann 1991).
Below the soil surface Prosopis plants have a zone of buds which remain
dormant as long as the primary bud or tree grows and develops normally. If
the growth of the seedling or tree trunk is destroyed by freezing, drought, fire,
cutting or trampling, underground buds will initiate new growth, and multistemmed plants will form (Fisher 1977). Prosopis trees produce large quantities of seeds (Zimmermann 1991) which facilitate their effective spread,
if there are no limiting factors. Elimination of natural periodic prairie fires,
reduction of natural grass cover, increased dissemination of seeds by large
herbivores, and reduction or absence of seed-feeding predators contributed
to the increased Prosopis densities in USA and South Africa (Fisher 1977,
Zimmermann 1991). Drought resistant acacias (e.g. A. holosericea, A. nilot-
15
ica, A. tortilis) can also become weedy when introduced out of its native
range, e.g. in more humid zones, or if their growth is not restricted (NAS
1979, Zimmermann 1991, Hocking 1993).
1.5.7. Systematics of Acacia spp.
The genus Acacia was first systematically described in 1745. The major classification of acacias occurred 1875, when Bentham divided the subfamily
Mimosoideae into five tribes, Ingeae, Mimoseae, Mimozygantheae, Parkieae
and Acacieae. Within the tribe Acaciae, the genus Acacia contained six
series (Table 1-5) Chappill and Maslin 1995, Robinson and Harris 2000).
The first major realignment to this classification occurred 1972, when Vassal
(1972) viewed Acacia as a single genus consisting of three subgenera,
Acacia, Aculeiferum and Phyllodineae (Table 1-5). This is perhaps the most
commonly referred classification of Acacia spp. so far, although numerous
studies on their phylogeny and taxonomy have followed. However, no bigger
rearrangements have been done, because the taxonomy of acacias as well as
other mimosoid legumes is in a state of flux. The systematics of Acacia spp.
is not simple, and their classification (and nomenclature) will change in the
future (Chappill and Maslin 1995, Robinson and Harris 2000).
There are inconsistent opinions, how the three subgenera, Acacia,
Aculeiferum and Phyllodineae, are related to each other. According to the
cladistic analysis by Chappil and Maslin (1995), based on morphological
and physiological properties, the subgenera Aculeiferum and Phyllodineae
were closely related, and subgenus Acacia differed clearly from them. Con-
Table 1-5. Outlines of the major classifications and distributions of Acacia spp. according to
Robinson and Harrison (2000).
Bentham (1875)
Distribution
Robinson and Harrison (2000) of Acacia spp.
Vassal (1972)
Subfamily Mimosoideae
Tribe Mimoseae
Tribe Mimozygantheae
Tribe Parkiae
Tribe Acaciae
Genus Acacia
series Gummiferae
Tribe Mimoseae
Tribe Acaciae
Genus Acacia
subgenus Acacia
subgenus Acacia
series Vulgares
series Filicinae
subgenus Aculeiferum
subgenus Aculeiferum
series Botrycephalae
series Phyllodineae
series Puchellae
subgenus Phyllodineae
tribe Ingae
subgenus Phyllodineae
Faidherbia albida
Faidherbia albida
Tribe Ingeae
16
America, Africa,
Asia and Australia
America, Africa,
Asia and Australia
Australia, New
Guinea, Hawaii
Mascarena Islands
Africa
tradictory, Bukhari et al. (1998, 1999) suggested according to the analysis of
seed storage protein size variations and chloroplast phylogeny that the subgenus Aculeiferum was ancestral to the subgenera Acacia and Phyllodineae.
Based on a plastid DNA phylogeny, Robinson and Harris (2000) recently
proposed, that the subgenera Acacia and Aculeiferum are sister taxa whilst
the subgenus Phyllodineae is sister to tribe Ingeae (containing e.g. Calliandra
spp.). Moreover, the tribe Acacieae may contain the genus Acacia only with
two subgenera, Acacia and Aculeiferum (Table 1-5). The differences in interpretation of the analyses might be due to that some plant characters included
in the tests have arisen more than once during the evolution, whereas others
have been lost (Chappill and Maslin 1995, Robinson and Harris 2000).
Faidherbia albida (synonym A. albida), is a popular acacia in agroforestry
because it is capable of maintaining leaves during the dry season. Its taxonomic status has been particularly equivocal. Vassal (1972) moved Acacia
albida to the monotypic genus Faidherbia within the tribe Acacieae (Table
1-5). Several subsequent studies supported this division while others did not
(Chappill and Maslin 1995, Bukhari et al. 1998, 1999). In the recent work of
Robinson and Harris (2000) F. albida appeared to be basal to the tribe Ingeae
and to the subgenus Phyllodineae (Table 1-5).
1.5.8. Systematics of Prosopis spp.
Burkart (1976) has recognised five distinctive groups of species (sections) in
the genus Prosopis (Mimosoideae). According to his view, Prosopis is an old
genus, which diverged early into several principal lineages, some containing
very similar species and others forming clearly distinct groups (sections). The
systematics of Prosopis spp. is otherwise confusing because hybridisations
are common (Allen and Allen 1981, Zimmermann 1991). As a consequence,
several Prosopis species have been confused in the literature and/or in practice: P. juliflora and P. glandulosa (NAS 1979), P. chilensis and P. alba ,
P. chilensis and P. juliflora (NAS 1979, Roshetko 2001). Many studies indicate that Acacia and Prosopis trees might be closely related (Chappill 1995,
Bukhari 1997a, Bukhari et al. 1998). For example, A. oerfota (former A.
nubica) was found to be distantly related to the subgenus Acacia (Bukhari et
al. 1999), and fitting better in Prosopis than in Acacia (Bukhari 1997b).
1.6. RHIZOBIA NODULATING ACACIA AND PROSOPIS TREES
Root- and stem-nodule bacteria belong almost exclusively to the α-subclass
of Proteobacteria. The major rhizobia are currently divided into about
30 species within six genera: Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium (Wang and MartinezRomero 2000). However, recently new types of rhizobia have been found
17
from tropical legumes. Bacteria belonging to the genus Methylobacterium,
exhibiting both nodulation ability and methylotrophic properties (grew facultatively e.g. on methanol), were isolated from Crotalaria legumes (Papilionoideae; herbs and shrubs; Sy et al. 2001). Devosia sp. was isolated from
stem-associated nodules of the aquatic legume Neptunia natans (Mimosoideae; Rivas et al. 2002). Moreover, nodulation of tropical legumes by
Burkholderia sp. (Moulin et al. 2001) and Ralstonia sp. (isolated from
Mimosa spp.; Chen et al. 2001), members of the β-subclass of Proteobacteria, indicated that wide-range of bacteria can form symbioses with legumes.
Traditionally, rhizobia have been divided into fast- and slow-growers (i.e.
rhizobia and bradyrhizobia) but even this general idea may change. The
term meso means intermediate growth habit between fast and slow-growers
and thus, the name Mesorhizobium denotes both phylogenetic position and
growth rate (Chen et al. 1995, Jarvis et al. 1997). Studies with Kenyan
rhizobia showed that strains belonging to Bradyrhizobium spp. exhibited
very slow to intermediate growth, those belonging to Mesorhizobium spp.
showed intermediate to fast growth and strains belonging to Rhizobium spp.
and Sinorhizobium spp. grew fast or very fast (Odee et al. 2002).
Compared to the taxonomy of well-known rhizobia nodulating crop legumes (one plant species usually is nodulated by one or few rhizobial species), the taxonomy of rhizobia nodulating woody legumes is generally
confusing, because there is no strict host specificity between the tree legume
and the microsymbiont. A wide range of rhizobia, including both fast- and
slow-growers, can induce nodules on them. However, the spectrum of rhizobia capable of forming effective nodules on their hosts, is more restricted
(Zhang et al. 1991, de Lajudie et al. 1994, Odee et al. 1997, Nick et al.
1999a, b, Lafay and Burdon 2001).
1.6.1. The most common species and genera among tree rhizobia
Present data suggest that in dry Africa, acacias (subgenera Acacia and
Aculeiferum) and the introduced Prosopis spp. are commonly nodulated by
fast or moderately fast-growing rhizobia, and especially, by those belonging
to the genera Sinorhizobium and Mesorhizobium (references in Table 1-6).
Slow-growing bradyrhizobia have been isolated only occasionally from root
nodules of African acacias (Dreyfus and Dommergues 1981, Odee et al.
1997). However, Bradyrhizobium species were capable of forming effective
nodules on A. seyal (Odee et al. 2002). F. albida, which is distantly related to
other acacias (Vassal 1972, Robinson and Harris 2000) seems almost solely
to prefer Bradyrhizobium spp. (Table 1-6).
Australian acacias belonging to the subgenus Phyllodineae (Vassal
1972, Robinson and Harris 2000) and occurring in more temperate climate
(Norris 1956) prefer slow-growing Bradyrhizobium species (Dreyfus and
18
Table 1-6. Rhizobial species isolated from the nodules of Acacia and Prosopis trees
growing in arid and semiarid regions of the tropics.
Species
Africa
Bradyrhizobium sp.
B. elkanii
B. japonicum
Mesorhizobium sp.
huakuii
loti
plurifarium
Rhizobium sp.
R. tropici
Sinorhizobium sp.
S. arboris
S. kostiense
S. saheli
S. terangae bv. acaciae
Host plants
References
F. albida (Se, Ke), A. nubica (Ke)
F. albida (Se, Ke)
F. albida (Se, Ke)
A. polycantha; A. xanthophloea,
A. tortilis (Ke)
A. arenaria (Ke)
A. senegal, A. tortilis subsp. heteracantha
heteracantha (Ke)
Acacia sp., A. senegal, A. seyal, A. tortilis
subps. raddiana, P. juliflora (Se, Su)
A. senegal (Su)
A. karroo (Ke)
A. cyanophylla, A. gummifera, A. horrida
A. tortilis subsp. raddiana (Mo);
A. senegal (Su), P. chilensis (Ke)
A. senegal. P. chilensis (Su, Ke)
A. senegal. P. chilensis (Su)
A. senegal. P. chilensis (Su)
A. horrida, A. mollissima, A. tortilis subsp.
raddiana, A. senegal (Se); A. senegal (Su),
P. chilensis ( Ke)
Dupuy et al. 1994; Odee et al. 2002
Dupuy et al. 1994; Odee et al. 2002
Dupuy et al. 1994; McInroy et al. 1999
Haukka et al. 1998; Odee et al. 2002
Haukka et al. 1998, McInroy et al. 1999
McInroy et al. 1999
de Lajudie et al. 1998
Nick et al. 1999a
McInroy et al. 1999
Khbaya et al. 1998; Nick et al. 1999b
Nick et al. 1999b
Nick et al. 1999b
Nick et al. 1999b
de Lajudie et al. 1994; Lortet et al.
1996; Nick et al. 1999b
Asia
Mesorhizobium loti
A. catechu, A. nilotica (North India)
Kumar et al. 1999
Australia
Bradyrhizobium sp.
B. elkanii
B. japonicum
A. obliquinervia
Several Acacia spp.
A. saligna; several Acacia species
Lafay and Burdon, 1998
Lafay and Burdon, 2001
Marsudi et al. 1999;
Lafay and Burdon, 2002
Marsudi et al. 1999
spp. (lupinus)
A. saligna;
Rhizobium
leguminosarumbv. phaseoli A. saligna
tropici
A. saligna; several Acacia species
Latin America
Mesorhizobium chacoense P. alba
Sinorhizobium sp.
Prosopis sp. (Mexico)
P. juliflora (Brazil)
Abbrevisions:
Marsudi et al. 1999
Marsudi et al. 1999;
Lafay and Burdon 2001
Velazquez et al. 2001
Haukka et al. 1998
Haukka et al. 1998; Moreira et al. 1998
A., Acacia; P., Prosopis;
Ke, Kenya; Mo, Morocco; Se, Senegal; Su, Sudan
Dommergues 1981, Barnet et al. 1985, Table 1-6). In a recent study, 96.6%
of the 118 bacteria isolated from the nodules of 13 different Acacia species belonged to Bradyrhizobium sp. (Lafay and Burdon 2001). The situation might be similar in other legumes since most of 735 rhizobial strains
(94.3%) isolated from shrubby legumes of temperate south-eastern Australia
were identified as Bradyrhizobium sp. (Lafay and Burdon 1998). However,
19
also fast-growing rhizobia, showing similarity with R. leguminosarum bv.
phaseoli and R. tropici have been identified from root nodules of Australian acacias (Table 1-6).
The taxonomy of rhizobia nodulating Prosopis trees on the American
continent is still poorly known. The first studies indicated that some dry
growth sites of Prosopis spp. were occupied only by fast-growing rhizobia
(Jenkins et al. 1988b, Milnitsky et al. 1997), whereas the other sites were
occupied by fast and slow-growers (Jenkins et al. 1987, 1989, Waldon et al.
1989). Two strains belonging to Sinorhizobium sp. have been identified from
Prosopis nodules in Brazil and Mexico (Table 1-6). Hence, a new Mesorhizobium species, M. chacoense has been characterised from the nodules of P.
alba trees grown in central Argentina (Velazquez et al. 2001).
1.6.2. Occurrence of rhizobia in deep soils
As discussed in Chapter 1.5.2, several Acacia and Prosopis species occupying arid and semiarid regions develop deep root systems, located at a
depth of 4-40 m. Apparently, deep-rooted trees develop two distinct lateral
root systems, one near the surface, the other exploiting deep-water resources
(Jenkins et al. 1987). Recovery of nodules in deep soils indicates that these
soils contain rhizobia. Indeed, under F. albida in Sahelia, 103 rhizobia g-1 of
soil were found as deep as 34 m below ground (Dupuy and Dreyfus 1992). In
the warm deserts of USA, rhizobia capable of inducing N2-fixing nodules on
Prosopis spp. were observed at a depth of 4-13 m (Jenkins et al. 1987, 1988a,
1989). Generally, population densities of Prosopis rhizobia were substantially greater (103-107 cells g-1 of soil) in deep soil layers than were at dry soil
surfaces (< 10 cells g-1 of soil; Virginia et al. 1986, Jenkins et al. 1988a). The
situation was similar with bradyrhizobia under F. albida grown either in dry
or moist sites (Dupuy and Dreyfus 1992).
Interestingly, rhizobial population at different depths under Prosopis trees
generally consisted of both fast-growers and slow-growers. Although the concentration of salts, P and N decreased with increasing depths (Virginia et al.
1986, Jenkins et al. 1988a, Ehleringer et al. 1992), no single factor (e.g. soil
moisture, nutrient levels) explained cell concentrations or the distribution of
fast- and slow-growers across the desert ecosystems (Jenkins et al. 1988a,
1989). According to the physiological tests, slow-growers from phreatic soils
and those from surface soils formed two distinct groups (Waldon et al. 1989).
Genetic studies indicated that the fast and slow-growers fell into soil zonecorrelated groups of divergent but related isolates (Thomas et al. 1994). The
recently characterised Argentinean species, M. chacoense, which effectively
nodulated at least P. alba, P. chilensis and P. flexuosa, was described as slowgrowing. Colonies (1-3 mm in diameter) appeared on yeast-mannitol (YEM)
agar after incubation for seven days at 28ºC; generation time 10-24 h in YEM
20
broth (Velazquez et al. 2001). Thus, perhaps the slow-growers found under
other Prosopis trees represented Mesorhizobium sp.
1.7. DEVELOPMENT OF SYMBIOSES BETWEEN RHIZOBIA AND LEGUMINOUS
PLANTS
The development of the symbiotic relationship between rhizobia and legumes
is a highly interactive process that include i) molecular communication
between the free-living organisms, ii) an infection phase when rhizobia enter
root or stem nodules and iii) a final symbiotic phase when rhizobia occupying
nodules fix N2. The dialogue between a legume and rhizobia starts when the
plant releases organic compounds that attract rhizobia near their roots. The
plant also produces fenolic compounds, flavonoids, which induce the expression of nodulation (nod) genes of rhizobia. The nod genes encode nodulation
factors (lipochitin oligosaccharides) which in return induce early events of the
infection process in the host plant, such as changes in the root epidermis,
deformation and curling of root hairs (if it has hairs), and cell divisions in the
root cortex (= initiation of nodule primordium). During formation and function
of the nodule, the host plant expresses nodule-specific or nodule-enhanced
genes, nodulin genes (Fisher and Long 1992, Kijne 1992, Phillips 1999).
Depending on the species, rhizobia can nodulate several different plant
species (broad host range) or only one or few plant species (narrow host
range). Some bacteria have even adapted to a particular plant cultivar. Both
rhizobial Nod factors and plant flavonoids are involved in the host specificity.
Each rhizobium/plant species produces several, various Nod factors/flavonoids,
and differences in their sets and amounts influence host specificity. Other host
recognition systems are also required to promote proper development of N2fixing nodules (Brewin 1998, Hadri and Bisseling 1998, Hadri et al. 1998).
In general, the plants try to protect themselves against attacks of pathogenic bacteria and other microbes, for example, by generation of H2O2 (called
oxidative burst). During a proper Rhizobium-legume association, this hypersensitive reaction is controlled by decomposing H2O2. (Bueno et al. 2001).
The sugars present on cell surfaces (i.e. exopolysaccharides, EPS; capsular
polysaccharides, CPS; lipopolysaccharides, LPS) are essential for rhizobia to
avoid host defence responses during the invasive phase (Brewin 1998, Hadri
and Bisseling 1998, Albus et al. 2001). Albus et al. (2001) proposed recently
that LPSs released from the bacterial surface function as specific signal to the
plant, and promote symbiotic interaction and suppress defence responses.
1.7.1. Colonisation and attachment of rhizobia on the plant root
Rhizobia are polarly or subpolarly flagellated (Bradyrhizobium) or peritrichously flagellated (Rhizobium, Sinorhizobium) and thus, they can move.
21
Non-motile and non-flagellated cells are common in laboratory grown cultures
(Yost and Hynes 2000). The motility is especially important when rhizobia
compete for nodule occupancy (De Troch and Vanderleyden 1996).
In the rhizosphere, rhizobia migrate toward plant roots by chemotactic
response. Rhizobia are nonspecifically chemotactic to a wide range of attractants (e.g. dicarboxylic acids, amino acids, sugars, aromatic and hydroaromatic acids). The flavonoids attract rhizobia more specifically (De Troch and
Vanderleyden 1996, Yost and Hynes 2000). Attachment of rhizobia on the
roots and root hairs is a complex process, including surface factors of bacteria
(e.g. rhicadhesin protein, Ca2+ ions, cellulose fibrils, CPS, LPS, EPS , cyclic 1-2
β-glucans) and the plant (lectins and other adhesins (De Troch and Vanderleyden 1996, Matthysse and Kijne 1998). According to the lectin recognition
hypothesis, plant lectins mediate specificity in the Rhizobium-legume symbiosis. Although this hypothesis was presented already in the early 1970s
Table 1-7. Major features of the three infection modes. Subfamilies of the plants
according to Allen and Allen (1981). M=Mimosoideae, P=Papilionoideae.
Penetration
into the root
Induced cell
divison
Penetration
in the cortex
Spread inside
nodule
Through root
hairs and via
infection
threads
Inner cortex
Infection
threads
Infection
threads
Outer cortex
Between cells
Short infection
threads, host
cell divisions
Middle and
inner cortex
Between cells
Infection
threads
2B
?
Between cells
Short infection
threads
2C
Inner cortex
Between cells
Though altered
cell walls
2D
Outer cortex
Between cells
Trough altered
cell walls, host
cell divisions
Inner cortex
Between cells
Infection thread
like structures
1A
1B
2A
3
Crack entry
Between cells
of intact
epidermises
22
(Hirsch 1999), it was shown only recently that a novel root lectin, present on
the root surface of the legume Dolichos biflorus, is capable of binding rhizobial Nod factors (Etzler et al. 1999). Closely related lectin homologs were also
found from other legumes (Roberts et al. 1999).
1.7.2. Infection and nodulation
The early stages of the symbiosis, when rhizobia enter the plant root, penetrate in the root cortex and spread inside nodules, vary between legume
species (Table 1-7). In general, rhizobia can enter the root i) through root
hairs via a plant derived tunnel, named as an infection thread, ii) by crack
entry, or iii) between cells of intact epidermises. In the root cortex, rhizobia
penetrate towards the nodule primordium through transcellular infection
threads or by colonisation intercellular spaces. Inside the nodule, bacteria
Nodule
type
Plant species
References
Indeterminate Medicago sativa, Pisum sativa
Trifolium. spp., Vicia hirsuta;
Galega spp.; (P) temperate
herbs
Determinate Glycine max; Lotus corniculatus;
Lotus japonicus; Phaseolus
vulgaris; Vigna radiata; (P)
tropical, subtropical
and temperate herbs
Intermediate
type
Sesbania rostrata (P)
tropical woody plant
Kijne 1992, Brewin, 1998 Hadri
et al. 1998; Lipsanen & Lindström
1988, Räsänen et al. 1991
Turgeon & Bauer 1982, 1985, Kijne
1992, Hadri et al. 1998; Vance et al.
1982; Schauser et al. 1999;
1994; Tate et al. 1994 Cermola et al.
2000; Newcomb & McIntyre 1981
Ndoye et al. 1994
Indeterminate Neptunia natans; N plena ; (M)
(aquatic herbs)
Subba-Rao et al. 1995; James et al.
1992
Indeterminate Chamaecytisus profilus (P),
temperate shrub
Vega-Hernandez et al. 2001
Determinate
Chandler 1978, Boogerd & van
Rossum 1997; Chandler 1982
Arachis hypogaea;
Stylosanthes spp.; (P)
tropical herbs
Indeterminate Mimosa scabrella (M)
tropical tree
de Faria et al. 1988, Sprent &
de Faria 1988
23
may spread via infection threads, by entering through altered cell walls
and/or together with the division of host cells (Figure 1-2, Table 1-7).
There are two main nodule types: elongated indeterminate nodules
and speherical determinate nodules (Figure 1-3). The indeterminate type is
characterised by a persistent apical meristem, while the determinate type
lacks such a meristem. Indeterminate nodules are elongated and cylindrical
because new cells are constantly being added to the distal end of the nodule.
The spherical form of determinate nodules mainly results from cell enlargement, while cell division ceases early (Hirsch 1992). Typically, indeterminate
nodules are induced in the inner cortex whereas determinate nodules originate in the outer cortex (Table 1-7).
It is generally assumed that the nodule morphogenesis, the mode of infection and the uptake of rhizobia in nodule cells are under the control of the
host plant (Dart 1977, Kijne 1992). According to Boogerd and van Rossum
(1997), this view is supported by the following evidences: i) nodulating
and non-nodulating phenotypes are conserved among taxonomically related
legume species. ii) The nodule type bears botanical taxonomic relevance.
iii) One and the same strain may infect different host legumes by different
pathways.
It appears that infection through root hairs is common for temperate leguminous herbs, whereas plants infected by crack entry grow mainly in the tropics (Table 1-7). Indeterminate nodules are common on woody legumes in
the Mimosoideae and on temperate herbaceous legumes, whereas determinate
nodules are found mostly among tropical leguminous herbs (Table 1-7).
1.7.3. A closer look at the infection process
This section is based on the data about the well-known hair infection route
(Figure 1-2), but similar features presumably occur also in other infection
modes. Roots and root hairs are only transiently susceptible to rhizobia
(Bhuvaneswari et al. 1981). The most frequently nodulated region is located
above the zone of rapid root elongation and below the smallest emergent hairs
present at the time of inoculation (Turgeon and Bauer 1985). Usually, not all
hairs are deformed but deformed hairs have a patchy distribution on the roots
(Vandenbosch et al. 1985). Infections also occur in localised groups (Dart
1977). Although numerous hairs deform, only few of them become infected,
and only a small percentage of the infection sites result nodules. For example,
10 M. sativa seedlings were estimated to have 80 000 hairs. From them, only
52 infection threads were found, and 27 nodules were formed (Wood and
Newcomb 1989). In the case of Trifolium spp. and Vicia hirsuta, 1.4 - 38% of
infected hairs resulted in nodules (Dart 1977). The host plant tends to restrict
the number of infections and nodulations by eliciting hypersensitive reactions,
such as oxidative burst (Vasse et al. 1993, Santos et al. 2001).
24
Figure 1-2. Diagrammatic presentations of the different infection modes among
legumes. Drawings are based on the references mentioned in Table 7-1. Figures are
not drawn to scale.
25
Root hairs occur only inconsistently. Rhizobia penetrate the mucigel m, and primary layers of radial
walls, pw, of epidermal cells, and enter between host cells possibly by digesting the middle lamella,
ml. The primordium is induced in the inner cortex (a). Rhizobia penetrate deeper within a matrix
(b). In the nodule, multiplication of bacteria results in cell walls being pushed inwards, allowing
intracellular penetration of bacteria (c). Inside the cell, bacteria pultiply, and remain enclosed by cell
wall material. Structures resembling infection threads cross cell boundaries, infecting adjacent cells
(d). pw = primary cell walls, sw = secondary cell walls.
26
27
In many cases, rhizobia can penetrate the hair cell or root epidermis,
when bacteria are compressed between two adjacent epidermal cells of the
hair or/and the root (Kijne 1992). Obviously, rhizobia are capable of entering the hair through a completely eroded hole, which they have created by
localised enzymatic hydrolysis of the host cell walls (Mateos et al. 2001). The
plant-derived infection thread consists of similar compounds as the plant cell
wall (cellulose, xyloglucan, methyl esterified pectins, non-esterified pectins)
but it is more resistant to cell wall-degrading enzymes. The infection thread
matrix contains at least proline-rich proteins and glycoproteins (Verma and
Hong 1996, Brewin 1998).
1.7.4. The structure of nodules
In fully developed nodules two major tissue types can be recognised: the
peripheral tissue and the central tissue (Figure 1-3). In general, the peripheral tissue consists of large vacuolated cortical cells (outer cortex), a layer of
small sclerenchymatic cells (nodule endodermis), and several layers of small,
densely packed, vacuolated cells (parenchyma or inner cortex). The vascular bundles and the vascular endodermis are completely surrounded by the
parenchyma (Figure 1-3). The cortical cells outside the nodule endodermis
might be modified in a variety of ways, e.g. by corky cells with thickened
walls or by lenticels, which play role in gaseous diffusion (Robertson and
Farnden 1980). In the determinate nodule, the endodermis is located around
the infected central tissue (Tate et al. 1994; Figure 1-4), whereas in the case
of indeterminate nodule with apical meristem the endodermis stop short
before the apex (Dart 1977).
The central tissue of the indeterminate nodules contains large infected cells
and smaller, vacuolated, uninfected cells (Brewin 1991, Hirsch 1992). In M.
28
sativa, the central tissue consists of several well-defined histological regions
(Figure 1-3), in which bacteroids are differentiated gradually by five steps.
From the apical part to the basal region, the following zones can be distinguished: the bacteria-free meristematic zone I, the infection zone II, in which
bacteria are released from infection threads, the narrow interzone, II-III, the
N2-fixing zone III and the senescent zone IV (Robertson and Farnden 1980,
Vasse et al. 1990), in which host cells autolysis leads to the senescence of
all bacteroids (Paau et al. 1980, Vance et al. 1980). Timmers et al. (2000)
recently described an additional, saphrophytic zone V, locating proximal to
the senescent zone IV. In this new zone, bacteria were released passively from
remaining infection threads and invaded plant cells, which were already completely senesced. These intracellular rhizobia did not resemble morphologically bacteroids but were rod shaped. They could not fix N2 and were not
subjected to the host cell autolytic processes. Thus, they behaved like saprophytic bacteria (Timmers et al. 2000). From five different types of bacteroids,
only one type, locating in distal zone III, can fix N2 (Vasse et al. 1990).
The zones above described have not been found in determinate nodules
(Dart 1977; Figure 1-4). Therefore, it has been supposed that the cells in the
central tissue are proximately in a similar developmental stage (Hadri et al.
1998). However, according to a recent study invasion of nodule cells is not
synchronous, and not all cells of the central tissue differentiate simultaneously (Cermola et al. 2000). The uninvaded cell aggregates are distributed
among infected cells so that they are in contact with all invaded cells (Tate
et al. 1994). The structure of the aeschynomenoid-type determinate nodule,
such as that of Arachis hypogaea, groundnut, differed from other determinate
nodules. The endodermis-like cell layer was absent in the nodule cortex, and
the infected central tissue lacked interspersed uninfected cells (Boogerd and
van Rossum 1997).
29
1.7.5. The symbiosome and the symbiosomal membrane
Inside nodules, rhizobia are taken into the host cytoplasm by endocytosis
(Robertson and Farnden 1980), Brewin 1998). When the thread reaches a
target cell, synthesis of the infection thread wall stops and the bacterium
being released is surrounded only by the host plasma membrane (= infection
droplets; Verma and Hong 1996, Brewin 1998; Figure 1-2). In some woody
legumes, such as in Andira spp. (Papilionoideae; Faria et al. 1986) and in
all genera examined in the subfamily Caesalpinioideae (Faria et al. 1987)
rhizobia are not released but develop the N2-fixing capacity within tubular
threads.
After bacteria have been released into the nodule cytoplasm, the plasma
membrane transforms to the symbiosomal (peribacteroid) membrane. The
entity, consisting of the bacteroid(s) and symbiosomal membrane is called
the symbiosome. Extension of the membrane, due to the subsequent growth
and division of bacteroids, is achieved by inclusions of vesicles derived from
the Golgi or endoplasmic reticulum (Roth and Stacey 1989, Verma and
Hong 1996, Brewin 1998). The symbiosomal membrane provides a physical barrier between the host cytoplasm and the bacterial cell and controls
the exchange of substrates and signal molecules between the two partners.
For example, it allows import of inorganic minerals and reduced carbon to
bacteroids and export of fixed N and heme for leghemoglobin synthesis to
the plant cytoplasm (Verma and Hong 1996).
After bacteroids have ceased division, they develop the capacity of N2
fixation. In indeterminate nodules, this phase is accompanied by enlargement and morphological differentiation of bacteroids. This phenomenon has
not been detected in determinate nodules and fixation threads (Vasse et al.
1990, Hadri et al. 1998). During the differentiation, bacteroids have to adapt
to new physiological conditions (i.e. low oxygen content, low pH; Brewin
1998). Each symbiosome contains various numbers (1-20) of bacteroids,
depending on the host species and age of the nodule (Mellor and Werner
1990). It seems that in indeterminate nodules of temperate legumes, bacteroids are singly enclosed by the symbiosomal membrane, whereas in determinate nodules each symbiosome contains groups of bacteroids (Dart 1977).
The fusion of small symbiosomes to form a large one partly explains the
existence of multi-bacteroid symbiosomes (Cermola et al. 2000).
1.7.6. N2 fixation
Nitrogenase is the enzyme that catalyses the reduction of N2 into NH3 in all
N2-fixing organisms. The rhizobial nitrogenase consists of two components,
dinitrogenase (FeMo protein) and dinitrogenase reductase (Fe) protein. The
nitrogenase catalyzes the following reaction:
30
N2 + 16 MgATP + 8e- + 8H+ ⇒ 2 NH3 + 16 MgADP + 16Pi + H2
N2 fixation is an extremely energy-consuming reaction. Therefore, symbiotic N2 fixers that benefit from plant photosynthesis, mainly contribute biologically fixed N2 on the earth (Kaminski et al. 1998). In isolated bacteroids,
95% of the fixed N2 was reported to be excreted into the medium as ammonia, the remainder being incorporated into bacteroids (Robertson and Farnden 1980). Fixed N2 is exported from bacteroid side across the symbiosome
membrane to the plant cytoplasm as ammonium (Tyerman et al. 1995) and
as amino acids (Rosendahl et al. 2001). From the nodules the fixed N is
exported to the plant as amides (e.g asparagine) or ureides (Boogerd and van
Rossum 1997). Nitrogenase catalyses the reduction of H2, which is an inhibitor molecule for N2 fixation. The bacteroids of some rhizobial strains are
capable of removing H2 by oxidation, with the help of uptake hydrogenase
(Mellor and Werner 1990, Bergersen 1997).
Nitrogenase is inactivated rapidly by excess O2 (Bergersen 1997). Therefore, several physiological and biochemical components regulate O2 supply
to nodules, such as a thin diffusion barrier in the inner nodule cortex, the
presence of O2-binding leghaemoglobins in the host cytosol, and respiration by large numbers of bacteroids and plant mitochondria (Bergersen 1997,
Kaminski et al. 1998). Leghemoglobin is the predominant plant protein of
the nodule (up to 30%), giving usually a red colour characteristic for the
effective nodule (Kaminski et al. 1998).
Symbiotic N2 fixation has a high demand for carbon sources. In addition
to nodule growth and bacterial proliferation, carbon compounds are essential for the generation of ATP and reducing power needed for nitrogenase.
Carbon skeletons are needed for the assimilation of fixed ammonia (Kaminski
et al. 1998). The photosynthates enter the nodule as sucrose via the phloem.
In indeterminate nodules, bacteroids take carbon mainly as dicarboxylic acids
(mainly succinate), apparently after conversion of sucrose through a fermentation pathway (Mellor and Werner 1990, Kaminski et al. 1998).
Free-living rhizobia have observed to fix N2 under special conditions.
Several Bradyrhizobium strains and some Rhizobium strains were able to fix
N2 (but did not grow) when the cells were cultivated under microaerophilic
conditions on defined medium containing approriate N2 source (Kaminski et
al. 1998).
1.7.7. Altruistic behaviour of N2-fixing bacteroids
In indeterminate nodules, the senescence of both nodules and bacteroids
has been considered as the end point of the symbiotic interaction. How then
can N2 fixation be evolutionary important for rhizobia, if they die when the
31
nodules degrade? According to a theory, rhizobia have an altruistic behaviour; they sacrifice their lives in order to help the reproduction of others
(Jimenez and Casadesus 1989), for example by enhancing production of root
exudates into the soil (Olivieri and Frank 1994). Because one or few cells can
initiate a nodule (Dart 1977), the rhizobial population within the nodule can
be considered as a genetically homogenous clone (Brewin 1998). It has been
shown that vegetative rhizobia, capable of saprophytic growth in soil and
nodule debris, were present in infection threads of the wild type nodules and
in inefficient nodules (Paau et al. 1980, Vance et al. 1980). Thus, although
bacteroids die when the nodule degrades, bacteroids benefit their undifferentiated relatives occupying the same nodule by producing more nodule
debris, i.e. food, through N2 fixation (Jimenez and Casadesus 1989, Olivieri
and Frank 1994, Brewin 1998). Recently, Timmers et al. (2000) suggested
that one function of “altruistic” bacteroids is to sustain nodule lifetime so that
nodule zone V forms and becomes occupied by intracellular, saprophytic
rhizobia. In all above-mentioned cases, the apparent suicide of N2-fixing
bacteria would result in the increase of the local rhizobial population when
rhizobia are released from degraded nodules (Olivieri and Frank 1994,
Timmers et al. 2000).
1.8. INHIBITION OF N2-FIXING SYMBIOSIS BY ABIOTIC FACTORS
The following two phenomena are familiar for the researchers working with
N2-fixing Acacia and Prosopis trees growing in arid and semiarid regions.
First, seedlings grow better and fix more N2 in optimal greenhouse conditions (but lacking mineral N) compared to seedlings of similar ages grown in
natural ecosystems (Hansen and Pate 1987). Secondly, although no nodules
are found on the root systems of the trees, soils sampled from their rhizosphere induce nodules on the same species in optimal greenhouse conditions (Barnet and Catt 1991). It is assumed that several abiotic factors in the
soil, such as water stress, high soil temperature, salinity, nutrient deficiencies,
alkalinity and acidity may limit growth, nodulation and N2 fixation of the
legumes grown in the tropics (Reddel 1993, Dakora and Keya 1997, Hungria
and Vargas 2000).
1.8.1. Abiotic constraints of arid and semiarid regions
Lack of water is the single most important factor that limits growth and
symbiotic N2 fixation of leguminous plants growing in arid and semiarid
regions. In areas where evapotranspiration exceeds rainfall, saline soils may
also become a problem due to the accumulation of salts into the topsoil. In
addition, high soil temperatures may have detrimental effects. In the sections
1.8, 1.9 and 1.10 effects of these major constraints on Acacia and Prosopis
32
trees, their symbiotic partners and the development of symbiosis between
them are discussed. In order to understand how the Acacia and Prosopis
trees can survive in harsh environments, the general mechanisms associated
with tolerance of plants to water, salt and heat stresses are also examined.
1.8.2. Movement of water in the plant and soil
In plants, water is continually lost from the leaf to the atmosphere when
stomata are open to allow the uptake of CO2 for photosynthesis. The water
lost by transpiration is replaced by water uptake from the soil through the
root, stem and leave via the xylem (Turner 1986). Transport of water is driven
by physical forces. Only the development and maintenance of structures
needed for water transport require an energy input.
Plant physiologists have used the water potential (ψw) concept to define
transport of water across plant membranes, and as a measure of the plant
water status. Several separable components can contribute to the plant water
potential:
ψw = ψs + ψp + ψg (+ ψm)
The osmotic (solute) potential ψs represents the effect of dissolved solutes
on the water potential. The term ψp is the hydrostatic pressure. When it refers
to the positive hydrostatic pressure within cells, ψp is usually called turgor
pressure. The gravity causes water to move downward unless the force of
gravity is opposed by an equal and opposite force. Thus, the potential for
water movement, ψg , depends on the height of plants. The matric potential
ψm has been used to describe the reduction of free energy of water when it
exists as a thin surface adsorbed onto the dry soil particles, seeds and cell
walls. At the plant cellular level, significant components of the water potential are the osmotic potential (due to dissolved solutes) and the hydrostatic
pressure (Taiz and Zeiger 1998).
The water potential of wet soils may be divided into two major components, the osmotic potential ψs and the hydrostatic pressure ψp. In general, ψs
is negligible because solute concentrations of the soil water are low. Instead,
ψs is significant in saline soils. The hydrostatic pressure ψp is close to zero
for wet soils, but when the soil dries, it decreases (getting negative values).
Dry soils contain so little free water that it is not practical to measure the soil
ψs+ψp. In that case the soil water potential is defined by the soil matric potential, caused by the capillarity and interaction of water with solid surfaces of
the soil (Taiz and Zeiger 1998).
Water flow, especially in rapidly transpiring plants, is a passive process.
Water moves toward regions of low water potential or free energy, from soil
and roots to the leaves and the atmosphere. Accordingly, the water potential
decreases consistently when water is moving from the soil to the atmosphere.
33
However, the components of the water potential can vary at different parts of
the pathway. Diffusion, bulk flow and osmosis across membranes also help
move water from the soil through the plant to the atmosphere (Turner 1986,
Taiz and Zeiger 1998).
1.8.3. Drought stress and drought tolerance strategies in plants
In meteorological terminology drought is an absence of rainfall for a period
long enough to cause depletion of soil moisture and damage to plants
(Kramer 1980). The drought is permanent in arid regions whereas in areas
with well-defined dry and rainy periods it is seasonal. Drought has also
been defined as an environmental factor that produces water deficit or
water stress in plants (Kozlowski and Pallardy 1997). As the soil dries, its
matric potential becomes more negative, decreasing the soil water potential. The plants can continue to absorb water only as long as their water
potential is more negative than that of the soil (Taiz and Zeiger 1998).
Water deficit initiates the development of a low plant water potential and
falling of cell turgor below its maximum value (Kozlowski and Pallardy
1997). Consequently, reduced cell turgor causes closure of stomata, and
reduction in cell enlargement, thereby affecting several structures and functions, e.g. decrease in leaf surface area, transpiration, photosynthesis and
plant growth (Kramer 1980, Taiz and Zeiger 1998).
Drought resistance mechanisms of the plants can be divided into three
types: i) drought-escaping plants complete their productive life cycle before
the dry season begins; ii) dehydration postponement occurs by means of
different morphological and physiological mechanisms that reduce transpiration or increase absorption of water, e.g. thick cuticle, leaf rolling,
responsive stomata and deep root systems (Kramer 1980, Turner 1986); iii)
dehydration tolerance, defined as the ability of cells to continue metabolism at low leaf water status, is considered to arise at the molecular level
and to depend on the membrane stability and enzyme activity (Turner
1986, Turner et al. 2000).
Maintenance of turgor during a change in the water status is essential
for maintenance of metabolic processes in plant cells. Turgor can be maintained i) by maintaining water uptake, ii) by reducing water loss, and/or iii)
by osmotic adjustment. Moreover, abscisic acid and other phytohormones
are connected to the control of transpiration and water loss (Turner 1986,
Turner et al. 2000).
1.8.4. Dehydration postponement in Acacia and Prosopis
Many Prosopis and some Acacia trees can maintain a constant water uptake
by developing deep taproots by which they are capable of reaching moist
34
soil layers or the ground water (Nilsen et al. 1983; see section 1.5.2). One
of the most general mechanisms to reduce water loss is a reduction in leaf
area, due to either reduction in leaf area development and/or leaf senescence (Turner 1986). African acacias (subgenera Aculeiferum and Acacia)
are generally deciduous trees, dropping their leaves during the dry season.
Australian acacias (subgenus Heterophyllum) produce bipinnate leaves when
they are seedlings, but later the leaves are replaced by variously shaped,
highly lignified phyllodes accompanied by thick cuticles. The advantage of
the phyllodes is supposed to reside in their capacity to persist during elongated periods of drought (Ullmann 1989). Then, the phyllodes can response
rapidly to the first rains and start photosynthesis without using resources for
the production of new foliage (Montagu and Woo 1999).
Stomatal closure is another way to reduce water loss through transpiration
(Turner 1986). Acacia and Prospis trees have been observed to have both diurnal and seasonal stomatal regulation (Nilsen et al. 1983, 1986, Ullmann 1989,
Montagu and Woo 1999). Some acacias exposed to drought stress were capable of developing a greater water use efficiency, the ratio between dry matter
produced and water consumption (Nativ et al. 1999). Mechanisms related to
the osmotic adjustment, which plays an important role in the drought tolerance are discussed more precisely in the section 1.8.7.
1.8.5. Salt stress and salt tolerance mechanisms of the plants
Salinity reduces plant growth and photosynthesis due to the complex effects
of osmotic, ionic, and nutritional interactions, which are, however, still
poorly understood (Shannon 1997). In A. cyanophylla, the salinity decreased
the concentrations of shoot N, P and K (Hatimi 1999). Saline soils may
impose specific ionic effects on plants because high concentrations of ions
(Na+, Cl-, SO42-) accumulate in cells, inactivating enzymes and inhibiting
protein synthesis and photosynthesis. High concentrations of dissolved salts
in the rooting zone also generate a negative osmotic potential that lowers
the soil water potential. The general water balance is then affected, because
leaves need to develop lower water potential in order to maintain a “downhill” gradient of water potential between the soil and the leaves. To prevent
loss of turgor, most plants are capable of adjusting osmotically (Taiz and
Zeiger 1998; see section 1.8.7). Other salt tolerance mechanisms may be
based on accumulation or exclusion of ions.
Salt sensitive plants (glycophytes) try to restrict ion movement from roots
to shoots whereas salt resistant plants (halophytes) tend to take up Na+ ions
(Hasegawa et al. 2000). Many Acacia and Prosopis trees can grow in saline
soils. Low foliar Na contents of a few salt tolerant species studied suggested
that the trees were capable of excluding Na (Virginia and Jarrel 1983, Marcar
et al. 1991).
35
1.8.6. Heat stress and heat tolerance mechanisms of the plants
Few higher plants can survive a steady temperature above 45oC. At high
temperatures both photosynthesis and respiration are inhibited, but photosynthesis is repressed before respiration. High temperature impairs the
thermal stability of membranes, breaking the balance between the relative
strengths of hydrophobic and hydrophilic interactions among proteins, lipids
and the aqueous environment. As a result, membrane compositions and
structures change, causing leakage of ions.
In environments with intense solar radiation and high temperatures,
plants avoid heating of their leaves by decreasing their absorption of solar
radiation (by leaf waxes, leaf rolling and vertical leaf orientation). The same
mechanisms protect plants from water deficit. Heat shock proteins are connected to the improved heat tolerance but their precise role is still unclear
(Taiz and Zeiger 1998).
1.8.7. Osmotic adjustment and compatible solutes in plants
Osmotic adjustment is a process by which the water potential of the plant
can be decreased without a decrease in accompanying turgor (Taiz and
Zeiger 1998). Turner et al. (2000) have defined osmotic adjustment as an
active accumulation of solutes by the plant in response to increasing water
deficits in the soil and/or plant, thereby maintaining turgor or reducing the
rate of turgor loss, as water potentials decrease. Osmotic adjustment enables
plants to survive and grow also in saline environments. The osmotic adjustment occurs then either through compartmentation of toxic ions away from
cytoplasm into the vacuole and/or through accumulation of organic solutes,
such as compatible solutes, in the cytosol (Hasegawa et al. 2000). With the
aid of the osmotic adjustment the plants exposed to water deficit or high
salinity can maintain photosynthesis, delay leaf senescence and death, and
improve root growth (Turner 1986, Turner et al. 2000).
Compatible solutes, accumulated during the osmotic adjustment, are
highly soluble compounds that carry no net charge at physiological pH,
and are non-toxic at high intracellular concentrations and at higher temperatures. Under unfavourable osmotic conditions, compatible solutes raise the
osmotic pressure in the cytoplasm and stabilise proteins and membranes
(Yancey et al. 1982, Csonka 1989, McNeil et al. 1999). The osmoprotective
mechanisms of the compatible solutes are supposed to relate to the absence
of perturbing effects on macromolecule-solvent interactions. Inorganic ions,
such as NaCl, interact directly with proteins, favouring unfolding and leading
to denaturation of proteins, whereas compatible solutes are excluded from
protein surfaces (Rhodes and Hanson 1993, McNeil et al. 1999).
The plants can synthesise three types of compatible solutes: i) betaines
36
or quaternary ammonium compounds (e.g. glycine betaine) and tertiary
sulfonium compounds (e.g. β-dimethylsulfonepropionate, DMSP in marine
micro-algae), ii) polyols and sugars (e.g. trehalose) and iii) amino acids (e.g.
proline) (McNeil et al. 1999). The plant can synthesise several various compatible solutes, the composition of the set varying between species. Proline
is the most common osmolyte among plants (Erskine et al. 1996). Glycine
betaine accumulation occurs in diverse marine algae and in about 10 distantly related flowering plant families. The family Chenopodiaceae has several species that accumulate glycine betaine, e.g. sugar beet, Beta vulgaris
and spinach (McNeil et al. 1999).
1.8.8. N2 fixation under stress
Numerous studies have shown that water stress, salinity and high soil temperature reduce dramatically N2 fixation and nitrogenase activity of nodulated
leguminous herbs (reviewed by Zahran 1999). Similar phenomena have
been observed in acacias and other woody legumes (Marcar et al. 1991,
Purwantari et al. 1995, Zou et al. 1995). Although environmental stress
factors disturb normal photosynthesis, nitrogenase activity may not be limited
by the lack of photosynthetates (Delgado et al. 1993, Serraj et al. 1998).
Nitrogenase activity can even be more sensitive to a decrease in soil water
potential than stomatal closure (Pararajasingham and Knievel 1990).
During stress experiments, several changes in nodule metabolism and
compounds were associated with the reduced N2 fixation or nitrogenase
activity, decreased hemoglobin content resulting reduction in O2 availability
being the most common observation (Table 1-8). It is not clear, how stress
conditions cause a decline in N2 fixation. One of the common hypotheses
is that the effects of environmental stresses are mediated through variations
in nodular gas diffusion resistance (Sutherland and Sprent 1993). More precisely, the rate of N2 fixation may be limited by the supply of O2 (Walsh
1995). In water-stressed nodules, O2 supply to bacteroids may be restricted
in two ways: i) packing of different layers of the cortical cells limits diffusion of O2 (Guerin et al. 1990), and ii) alkaline proteases degrade O2-binding
leghemoglobin (Guerin et al. 1991). P enriched in the nodule cortex may
also associate with part of the O2 barrier (Franson et al. 1991).
A decrease in N2 fixation may involve variation in the balance between
the export of nitrogenous solutes via the xylem and the import of photosynthetates via the phloem (Walsh 1995). According to the proposal of Walsh
(1995), stress events modify the content of soluble sugars and nitrogenous
solutes in the nodule cortex, resulting in changes in cortical cell turgor. Subsequently, this event affects for example the shape of the cortical cells and
the intercellular water content, further changing permeability of the nodule
cortex to gases. Hence, because nodule anatomy and physiology are hetero-
37
Table 1-8. Effects of drought, salinity and high temperatures on nodule compounds
and structures in herbaceous legumes.
Symptoms
Nodule compounds
Decrease in leghemoglobin content
Decreased
nitrogenase
activity a)
Stress
Increase in cytosolic PEPC f)
Increase in bacteroid MDH g)
+
+
+
+
+
+
ND
+
+
+
ND
+
+
+
ND
ND
ND
+
+
Nodule structure
Disturbances in bacteroid development
ND
Temperature
ND
+
Temperature
Drought
Decrease in water potential and content
Decrease in lipids & proteins
Decrease in organic acids
Decrease in P conc. & P-use efficiency
Decrease in bacteroid respiration
Increase in proline content
Increase in alkaline proteolysis
Increase of nodule intracellular pH
Increase in soluble sugars
Increase in ureide concentration
Increase in total carbohydrates
Drought
Salinity
d)
Drought
Drought
Salinity
Drought
Salinity
Drought
Salinity
Drought
Salinity
Nodule senescence
Oxidative damage in lipids and proteins
a)
Nitrogenase activity has been determined by acetylene reduction method, 15N dilution
method or by measuring N content. b) Conditions with harmful effects are indicated in boldface.
c)
Whw = With holding water. d) ND = not determined. f) PEPC, nodule cytocolic
phosphoenolpyruvate carboxylase. g) MDH, bacteroid malate dehydrogenase
geneous among legume plants, the responses of N2 fixation to environmental
stresses may be a species-specific property (Walsh 1995).
1.8.9. Nodulation and infection under stress
Several studies both on herbaceous and woody legumes have shown that
drought, salinity and heat stresses cause decrease in nodule numbers and
nodule dry weight (e.g. (Singleton and Bohlool 1984, Zahran and Sprent
1986, Arayangkoon et al. 1990, Marcar et al. 1991, Purwantari et al. 1995,
Hatimi 1999). At least in the case of salt stress, symbiotic properties of the
legumes (N2 fixation, nodule dry weight, nodule number) were more sensitive to salinity than plant growth (Marcar et al. 1991). If the stress conditions
38
Growth conditions b) c);
growth medium
Plant species
References
-0.04 & -0.17 MPa; soil
Whw 2-9d: -0.8,- -2.4 MPa; sand
0.05 M NaCl; Leonard jars
0.05, 0.1 M NaCl; Leonard jars
Whw 0, 2, 4-8 d; 20-4%; soil
-0.04 & -0.17 MPa; soil
0.1, 0.15 M NaCl, solution
Whw 12-13d; soil
0.05, 0.1 M NaCl; Leonard jars
-0.04 & -0.17 MPa; soil
0.1, 0.15 M NaCl, solution
Whw 2-9d: -0.8,- -2.4 MPa; sand
“
“
“
“
-0.04 & -0.17 MPa; soil
Dehydration of the soil mixture
“
“
“
“
0.1, 0.15 M NaCl, solution
0.05 M NaCl; Leonard jars
“
“
“
“
Glycine max
Vicia faba
Pisum sativum
Pea, faba bean, bean
Vigna unguiculata
Glycine max
Medicago sativa
Glycine max
Pea, faba bean
Glycine max
Medicago sativa
Vicia faba
“
“
Glycine max
Ruiz-Lozano et al. 2001
Guerin et al. 1991
Delgado et al. 1993
Delgado et al. 1994
Pararajasingham & Knievel 1990
Ruiz-Lozano et al. 2001
Fougere et al. 1991
Franson et al. 1991
Delgado et al. 1994
Ruiz-Lozano et al. 2001
Fougere et al. 1991
Guerin et al. 1991
“
“
Ruiz-Lozano et al. 2001
Glycine max Serraj et al. 1998
“
“
Fougere et al. 1991
Delgado et al. 1993
“
“
22, 30oC; agar slopes
Trifolium subterraneum Pankhurst & Gibson 1973
22, 30oC; agar slopes
-0.04 & -0.17 MPa; soil
Trifolium subterraneum Pankhurst & Gibson 1973
Glycine max
Ruiz-Lozano et al. 2001
“
“
Medicago sativa
Pisum sativum
“
“
are long or/and strong enough, formation of nodules will cease completely.
It is generally believed that the infection process is the most sensitive phase
during nodule development (Singleton and Bohlool 1984). Once nodules
have formed, they are less affected by stress factors than the initial nodule
formation process (Purwantari et al. 1995). Studies with herbaceous legumes
having the root hair infection mode indicated that drought and salinity
stresses cause changes in root hair morphology and decrease the numbers of
markedly curling hairs (Table 1-9).
In mildly water-stressed Vicia faba nodules, the loss of turgor induced a
reduction in the peribacteroid space, the vesicle membrane became sinous,
and the cell cytoplasm appeared granular. Under more severe water stress, a
total disappearance of peribacteroid spaces was associated with the rupture
39
Table 1-9. Effects of drought, salinity and high temperature on the infection process
and nodulation in herbaceous legumes (PEG, polyethylene glycol).
Symptoms
Root hairs
Decrease in hair numbers
Decrease in rhizobial colonisation
Decrease in deformation
Decrease in mark curling
Stress
Short and swollen
Salinity
Salinity
Salinity
Osmotic
Salinity
Drought
Short, swollen, not deformed
Shrinkage of hairs
Salinity
Salinity
Infection process
Decrease in infection thread numbers
Drought
Osmotic
Salinity
Multiple branching & distortion
a)
Temperature
Growth conditions a) and
medium used
0.2, 0.4, 0.6% NaCl; Jensen tubes
1.0% NaCl; silica sand
1.0, 1.2, 2% NaCl; silica sand
0.1, 0.2 M PEG; sand
0.1 M NaCl, sand
Soil moisture 5.5-3.5%
(-0.36 to -3.6 105 Pa); sand
0.2, 0.4, 0.6% NaCl; Jensen tubes
1% (0.15 M) NaCl; silica sand
Soil moisture 5.5-3.5%
(-0.36 to -3.6 105 Pa; sand
0.2 M PEG, sand
0.2, 0.4, 0.6% NaCl; Jensen tubes
0.1 M NaCl; sand
22, 30oC; agar slopes
Conditions with harmful effects are indicated in boldface.
of vesicle membranes. The bacteroid content appeared to be very heterogeneous (Guerin et al. 1991). Use of mineral N may improve tolerance to some
stresses. For example, N2-fixing acacias were more sensitive to salinity than
N-fertilised plants (Marcar et al. 1991).
1.9. Abiotic stresses and free-living rhizobia
Desiccation of bacteria
Rhizobia living in the rhizosphere of Acacia and Prosopis trees are obviously
exposed to similar environmental stresses as their hosts. When the soil dries,
the availability of water is assumed to be reduced at least due to increased
solute concentrations of the soil water (Miller and Wood 1996). According to
Potts (1994), the immediate environment of the cell, being either the atmosphere or aqueous solution, distinguishes matric and osmotic systems. He has
defined desiccation as the removal of a substantial fraction of the cell water
through matric water stress. Desiccation can also be designated as air drying,
because bacterial cells lose water when they are exposed to a gas phase
with a water activity that is lower than that of the cell compartment. Desiccation plays a decisive role in bacterial communities that are found in aerophytic environments, on and in rocks and soils, in the phyllosphere, dusts
and aerosols (Potts 1994). The responses of bacterial cells to desiccation
can be: shrinkage of the bacterial cytoplasm and capsular layers, increase
40
Plant
species
References
Medicago sativa
Glycine max
Glycine max
Vicia faba
Vicia faba
Lakshmi-Kumari et al. 1974
Tu 1981
Tu 1981
Zahran & Sprent 1986
Zahran & Sprent 1987
Trifolium subterranum
Medicago sativa
Glycine max
Worral & Roughley 1976
Lakshmi-Kumari et al. 1974
Tu 1981
Trifolium subterranum
Worral & Roughley 1976
Vicia faba
Medicago sativa
Vicia faba
Trifolium subterraneum
Zahran & Sprent 1986
Lakshmi-Kumari et al. 1974
Zahran & Sprent 1986
Pankhurst & Gibson 1973
in intracellular salt levels, crowding of macromolecules, damage to external
layers (pili, membranes), changes in ribosome structure, and decrease in
growth. Reactive oxygen species can also damage proteins and DNA, leading
to accumulation of mutations (Potts 1994).
Death of rhizobial cells during desiccation was suggested to associate
with changes in membrane permeability (Bushby and Marshall 1977). It has
been hypothesised that during dehydration, the removal of water hydrogen
bond to the phospholipid headgroups of the membrane decreases the spacing between adjacent lipids. This leads to increased van der Waals interactions between the lipid side chains, resulting in an increase in the membrane
phase transition temperature. Then, the membrane is converted from the
liquid crystalline into the gel phase already at room temperature. Subsequent
rehydration results in a further phase transition of the membrane back to the
liquid crystalline phase. As consequence, the membrane barrier is disrupted,
leading to leakage of membranes (Potts 1994, Welsh 2000).
Bacteria under osmotic stress
Several reviews have focused on the responses of bacteria to variations in
the solute content of their aqueous environment (e.g. Csonka 1989, Galinski 1995, Miller and Wood 1996, Welsh 2000). Hyperosmotic conditions
cause rapid efflux of water and loss of turgor, and the cells may plasmolyse. Upon hypo-osmotic shock, water flows into the cell and increases
41
the cytoplasmic volume and/or cell turgor (Csonka 1989, Poolman and
Glaasker 1998). Most of the osmotic stress studies have been performed
with NaCl that, however, influences bacterial cells trough both ionic and
osmotic phenomena. In soils, the salinity and moderate drought stress
cause osmotic stress. The osmolarity of the rhizosphere, extending a few
millimeters from the surface of the plant root or rhizoplane, has been
assumed to exceed that of bulk soil water, because the root tissue excludes
salts, and both the plant roots and bacteria secrete organic nutrients and
mucilage (Miller and Wood 1996).
High growth temperature
All organisms respond to a sudden increase in growth temperature by inducing the synthesis of a number of heat shock proteins (Hsps). Hsps consist
of chaperons (such as GroEL, DnaK, DnaJ), small heat shock proteins
(sHsps) and proteases. Chaperons are involved in the proper folding of denaturated proteins, and proteases are required for the degradation of irreversibly damaged proteins. Many chaperons and proteases are also important
during normal growth but sHsps presumably function only under heat
stress. According to the present model sHsps bind to denaturated proteins
accumulated under stress and maintain them in a folding-competent state
(Munchbach et al. 1999, Nocker et al. 2001). When bacteria generally have
1-2 Hsps, rhizobia were found to contain multiple Hsps. For example, a
Bradyrhizobium strain had even 12 sHsps (Munchbach et al. 1999). Reason
for this phenomenon is unclear. It is also not known why a heat-sensitive
bean-nodulating Rhizobium strain had several Hsps but a heat-tolerant one
contained only two sHsps (Michiels et al. 1994).
1.9.1. Osmoadaptation in rhizobia
In the same way as plants, bacteria exposed to the salts can maintain osmotic
equilibrium across the membrane by allowing influx of salt or solutes (mainly
Halobacteriaceae) or by exclusion of salts via the production of compatible
solutes or other organic osmolytes (other bacteria). However, if organic
osmolytes are present in surrounding medium, bacteria prefer uptake over
synthesis de novo. Exogenous osmolytes that improve cell growth under
adverse osmotic conditions are referred to osmoprotectans (Galinski 1995).
Apparently, bacteria also adjust to moderate desiccation by synthesising
compatible solutes (Potts 1994).
Endogenous osmolytes
The responses of bacteria to growth-inhibiting osmolarity have been studied
extensively in the family Enterobacteriaceae and in the case of Rhizobiaceae, in S. meliloti. Under elevated salinity S. meliloti can accumulate K+
42
-ions and an organic anion, glutamate, and synthetise the following compatible solutes: N-acetylglutaminylglutamine amide (NAGGN), trehalose, and
glycine betaine, if the medium contains its precursor, choline (Table 1-10).
Recent data indicate that the set of endogenous osmolytes produced by
rhizobia can vary at least at the species level (Table 1-10). For example,
NAGGN has been found only in S. meliloti (Table 1-10). The types of the
accumulating osmolytes also depend on the stress level and on the growth
phase of the cell culture. In S. meliloti, glutamate accumulated at low salt
concentrations, but at higher levels, glutamate and NAGGN were observed.
All three osmolytes, glutamate, NAGGN, and trehalose accumulated only
at extremely high NaCl concentrations (Smith et al. 1994). When glycine
betaine was exogenously supplied in the growth medium, it accumulated as
the major osmolyte during the lag and early exponential phases, whereas
glutamate and NAGGN prevailed at the late exponential phase (Talibart et al.
1997). Trehalose was the major osmolyte of the stationary phase (Smith et al.
1994, Talibart et al. 1997). In conclusion, osmoregulation is very complex. A
number of different environmental factors, such as the level of osmotic stress,
growth phase of the culture, carbon source and osmolytes of the growth
Table 1-10. Accumulation of ions, endogenous osmolytes and compatible solutes
in Rhizobiaceae under osmotic stress (mostly in NaCl).
Compound
Iones
K+
Endogenous osmolytes
glutamate
Bacterial species
B. japonicum, Rhizobium sp. Miller and Wood 1996
S. fredii, S. meliloti
Rhizobium sp. (Prosopis)
S. meliloti
A. tumefaciens, S. fredii,
Rhizobium sp. (Acacia)
Endogenous compatible solutes
glycine betaine
- by direct uptake
A. tumefaciens
S. meliloti
- via synthesis from
S. meliloti
its precursor choline
mannosucrose
A. tumefaciens
N-acetylglutaminylglutamine S. meliloti
amide (NAGGN)
trehalose
References
Hua et al. 1982
Botsford & Lewis 1990, Smith et al.
1994, Talibart et al. 1994, 1997
Smith et al. 1994
Smith et al. 1990
Talibart et al. 1997
Smith et al 1988
Smith et al. 1990
Smith and Smith 1989
Smith et al. 1994
Talibart et al. 1994, 1997
S. meliloti
Smith and Smith 1989
Breedweld et al. 1990, 1993
Rhizobium sp. (Acacia)
Smith and Smith 1989
R. leguminosarum bv. trifolii Breedweld et al. 1991, 1993
Smith et al. 1994
Talibart et al. 1994
Rhizobium sp. (peanut)
Ghittoni and Bueno 1995
43
medium control the combination of naturally occurring endogenous osmolytes in rhizobial cells (Smith et al. 1990, 1994).
Exogenous osmolytes (osmoprotectants)
As indicated above, bacteria prefer uptake of compatible solutes over synthesis de novo. The transport systems for external osmolytes (osmoprotectants)
are relatively unspecific, and bacteria accept components of plant and animal
origin (Galinski 1995). Present data indicate that compounds, which can act
as osmoprotectants, vary between different rhizobial species (Table 1-11).
In contrast to Esherichia coli, S. meliloti and many other rhizobial species can use glycine betaine as a C and/or N source under low osmotic
stress but as an osmoprotectant in high osmolarity. Only the slow-growing B.
japonicum, which is considered as an osmosensitive species, is incapable to
transfer glycine betaine and its precursor choline (Boncompagni et al. 1999).
Under low osmotic stress glycine betaine is degraded via successive demethylation to glycine, while at elevated osmolarity the catabolism is blocked
and glycine betaine is accumulated in cells (Sauvage et al. 1983, Bernard et
al. 1986, Smith et al. 1988, Boncompagni et al. 1999).
Table 1-11. The effect of exogenously supplied osmolytes (osmoprotectants) on the
growth of rhizobia under osmotic stress. The tests were mostly performed in NaCl
(DMSP = b-dimethylsulfonipropionate).
Compound
Accumulators
choline
glycine betaine
DMSP
-pipecolic acid
DL
Growth stimulation
No growth stimulation
References
S. meliloti, A. tumefaciens,
M. huakuii, R. galegae,
R. tropici, S. fredii,
A. rhizogenes, B. japonicum
R. etli, all biovars of
R. leguminosarum
Boncompagni
et al. 1999
S. meliloti, A. tumefaciens,
M. huakuii, Rhizobium sp.
(Hedysarum), R. galegae
R. tropici, S. fredii, M loti
S. meliloti
S. meliloti
A. rhizogenes, B. japonicum
R. etli, Rhizobium sp.
(Sesbania), all biovars of
R. leguminosarum
Sauvage et al. 1983,
Bernard et al. 1986,
Boncompagni
et al. 1999
Pichereau et al. 1998
Gouffi et al. 2000
Rhizobium sp. (Hedysarum)
Talibart et al. 1994
B. japonicum, M. huakuii,
R. leguminosarum bv. viciae,
S. fredii
Gouffi et al. 1998,
Gouffi et al. 1999
Chemical mediators
ectoine
S. meliloti, B. japonicum,
Rhizobium bv. trifolii and
bv. viciae
Disaccharides
sucrose
S. meliloti,
R. leguminosarum
bv. phaseoli and trifolii
trehalose
B. japonicum
S. meliloti
R. leguminosarum
bv. phaseoli and trifolii
B. japonicum, M. huakuii,
R. leguminosarum bv. viciae
S. fredii
44
Elsheikh and Wood
1990
Gouffi et al. 1999
Intracellular trehalose was also observed to be metabolised by S. meliloti
and R. leguminosarum bv. trifolii at low osmolarity (Breedveld et al. 1993).
Under osmotic stress, trehalose is not accumulated in the cytosol of rhizobia but after degradation during the early exponential phase it is indirectly
contributed to enhance levels of two endogenous osmolytes, glutamate and
NAGGN (Gouffi et al. 1999). Trehalose is usually broken down into two glucose units (Galinski 1995).
It is supposed that glycine betaine is released into ecosystems by primary microbial producers upon dilution stress (rain fall, flooding), by decaying plant and animals, or by mammals in the form of excretion fluids (e.g.
urine Galinski and Truper 1994, Sleator and Hill 2001). The precursor of
glycine betaine, choline, is a common constituent of the eukaryotic membranes (phosphatidylcholine) and should therefore be widespread in the soil
(Boncompagni et al. 1999). Plant polysaccharides, cellulose and starch, after
degradation by bacteria and fungi are presumably natural sources of disaccharide osmoprotectants (Gouffi et al. 1999). Gouffi et al. (1999) suggested
that the plant-derived disaccharides are highly osmoprotective for plantbeneficial bacteria, such as for rhizobia, but are not osmoprotective for other
soil bacteria, e.g. for B. subtilis and P. aeruginosa.
Glycine betaine and bacteroids
Glycine betaine may protect rhizobia occupying salt-stressed nodules.
Isolated S. meliloti bacteroids were able to transport glycine betaine, and
the uptake activity was increased after addition of NaCl (Fougere and Le
Rudulier 1990b). The bacteroids also transported choline, which was further
converted to glycine betaine when the bacteroids were subjected to salt
stress. Generally, bacteroids/nodules degraded glycine betaine at low salinity
but the catabolism was blocked by increasing salinity, allowing accumulation of glycine betaine to the bacteroids/nodules (Fougere and Le Rudulier
1990a, Pocard et al. 1991). The high level of glycine betaine presumably
maintained better water status in the nodule and protected N2 fixation against
salt stress (Pocard et al. 1991). Increased levels of other osmolytes, proline
and pinitol (a carbohydrate) have been detected in the cytosol and bacteroids
of salt-stressed M. sativa nodules (Fougere et al. 1991) but their function has
remained unknown.
1.9.2. Role of trehalose during desiccation
Many bacteria, yeast, invertebrates and some plants are able to survive long
periods in a desiccated state, where even more than 99% of the water has
been lost. A common feature for all these organisms is the presence of
high concentrations (< 20%) of disaccharides, trehalose (microbes, invertebrates) and/or sucrose (plants) in their dried tissues (Welsh 2000). It has been
45
hypothesised that compatible solutes are important components to maintain
viability under moderate water deficit, whereas the disaccharides, trehalose
and sucrose, offer protection under extreme water deficit (Potts 1994).
Trehalose has been proposed to stabilise dried biological membranes and
preserve the integrity of proteins and enzymes (Potts 1994). Obviously, direct
hydrogen bonding of trehalose replaces water and maintains the normal
packing of the phospholipid headgroups. Thus, the membrane is maintained
in the liquid crystalline phase and does not undergo damaging transition
to the gel phase or leakage of its contents upon rehydration (Welsh 2000).
According to another hypothesis, the property of dried sugar solution to
form amorphous glasses is the critical factor. Biological molecules entrapped
within glass would be stable at all temperatures below the glass-liquid transition temperature. The chemically inert nature of trehalose may also prevent
deleterious chemical reactions between dried molecules and the glass matrix
(Welsh 2000). In addition to osmotic stress (as compatible solute) and desiccation, trehalose also protects bacterial cells from heat and low O2 levels
(Potts 1994, Welsh 2000).
1.10. INHIBITION OF N2-FIXING SYMBIOSES BY BIOTIC FACTORS
Not only the abiotic constraints of arid and semiarid regions can inhibit
formation of the N2-fixing symbiosis between rhizobia and leguminous plants
but the factors associated with the bacterial population in the soil may cause
similar results. The main reasons for the lack of nodules or formation of ineffective ones on Acacia and Prosopis roots may be: i) the soil is lacking suitable rhizobia or there are too few of them; ii) indigenous rhizobia induce
ineffective nodules; iii) the nodules are induced by Agrobacterium sp.
The total absence of rhizobia is probably very rare in soils because rhizobia seem to be capable of persisting in harsh soil conditions even without the
host plant (Barnet et al. 1985, Odee et al. 2002). Instead, the lack of compatible rhizobia for a given host plant is probably a common phenomenon.
Rather low numbers of compatible rhizobia are needed for the nodulation,
at least in favourable conditions. Inoculation of woody legumes significantly
increased shoot N, when the soils contained as few as 50 cells g-1 soil (Turk
et al. 1993). However, almost one-half of the tropical soil samples contained less than 100 rhizobia g-1 of soil (Singleton and Bohlool 1984). In
Kenyan soils collected from different ecological zones, the size of the rhizobial population was very varied, ranging from 3.6 to 2.3 x105 cells g-1 soil
(Odee et al. 1995). Thus, low amounts of compatible rhizobia present in soil
may cause impaired nodulation.
Several experiments with acacias have shown that although nodules were
formed on the roots, not all were capable of fixing N2 (Lawrie 1983, Barnet
et al. 1985, Odee et al. 2002). In Australia, only 36% of the strains isolated
46
from Acacia spp. significantly increased plant growth (Barnet and Catt 1991).
Also a later study with rhizobia isolated from Australian acacias showed a
very wide variation in effectiveness, some host-rhizobia combinations being
close to parasitic (Burdon et al. 1999).
Bacteria belonging to the genus Agrobacterium are closely related to rhizobia but they induce tumours on plant roots or shoots. Agrobacteria are
common soil and rhizophere organisms. Strains resembling or belonging
to Agrobacterium sp. have often been identified from the root nodules of
Acacia and Prosopis trees grown in arid and semiarid Africa (Khbaya et al.
1998, Nick 1998, de Lajudie et al. 1999, Odee et al. 2002). Nevertheless,
when reinoculated in laboratory conditions, they did not induce nodules
(Nick 1998, Odee et al. 2002). Only after massive inoculation some strains
induced small number of ineffective nodules on different Acacia species (de
Lajudie et al. 1999). The explanation for the presence of agrobacteria in the
nodule is still unknown, but it has been speculated that either agrobacteria
enter nodules together with effective rhizobia, or that agrobacteria carrying
some symbiotic genes can infect legume roots (de Lajudie et al. 1999). Consequently, agrobacteria represent one group of bacteria that can induce nonfixing nodules on Acacia and Prosopis roots.
47
2. AIMS OF THE STUDY
N2-fixing, drought tolerant and multipurpose Acacia and Prosopis species
are appropriate trees for reforestation of degraded areas in arid and semiarid regions of the tropics and subtropics. Inoculation of tree seedlings with
tested, effective rhizobia already at the nursery stage may be crucial in
order to exploit their N2-fixing capacity after transplantation to the field. The
collection of Sudanese tree rhizobia, gathered in the Sudan 1987-1988 with
a Finnish Sudanese Forestry Program (Karsisto and Lindström 1992), is well
characterised (Zhang et al. 1991, 1992, Zahran et al. 1994). De Lajudie et al.
(1998) and Nick et al. (1999b) described three new species among them (S.
arboris, S. kostiense and M. plurifarium), and Haukka et al. (1998) studied
their genetic diversity and phylogeny. However, very little was known about
the symbiosis between the rhizobia and Acacia/Prosopis trees. Consequently,
it was not known which factors in arid and semiarid soils on one hand
favour and on the other hand impair or prevent the development of effective symbiosis. This knowledge is important when developing effective inoculants. Therefore, this thesis has following objectives (corresponding papers
indicated in parentheses):
1. Investigation of symbiotic properties of five putative inoculant strains,
belonging to Sinorhizobium sp. The strains were inoculated on nine
Acacia and five Prosopis species, characteristic for semiarid and arid
regions in Africa and Latin America. The symbiotic properties studied
included host specificity, infection mode, nodule type, and the effectiveness of the symbiotic association (I).
2. Characterisation of effects of thermal, salt and osmotic stresses on the
growth (II, III) and activity (IV) of free-living Sinorhizobium cells.
3. Investigation of infection and nodulation of inoculated A. senegal seedlings exposed to heat (II) and drought stresses (III), and evaluation of the
potential factors explaining failures in the nodulation process.
4. Evaluation of the roles of two compatible solutes, glycine betaine and
trehalose, for the Acacia senegal–Sinorhizobium symbiosis exposed to
drought stress (III).
5. Examination of potential adaptation mechanisms connected to several
peculiar responses of S. arboris populations or cells during the stress
experiments (II, III, IV).
48
3. MATERIALS AND METHODS
3.1. BIOLOGICAL MATERIAL
Bacterial strains used in this study are listed in Table 3-1. Tree species used
are listed in Table 3-2 according to their taxonomic position.
3.2. PARAMETERS AND STRESS CONDITIONS STUDIED
Methods used and parameters examined are described in detail in the original publications and summarised in Table 3-3. Stress experiments performed
are gathered in Table 3-4.
Table 3-1. Bacterial strains and plasmids used in this study.
Species
Host plant
Geographical
origin
Sinorhizobium sp.
HAMBI 1480
A. senegal
Sudan, Kosti
P. chilensis
Sudan, Kosti
“
I, II, III, IV
P. chilensis
A. senegal
P. chilensis
Sudan, Kosti
Sudan, Kosti
Sudan, Khartum
“
“
“
I
I
A. senegal
Sudan, El Fau
“
I, III
A. senegal
A. senegal
Sudan, El Obeid
Sudan, El Fau
“
“
I
I
A. mollisima
Senegal
S. arboris
HAMBI 1552
S. kostiense
HAMBI 1483
HAMBI 1489
HAMBI 1493
S. saheli
HAMBI 1496
S. terangae
HAMBI 1550
HAMBI 1551
S. terangae bv. acaciae
ORS 1058
Modified strains
S. arboris
HAMBI 1552 R
HAMBI 2180
HAMBI 2190
Plasmids
pCAM121
pAM102
pRK2013
Properties
Reference
Zhang et al. 1991,
Nick et al. 1999ab
Used in
paper
I
de Lajudie et al. 1994,
Lortet et al. 1996
I
IV
II
IV
IV
II, III
IV
Wilson et al. 1995
II
Möller and Jansson,
1998
Ditta et al. 1980
IV
Parental strain
smr
HAMBI 1552
containes gusA21 HAMBI 1552
containes luc
HAMBI 1552
Description
Paph-gusA-TrpA ter translational
fusion with adjacent unic SpeI
site in mini Tn5 Sm/Sp
lacI, luc,ori R6K, oriT RP4, Ptac,
NotI, bla, Kmr
repcolE1, Kmr, Nmr
49
IV
Table 3-2. Generic names, original distribution and geographic origin of the seeds
of the tree species used in this work.
Tree species
Tribe Acaciae
Subgenus Acacia
Acacia nilotica (L.) Del.
Acacia oerfota (Forssk.) Schweinf.
(synonym A. nubica Benth.)
Acacia seyal Del.
Acacia sieberiana DC.
Acacia tortilis subsp. raddiana
(Savi) Brenan.
Subgenus Aculeiferum
Acacia angustissima (Mill.) Kunze
Acacia mellifera (Vahl.) Benth.
Acacia senegal (L.) Willd.
Subgenus Phyllodineae
Acacia holosericea G. Don
Tribe Mimoseae
Prosopis africana (Guill. & Perr.) Taub.
Prosopis chilensis (Molina) Stunz
Prosopis cineraria (L. Druce)
Prosopis juliflora (Sweet) DC.
Prosopis pallida (Willd.) Kunth.
Subfamily PAPILIONOIDEAE
Sesbania rostrata Bremek & Oberm
a)
Geographic Original
origin of
geographic
seeds
distribution a)
Sudan
Sudan
Africa, Arabian Peninsula, India
Africa
Sudan
Sudan
Senegal
Africa
Africa
Africa, Near and Middle East
Chile
Sudan
Kenya
Senegal
Sudan
Central America
Africa, Middle East, India, Pakistan
Senegal
Northern Australia
Senegal
Chile
Sudan
Senegal
Senegal
Peru
Africa
South America
India, Pakistan, Middle East
South and Central America, Mexico
South America
Senegal
Allen and Allen 1982, Roshetko 2001.
3.3. STATISTICAL ANALYSIS
In papers I and II, analysis of variance was performed with the Excel program
package version 2000 for Windows 98 (Microsoft Corporation) and with
the SAS System program package version v.6.12 for Windows (SAS Institute
Inc.). Tukey’s test was applied to compare means at P < 0.05 with the SAS
system. In paper III, comparisons between treatments were carried out with
the program SPSS 10.0 for Windows by using either one-way ANOVA or a
non-parametric Kruskal-Wallis test. To compare means at P < 0.05, Tukey’s
test was applied after ANOVA and t’-test was applied after the Kruskal-Wallis
test. Principal component analysis (PCA) was performed with the Matlab
program package version 42.c1 for Windows (The Mathworks, Inc) equipped
with the Data analysis Toolbox (ProfMath Oy, Helsinki, Finland; paper I).
50
Table 3-3. Parameters and methods used in this study, and the respective papers
where they appear and references.
Used
in paper
Parameter
Method
Cell morphology
Epifluorescence microscopy
after staining with acridine orange
Culturable cells
Plate counts (cfu)
Esterase active cells
Epifluorescence microscopy
after staining with SFDA
IV
Tsuji et al. 1995, IV
Glycine betaine
content of the plants
HPLC
III
Rajakylä and Paloposki
1983
Growth of bacteria
Plate counts
Optical cell density (A 600 nm),
Culturing on Bioscreen
Growth of plants
Determination of shoot length.
number of nodules and dry
weights of shoots and roots
I II III
I II III
I II III
Infection process
Light microscopy after staining
plant roots with methylene blue
and GUS substrate X-glc-A
I
Luciferase activity
References
IV
II III IV
II III IV
III IV
III
III
II III
Light output measured
with the luminometer
Vasse and Truchet 1984
Wilson et al. 1995
IV
Maximum temperature Temperature-gradient
for bacterial growth
incubator
and GUS production
II
II
Möller and Jansson
1998, IV
Andersson et al. 1995,
Nitrogen fixation
Acetylene reduction method
I II III
Lindström 1984a, b
Räsänen et al. 1991
Nodule structure
Semi-thin nodule sections
I
Sprent et aI 1989, I
Number of culturable
bacteria in the
rhizosphere
CFU count
Permabilised (dead)
cells
Epifluorescence microscopy
after staining with SYTOX green
Soil moisture
Moisture fraction
Total cell number
Epifluorescence microscopy
after staining with acridine orange
Transformation
of marker genes
Plate mating and
triparental mating
II III
IV
Roth et al. 1997, IV
IV
Bloem 1995, IV
IV
Wilson 1996
Maniatis et al. 1982
III
II
51
Table 3-4. Stress experiments performed in this study (GB, glycine betaine;
PEG, polyethylene glycol).
Marker
gene
Paper
Root temperature of
28, 36, 38 , 40, and 42oC
gusA
II
Growth, activity and
physiological status
of the cell cultures
Culturing of cells at 28, 37,
and 40oC
luc
IV
Drought
stress
Growth of plants
and bacteria,
symbiotic interactions
Well and less watered
seedlings without or with
glycine betaine and trehalose
gusA
III
Salt and
osmotic
stress
Growth of liquid
cell cultures
Culturing of cells in
NaCl ( 0.1, 0.5, and 1.0 M)
PEG 6000 (9, 17, and 24%)
with or without osmoprotectants
GB (0.01, 0.1, and 1.0 M)
trehalose (0.01, 0.1, and 0.9 M)
Stress
Reseach subject
Conditions
Heat stress
Growth of bacteria
and plants,
symbiotic interactions
Heat stress
52
III
4. RESULTS AND DISCUSSION
4.1. SYMBIOTIC PROPERTIES OF AFRICAN SINORHIZOBIA
4.1.1. Host specificity
Few studies have so far been reported on the host specificity of the rhizobia nodulating Acacia and Prosopis trees. Extensive cross nodulation tests
performed between 15 Australian Acacia species and about 40 rhizobial
strains isolated from different Australian acacias indicated that isolates that
performed well on one Acacia species often performed well on others as
well (Thrall et al. 2000). A similar phenomenon could be detected in this
work. The five African Sinorhizobium strains tested, namely S. arboris strain
1552T, S. kostiense strains 1489 T and 1493, S. saheli strain 1496 and S.
terangae bv. acaciae ORS 1058 (I: Table 1), were capable of effectively
nodulating a wide range of African acacias (A. mellifera, A. nilotica, A.
oerfota (synonym A. nubica), A. nilotica, A. senegal, A. seyal, A. sieberiana, A. tortilis subsp. raddiana) and several Latin American Prosopis spp. (I:
Table 2).
Although a wide range of rhizobia are capable of inducing N2-fixing
nodules on Acacia and Prosopis trees, there seems to be a certain degree
of host specificity between rhizobia and tree species (Khbaya et al. 1998,
Zerhari et al. 2000). Thrall et al. (2000) found that in Australian acacias,
species with more limited distribution or tighter ecological requirements
have a greater degree of specificity than widespread species. A similar specificity seems to occur within rhizobial species. In African S. terangae,
two biovars, sesbaniae and acaciae have been distinguished according to
which plants the strains induce effective nodules on (Lortet et al. 1996).
Haukka et al. (1998) suggested that an equal division could be done in S.
saheli, because S. saheli strains isolated from Sesbania spp. induced effective nodules on several Sesbania spp. but ineffective ones on Acacia spp.
(de Lajudie et al. 1994), whereas the S. saheli strain isolated from Acacia
sp. and used in this thesis work behaved in an opposite way (I: Tables 1,
2). Interestingly, the nodA sequences were clearly different depending, on
whether S. saheli strains were isolated from Acacia spp. or Sesbania cannabina (Haukka et al. 1998). Unlike acacias, trees and bushes of the papilionoid genus Sesbania grow in moist and waterlogged soils (Allen and Allen
1981, Roshetko 2001). Thus, taxonomically similar Sinorhizobium species
appear to have adapted to different environments and adjusted to nodulate
different host plants, probably, through lateral transfer of symbiotic genes
(Haukka et al. 1998).
The fact that how Acacia and Prosopis trees belonging to different
genera share same rhizobia might be explained by these trees being closely
53
related (Chappill 1995, Bukhari 1997, Bukhari et al. 1998). Comparison
of symbiotic genes (nodA, nifH) revealed that Latin American sinorhizobia,
which were isolated from Prosopis nodules, differed from African sinorhizobia (Haukka et al. 1998). Thus, it would be interesting to know if Latin
American sinorhizobia are also capable of effectively nodulating African
acacias.
It was surprising that the African sinorhizobia induced only ineffective
nodules on African P. africana although they were capable of forming N2fixing nodules on Latin American Prosopis spp. (P. chilensis, P. juliflora
and P. pallida) and on Afro-Asian P. cineraria (I: Table 2). P. africana may
grow in slight moister environments, with annual rainfall over 600 mm
(Keay 1989, Bellefontaine et al. 2000), but otherwise it should share similar growth sites with several Acacia species studied here. P. africana was
found to be ancestral to other Prosopis trees (Bukhari et al. 1998) forming
a separate series (Burkart 1976). The growth habit of P. africana seedlings
differed from that of other Prosopis species; P. africana had a sturdy but
smooth stem whereas the stem of other Prosopis (and Acacia) seedlings
became woody early (I). Perhaps P. africana has diverged from other Prosopis species a so long time ago that they do not share similar rhizobia
any more.
As discussed in the section 1.5.7, African and American acacias belonging to the subgenera Acacia and Aculeiferum are more closely related
than they are to the Australian acacias belonging to the subgenus Phyllodineae (Robinson and Harris 2000). This might explain why the African
sinorhizobia induced only small, ineffective nodules on A. holosericea (I:
Table 2). Probably A. holosericea, like other Australian acacias, prefers
slow-growing bradyrhizobia (Dreyfus and Dommergues 1981, Marsudi et
al. 1999, Lafay and Burdon 2001). Interestingly, the nodulation pattern of
the broad host range Rhizobium sp. strain NGR 234, originating in Papua
New Guinea, distinguished Australian acacias from other Acacia species.
The strain effectively nodulated Australian acacias but not African and
South American ones, and neither Prosopis spp. (Pueppke and Broughton
1999).
4.1.2. Variation in the effectiveness of the symbiotic association
Except for A. holosericea and P. africana, inoculation of Acacia and Prosopis
with effective Sinorhizobium strains improved plant yield compared to that
of uninoculated seedlings (I: Fig. 5). However, no strain appeared to be superior to the others (I: Fig. 6B), and there were no differences between any
rhizobia-Acacia or rhizobia-Prosopis combinations (I: Fig. 5).
The effectiveness of symbiotic associations (determined mostly as growth
performance) between native rhizobia and acacias can vary a lot (Barnet
54
et al. 1985, Barnet and Catt 1991, Burdon et al. 1999, Thrall et al. 2000).
Parallel results were obtained in this work when A. senegal seedlings were
inoculated with seven Sudanese strains. Based on the ability to improve plant
yield, the strains could be classified as effective, poorly effective and ineffective (I: Fig. 7).
It is a generally accepted theory that rhizobia have adjusted to nodulate
various legumes through lateral transfer of symbiotic genes (Haukka et al.
1998). Consenquently, rhizobia may also lose symbiotic genes, especially, if
they are located on symbiotic plasmids, which are easily movable genomic
elements. Indeed, many rhizobial strains have been observed to lose their N2
fixation or nodulation ability during storage (Sutherland et al. 2000, Odee et
al. 2002a). In our laboratory, Sudanese sinorhizobia were observed to carry
several large plasmids, one of those containing symbiotic genes (Haukka et
al. 1998). According to earlier studies, Sinorhizobium sp. strain 1480 formed
effective nodules on A. senegal and P. chilensis plants (Zhang et al. 1991).
Later, no plasmids were detected (Haukka et al. 1998), and in this work strain
1480 induced small, white and ineffective nodules on A. senegal (I: Table 1;
Fig. 7). In laboratory conditions, the heat treatment can trigger elimination of
plasmids from rhizobial cells (Baldani and Weaver 1992), but it is still unknown to which extent loss of symbiotic plasmids occurs among indigenous
or inoculant rhizobia in the soil.
Both herbaceous and woody legumes have exhibited genetic variability
in the properties related to growth and N2 fixation (Sanginga et al. 1990,
Vance 1998). In this work, the Sudanese provenance of A. senegal showed
better growth and symbiotic performance than Kenyan and Senegalese A.
senegal (I: Fig. 6B). Thus, by taking into consideration symbiotic and N2fixing properties when breeding or selecting woody legumes, it would be
possible to enhance biological N2 fixation.
4.2. Development of the symbiosis between sinorhizobia and Acacia
and Prosopis trees
As discussed in Section 1.7.2., depending on the legume species, rhizobia
can enter plant roots either via wounds (crack entry), intact epidermis, or by
the classic root hair pathway. Both the crack infection and root hair infection may give rise to either determinate or indeterminate nodules (Table 1-4).
Except for the extensive work of Corby (Corby 1988), who classified legumes
according to the nodule morphology, there are only few reports in which
infection or nodulation on Acacia/Prosopis trees have been examined (P.
glandulosa; Baird et al. 1985). This work showed that, except in A. seyal, the
infection mode between sinorhizobia and Acacia or Prosopis species studied
resembled that of classical root hair infection, and that the nodules formed
shared features with indeterminate nodules (I).
55
4.2.1. Infection through the root hairs
The infection process between S. arboris and Acacia/Prosopis seedlings had
several common features with the classical root hair infection: presence of
root hairs, deformation of hairs, and formation of infection threads (I: Table 2,
Fig. 1 and 2). Infection threads were also found in P. glandulosa hairs (Baird
et al. 1985).
As in herbaceous legumes (Dart 1977, Vandenbosch et al. 1985, Räsänen
et al. 1991), inoculated Acacia/Prosopis hairs were shorter and broader than
uninoculated ones (I: Fig. Ia, b), markedly curled hairs occurred in patches
(I) and infection occurred in localised groups (L. Räsänen, unpublished). The
latter phenomenon was also observed on P. glandulosa (Baird et al. 1985).
P. chilensis, P. juliflora and P. pallida had long deformed hairs with knob
or shepherd’s crook-like structures (I), similar to the characteristics of alfalfa
hairs (Räsänen et al. 1991).
There were some features which were characteristic of the early infection process of Acacia/Prosopis trees. In herbaceous papilionoid legumes,
the hairs, which mainly are long, form a continuous cover in the elongation region of the roots, and hairs of different sizes are deformed (e.g. Räsänen et al. 1991). In Acacia spp., the short hairs grew on lateral roots in
patches, and there were many bare roots. Prosopis species had a denser
cover of longer hairs on the lateral roots (I). Deformed hairs of Acacia
and P. chilensis were usually dwarfed, swollen and bent against the root
surface (I: Fig.1b-d). In longer hairs of all species, infection pockets, i.e.
intercellular spaces for rhizobia, were detected near the site of rhizobial
penetration (I: Fig. 1f). Later, when bacteria had proliferated, the pockets
were enlarged, appearing as sac-like structures, from which the infection
threads originated (I: Fig. 2a-c, f). Studies with the papilionoid S. rostrata
indicated that the formation of infection pockets, which is a prerequisite
for the nodule initiation, was accompanied by local plant cell death. Probably, Nod factors induced an oxidative burst generating reactive oxygen
species and local ethylene production, subsequently causing death of
plant cells (D’Haeze et al. 2002).
In the case of A. seyal only single hairs developed at lateral root junctions and no proper infection threads were found on them (I). Thus, it is
possible that this species is infected for example by crack entry (Fig. 1-2).
In optimal laboratory conditions the infection process between sinorhizobia and Acacia/Prosopis seedlings proceeded almost as fast as in herbaceous legumes. Although the lateral roots were still short or developing
and hairs were uncommon four days after inoculation, deformed hairs
were common three days later. The first infection threads appeared seven
days and first nodules 11 days after inoculation, respectively.
56
4.2.2. The structure of effective indeterminate nodules
In Acacia and Prosopis, the nodules developed singly or grouped mostly
on lateral roots and occasionally, on taproots, or on short and thin rootlets,
which arose from the taproot. Young nodules were spherical, but later they
became elongated and often branched, being able to grow from several
points (I: Figures 3C, 4C). The nodules showed typical indeterminate arrangements of tissues: an outer cortex, nodule endodermis, several vascular
bundles, and a central tissue composed of bacteroid containing cells and
uninfected cells (I: Figures 3, 4A, B). The cortex layers of A. nilotica and
A. sieberiana nodules were especially thick (I: Figures 3A, B). Except P. africana nodules, those of other Prosopis species, A. nilotica and A. sieberiana
contained dark stained depositions, presumably tannins in the nodule cortex
(I: Fig 3C). Both the rigid cell layers and tannin inclusions have been reported
to be features of perennial nodules (Fred et al. 1939, Allen and Allen 1981).
Though Acacia and Prosopis nodules were basically indeterminate, and
in common with the descriptions of Baird et al. (1985) and Corby (1988), the
following evidence suggests that the nodules diverge to some degree from the
classical indeterminate nodule (Vasse et al. 1990). P. glandulosa, a common
tree in warm deserts of North America, usually forms elongate, indeterminate
nodules. The morphology of nodules showed, however, high variability when
the plants were inoculated with cowpea (Vigna unguiculata) rhizobia (Virginia et al. 1984, Baird et al. 1985) or with soil collected from various depths
under Prosopis canopies (Johnson and Mayeux 1990). P. glandulosa seedlings produced two types of N2-fixing nodules: typical indeterminate nodules and spherical ones that resembled the determinate type lacking a clear
meristemic/invasion zone (Virginia et al. 1984). It was assumed that the variability in nodule morphology represented different stages of maturity (Johnson and Mayeux 1990). Also in my thesis work, the apical meristem was not
always easily detectable (I: Fig. 3A). Like in Australian A. saligna (Marsudi et
al. 1999), a low proportion of bacteroid filled cells were characteristic for the
central tissue of Acacia and Prosopis nodules studied here (Fig. 3B, 3C).
4.2.3. Ineffective nodules of A. holosericea and P. africana
The internal structure of ineffective nodules may vary a lot, being like effective
ones or resembling more a tumour than a nodule. Obviously, the more the
ineffective strain is related to those rhizobia that induce N2-fixing nodules on
the given plant, the more nodules formed resemble characteristic ones. In P.
glandulosa, ineffective nodules contained also bacteroids (Virginia et al. 1984,
Baird et al. 1985). In this thesis work, the five African sinorhizobial strains
induced hair deformation and ineffective nodules on the roots of Australian A.
holosericea and African P. africana (I: Table 2). This indicated that some recog-
57
nition occurred between bacteria and plants. Nevertheless, the strains tested
only distantly resembled compatible rhizobia of A. holosericea or P. africana,
because tumour-like structures with undifferentiated cell tissue formed on A.
holosericea roots (I: Fig. 4D) and nodule-like structures, having some organisation of cell layers, developed on P. africana roots (I: Fig. 4E).
Acacia and Prosopis trees seem on one hand to be capable of forming
N2-fixing nodules with a wide range of rhizobia and on the other hand they
are not able to exclude nodulation by incompatible rhizobia. Vegetative rhizobia, being capable of saprophytic growth in soil and nodule debris, has
been found both from effective and ineffective nodules (Paau et al. 1980,
Vance et al. 1980, Timmers et al. 2000). Sprent suggested (2001) that at the
community level both legumes and rhizobia gain advantage from promiscuity. Ineffective (as well as effective) Acacia nodules may be a way of maintaining rhizobial populations under stress conditions. Rhizobia ineffective
with Acacia sp. may be effective on other legumes growing in the same area.
Subsequently, in the wet season for example annual herbs benefit from rhizobia released from the ineffective nodules.
4.3. TOLERANCE OF SINORHIZOBIA TO ABIOTIC STRESSES
4.3.1. Properties of two potential inoculant strains
The two major strains, S. arboris 1552 and S. saheli 1496, used in this work
differed positively from other strains due to their competent symbiotic properties already during the first nursery tests performed in the Sudan (Karsisto and
Lindström 1992). Later studies together with this work indicated that they also
possess other favourable properties (Table 4-1). Strain 1552 can be considered
as salt and osmotolerant, whereas strain 1496 is sensitive to salt but tolerated
higher growth temperatures (Table 4-1). According to the nodulation tests, both
strains were competitive against an ineffective strain, but strain 1496 was a
better competitor when matched with strain 1552 (Zhang et al. 1992).
Trehalose appeared to be a good carbon source for the strains 1552 and
1496 (III). When the growth of the strains was tested on minimal medium
with six sugars (e.g. glucose, mannitol, trehalose), trehalose also caused the
most rapid growth (L. Räsänen, unpublished).
4.3.2. Tolerance of heat
Tropical rhizobia isolated either from herbaceous or woody legumes can
grow at around 40˚C (e.g. Hafeez et al. 1991, Baldani and Weaver 1992,
Surange et al. 1997, Hashem et al. 1998, Zerhari et al. 2000). Similar observations were done with the Sudanese tree rhizobia (Zhang et al. 1991,
Zahran et al. 1994). Extremely thermotolerant Pakistan and Indian strains,
58
Table 4-1. Properties of two major strains, Sinorhizobium arboris HAMBI 1552
and S. saheli HAMBI 1496, used in this work.
Strain
Parameter
1496
1552
References
Tmax; YEM agar
Tmax; TY agar
44.2oC
41.2-41.7oC
41.5oC
40.8oC
Zahran et al. 1994
L. Räsänen, II
NaCl tolerance in YEM broth
Growth in 0.5 M NaCl; YEM agar
Growth in 0.1 M NaCl; BD broth
Growth in 0.5 M NaCl; BD broth
1.5% (0.26 M)
+++
+
3% (0.5 M)
++
+++
++
Zhang et al. 1992
Zahran et al. 1994
III
III
Tolerance of sucrose; YEM agar
0.34 M
1.0 M
Zahran et al. 1994
Growth in 9% PEG 6000
Growth in 24% PEG 6000
++
not determined
++
+
III
III
Colony morphology
small, compact
large, mucuous II, III, IV
- no growth; + weak growth; ++ moderate growth; +++ good growth
such as a strain isolated from the tree Albizia lebbek, could grow at around
50°C (Hafeez et al. 1991, Surange et al. 1997). With reference to data
above, S. arboris strain 1552 and S. saheli strain 1496 are well adapted to
tropical soils, with Tmax around 41°C and 41.5-44°C, respectively (Table 4-1).
However, the study with the heat-tolerant rhizobial strain indicated that it
did not possess better abilities to fix N2 at high temperature. Probably, the
advantage of the thermotolerance is connected to the fact that the strain has
capacity to survive the periods of thermal stresses and to recover afterwards
(Michiels et al. 1994).
When strain 1552 was grown as liquid cultures at 40ºC, growth was
instable (IV: Fig. 1A), indicating that it suffered from thermal stress. Interestingly, strain 1552 probably experienced some stress already at 37ºC because
the luc-marked derivative of strain 1552 showed large variation in optical
density, i.e. EPS production. In addition, luciferase activity decreased rapidly
at the end of the incubation, revealing that energy reserves were exhausted
(IV: Tables 1 and 2).
4.3.3. Tolerance of salt
Arid and semiarid soils often suffer from elevated salinity due to high evaporation. Thus, it was not surprising that like S. arboris strain 1552 (Table 4-1),
many rhizobia isolated from Acacia and Prosopis trees were moderately salt
tolerant, capable of growing in 0.3-0.5 M (2-3%) NaCl (Craig et al. 1991,
Zhang et al. 1991, Zahran et al. 1994). Several very salt tolerant strains
(0.6-0.86 M or 3.5-5% NaCl) were isolated from the nodules of Indian,
Pakistani and Moroccan acacias acacias (Surange et al. 1997, Kumar et al.
59
1999) Zerhari et al. 2000). About a third of the strains isolated from different
legumes growing in salt-affected soils in Morocco were very (0.86 M NaCl)
or extremely salt tolerant (1.7-2.4 M, 10-14% NaCl) (Abdelmoumen et al.
1999). The growth of the salt sensitive S. saheli 1496 strain was stimulated
by 0.1 M NaCl (III: Fig. 2). Perhaps this phenomenon was due to that NaCl
influences trough ionic and osmotic factors and/or rhizobia occupying arid
and semiarid soils are generally adapted to grow with some salts.
There is a couple of studies, in which the salt sensitive strain was more
affected than the salt tolerant one or failed in nodulation of salt tolerant
Acacia seedlings grown under salt stress (Zou et al. 1995, Kumar et al. 1999).
However, the salt tolerance in the laboratory does not necessary indicate salt
tolerance in the field.
Subsequently, it is not known whether the salt tolerance is a favourable property only for free-living bacteria occupying saline soils or does it play some
role also during the infection process and symbiosis when the host legume
grows in saline soil. In the symbiotic state rhizobia live in a cell where the
solute concentration is high, approximately 0.3-0.4 M (Sprent 1984). When
free-living rhizobia differentiate into bacteroids, LPSs located in the outer surface undergo structural modifications that are somehow involved in the changes in environmental conditions (Kannenberg et al. 1998). Possibly, bacteroids
in any case adapt to high osmolarity through LPS modifications, and therefore
the salt tolerance of a strain may not be relevant during the symbiosis.
The model organisms Escherichia coli, Salmonella typhimurium and S.
meliloti reacted to salt stress mainly by increasing levels of K+ and glutamate
(Galinski 1995, Miller and Wood 1996). Interestingly, rhizobial strains that
were isolated from the nodules of Acacia sp. growing in salt-affected soils
behaved like halobacteria, by accumulating NaCl but not KCl when grown
in NaCl. The relative proportion of glutamate also remained fairly constant
irrespective of the salt treatment (Craig et al. 1991). Thus, rhizobia may also
have other kinds of salt tolerance mechanisms in addition to the presently
known ones.
Salt tolerance of rhizobia may correlate with drought tolerance (Athar
1998). This information can help when looking for drought tolerant strains.
Recently, eight gene loci were found to be involved in salt tolerance of R.
tropici. Most of these genes were also required for adaptation to hyperosmotic media, and all genes were important for the bacteroid development or
function (Nogales et al. 2002).
4.3.4. Roles of glycine betaine and trehalose for sinorhizobia
Compatible solutes as a carbon source. Contrary to E. coli and S. typhimurium, rhizobia have a capacity to utilise glycine betaine as a carbon and/or
nitrogen source at low osmolarity (Miller and Wood 1996). S. arboris strain
60
1552 and S. kostiense strain 1496 could use 0.01 and 0.05 M glycine
betaine as a sole carbon source but the growth was delayed compared to that
with 0.006 M glucose or 0.01 M trehalose (III: Fig. 1A, 2A. This suggested
that glycine betaine is not a primary carbon source and/ or it is degraded
through a secondary pathway.
Compatible solutes as osmoprotectants. Although the potential to transport glycine betaine is widespread among rhizobia (Sauvage et al. 1983,
Bernard et al. 1986, Smith et al. 1988, Boncompagni et al. 1999), not all rhizobia are able to use glycine betaine as an osmoprotectant (Boncompagni et
al. 1999). In the case of the tropical Sinorhizobium strains used in this work,
0.01, 0.05 and 0.1M glycine betaine and trehalose functioned as osmoprotectants under osmotic stress induced by 9 and 17% PEG but only trehalose
improved cell growth under salt stress (0.5 M NaCl; III: Fig. 1, 2) According
to Gouffi et al. (1999) exogenous trehalose serves under osmotic stress both
as a carbon source and as an osmoprotectant. This theory may explain why
the stressed Sinorhizobium cultures produced better a growth response with
trehalose than with glycine betaine (III: Fig. 1, 2).
4.4. THE A. SENEGAL-S. ARBORIS SYMBIOSIS UNDER HEAT AND DROUGHT
STRESS
4.4.1. Rhizobial population in the soil
Dry and hot seasons restrict N2 fixation and nodulation of leguminous trees
and bushes (Barnet et al. 1985, Hansen and Pate 1987). In arid and semiarid
regions of the tropics, the soil temperatures near the surface can be very high.
In Australian sandy soils, the temperature near the soil surface was 59°C at
the air temperature 39°C. However, the soil temperature decreased rapidly
with depth, being moderate 35°C, at 15 cm (Fig. 4-1).
There is no information available, how rhizobia tolerate high temperatures in natural desert soils. It appears, however, that rhizobia are more resistant to high temperatures in soil than in laboratory medium (AbdelGadir
Fig. 4-1. Soil temperatures in bare
sandy soils of Western Australia
according to Chatel and Parker
(1973). The maximum air temperature was 39°C. At 5.1 cm the temperature was above 40°C for seven
hours and at 10.2 cm above 35°C for
eight hours.
61
and Alexander 1997). A similar phenomenon was observed in this work.
Although the Tmax for S. arboris strain 1552 was around 41°C in laboratory
conditions (Table 1-4), the numbers of culturable cells in the A. senegal rhizosphere were similar (107 cfu g-1) irrespective of the daily maximum soil
temperature (30°C or 42°C; II).
The study performed with Indian desert soils suggested that not the high
soil temperature but the low organic matter and poor soil moisture were the
major factors that reduced the numbers of different micro-organisms (Rao
and Venkateswarlu 1983). Indeed, in drought-affected A. senegal soils the
numbers of culturable rhizobia were significantly reduced, approximately
from 107 to 106 cfu g-1 (III: Table 1). In conclusion, one can assume that during
the dry season, the water deficit together with the high soil temperature will
considerably decrease rhizobial numbers or cause a lack of rhizobia in the
surface soils (0-10 cm).
4.4.2. Adaptation of A. senegal seedlings to heat and drought
Adult A. senegal trees tolerate high daily temperatures, the mean maximum
temperatures being around 48ºC (Table 1-3). A. senegal seedlings also
appeared to be resistant to high temperatures, by surviving several weeks at
the maximum root temperature of 42ºC (II). Nevertheless, seedlings responded
to the thermal stress by stopping shoot growth and nodulation (II: Fig. 2, 3).
Unlike uninoculated, non-stressed plants, thermally stressed seedlings lacking
nodules did not suffer from a shortage of N, although they were grown in
nitrogen-free media (II). Due to the big size, A. senegal seeds are capable of
germinating under drought stress. Their seeds are also a large source of energy
and nutrients (Khurana and Singh 2001). These properties might explain how
thermally stressed A. senegal seedlings with limited nutrient sources could
survive a prolonged period on the seed reserve (II).
Adult A. senegal trees are very drought resistant, having several dehydration postponement strategies (e.g. deep rooting system, reduction in leaf
area, drought-deciduous growth habit; section 1.8.4). Formation of gum
arabic is also supposed to be a natural response of A. senegal to store
strongly hydrophilic carbohydrates under drought stress (Roshetko 2001). A.
nilotica and A. tortilis seedlings were able to increase the root:shoot ratio
under drought stress, resulting in enlargement of the absorptive root biomass
(Michelsen and Rosendahl 1990, Otieno et al. 2001). When A. senegal were
grown under favourable watering conditions, root hairs usually occurred in
patches and there were many bare roots (I). However, under water deficit
both inoculated and uninoculated seedlings developed more hairs on their
roots than unstressed ones (III), obviously, in order to enhance efficient water
uptake.
62
4.4.3. Infection and nodulation under stress
Use of the GUS-marked S. arboris strain 2180, which expressed ß-glucuronidase (GUS) both in free-living cells and during the early stages of symbiosis,
turned out to be a sensitive and rapid tool for following and comparison of
infection and nodulation between stressed and non-stressed roots. Contrary
to the traditional stain methylene blue, only areas colonised or occupied by
GUS-marked strain were stained blue. This was important in the case of A.
senegal roots, because the hairs were short and unevenly distributed, and
infection threads occurred in very short hairs.
The unfavourably high root temperature and drought stress caused A.
senegal hairs to be partly abnormally deformed. Short and swollen bottlelike hairs were typical under heat stress (II: Fig. 4b-d), and very short or
dwarfed, swollen hairs were characteristic of water-stressed hairs (III: Fig.
8d). Under both stresses, infection threads were rare, and they seemed to be
either disintegrated (II: Fig. 4e) or they had weak glucuronidase activity (III
Fig. 8e). Similar observations were made in Trifolium subterraneum nodules,
where the high root temperature disrupted growth of infection threads and
disturbed transformation of bacteria into bacteroids. Thermal stress may also
accelerate nodule senescence (Pankhurst and Gibson 1973).
In heat stress experiments, nodulation of A. senegal seedlings was impaired at 38°C, reduced to 50% at 40°C and completely inhibited at 42°C (II:
Fig. 2).
The water stress retarded or stopped the normal nodule development so
that the numbers of nodule initials were higher on water-stressed plants than
on well-watered plants. For the numbers of true nodules the situation of was
reversed (III: Fig. 7). The nodules formed showed signs of premature senescence. After incubation with GUS substrate buffer, nodules displayed weak
glucuronidase activity (III: Fig. 8e), indicating low cell numbers and/or low
activity of rhizobia occupying nodules.
In T. subterraneum, water deficit was also observed to prevent the development of nodules (Worral and Roughley 1976).
In many tropical crop legumes, a root temperature of around 40°C (5-8
h per day) inhibited development of new nodules (Arayangkoon et al. 1990,
Kishinevsky et al. 1992, Hungria and Franco 1993). Of the tropical woody
legumes, Sesbania sesban still nodulated at 40°C but Calliandra calothyrsus
did not (Purwantari et al. 1995). In the case of A. senegal seedlings, nodulation was completely inhibited at 42°C (II). However, the inhibition of nodulation was reversible. After A. senegal seedlings were transferred from 42°C
to 30°C, N2-fixing nodules developed on those parts of lateral roots that were
developed after completion of thermal stress (II).
When Acacia and Prosopis seedlings were grown in optimal growth
chamber or greenhouse conditions, big nitrogen-fixing nodules were gene-
63
rally formed just below the soil surface (0-5 cm), on the hairs located at the
root base (L. Räsänen, unpublished results). Data of Chatel and Parker (1973)
suggest that the temperature in the surface soil (0-10 cm) can be so high, ranging between 60 and 39°C (Fig. 4-1) that the nodulation of the upper roots
of the tree seedlings could be prevented in the field.
In spite of the seedlings being exposed to the high root temperature of 42°C
or severe drought stress, it appeared that the stressed A. senegal seedlings had
the most important elements that were needed for proper infection. The plants
had lateral roots and root hairs (II: Fig. 4; III: Fig. 8), although their development
was delayed at high root temperature (II). The hairs were colonised by rhizobia,
and they were partly deformed (II: Fig. 4b; III), indicating that S. arboris cells
were capable of producing Nod factors. Both the thermally stressed and waterstressed soils contained sufficient numbers of rhizobia for nodulation, 105 -107
cfu g-1 (II, III). Also according to Williams and de Mallorca (1984), the reduction in nodule numbers on soybean roots under water stress was not associated
with a reduced number of rhizobia in the rhizosphere, nor due to an effect on
root growth or hair formation. What could then explain why nodulation was
inhibited? Generally, plants are more sensitive to environmental stresses than
rhizobia, and it is usually the plant that restricts nodulation. The plant hormone
abscisic acid (ABA) is one of the major signals operating during drought and
saline stress (Bray 1997). Foliar application of ABA to unstressed soybean roots
was found to inhibit nodulation. In addition, under leaf moisture stress the
endogenous content of this plant hormone was increased in the roots. Thus,
ABA may contribute to the extensive inhibition of nodulation observed under
water stress (Williams and Sicardi de Mallorca 1984).
4.5. ROLE OF COMPATIBLE SOLUTES ON THE A. SENEGAL-SINORHIZOBIUM
SYMBIOSIS
4.5.1. Endogenous glycine betaine in A. senegal
Glycine betaine is a compatible solute, which enables some flowering plants
to grow in salt-affected soils. It appears that the capacity to synthesise glycine
betaine is widespread, being characteristic for angiosperms but expressed
strongly by some and weakly by others (Rhodes and Hanson 1993). According to Rhodes and Hanson (1993), under non-stressed condition glycine
betaine levels are for accumulating and nonaccumulated species 5-100
µmol g-1 dry wt and <1 µmol g-1 dry wt, respectively. Under natural or
experimental saline or dry conditions accumulators contain glycine betaine
40-400 µmol g-1 dry wt. According to this definition, acacias do not accumulate glycine betaine because the glycine betaine content was less than 1
µmol g-1 dry wt for non-stressed plants (Rhodes and Hanson 1993; III) and
8.5 µmol g-1 for drought-stressed Australian acacias (Erskine et al. 1996).
64
Proline and polyols (pinitol, mucoinositol) are the most common compatible
solutes detected in acacias so far (Erskine et al. 1996).
Many crop plants are not able to synthesise glycine betaine but they
can translocate foliar-applied glycine betaine from leaves to other organs. In
addition, plants usually are incapable of degrading glycine betaine (Mäkelä
et al. 1996, McNeil et al. 1999). Therefore, foliar application of glycine
betaine, which is a major by-product when sugar is manufactured from sugar
beet (Beta vulgaris), has been used to improve drought tolerance and subsequently, to increase crop production of drought-sensitive plants (Agboma
et al. 1997, Diaz-Zorita et al. 2001). Addition of glycine betaine to the soil
has functioned similarly to the foliar application (Blunden et al. 1997, Xing
and Rajashekar 1999). The suitability of foliar application was also tested
on Sudanese A. senegal seedlings (III). Then, the foliage could absorb and
translocate 0.01 M glycine betaine into roots but the leaves were sensitive to
0.01 M glycine betaine, tolerating concentrations of 0.001 M.
4.5.2. Effects of exogenous compatible solutes on A. senegal seedlings
Moisture stress is considered as the major factor that causes mortality among
tree seedlings during the dry period (Bukhari 1998, Khurana and Singh
2001). Seedlings that develop some physiological and/or morphological
adaptive characteristics to water stress can account for greater success in
restoration efforts (Khurana and Singh 2001). To understand mechanisms
that presumably function when A. senegal seedlings adapt to the drought
stress, roles of two compatible solutes, glycine betaine and trehalose, were
evaluated (III).
0.0003 M glycine betaine in the soil mix helped A. senegal seedlings,
so that 60% of the plants were still alive 42 days after inoculation whereas
the drought-stressed plants grown without any increments were wilted 30
days after inoculation (III: Fig. 6). The daily transpiration rates of the glycine
betaine-treated soybeans were relatively lower than those of plants not treated (Agboma et al. 1997). Application of glycine betaine allowed also beans
(Phaseolus vulgaris) to maintain a better water status during water stress
so that they developed wilting symptoms much later compared to stressed
plants without glycine betaine (Xing and Rajashekar 1999). In this work,
drought-affected soils containing glycine betaine tended to keep more water
than non-treated soils (III: Table 1). Taken together these data suggest that
application of glycine betaine enabled A. senegal seedlings to control water
uptake and transpiration more efficiently.
0.0003 M trehalose slightly increased the numbers of A. senegal plants
survived (III: Fig. 6). However, plants do not accumulate trehalose but an
another disaccharide, sucrose (Goddijn and van Dun 1999). Thus, whether
the slightly improved survival of the seedlings was for example, due to the
65
capability of trehalose to preserve biological structures against desiccation
(Potts 1994, Goddijn and van Dun 1999, Welsh 2000), or based on its ability
to absorb water, remained unknown.
The advantageous effect of compatible solutes may be also due to the
enhanced EPS production or other activity of rhizobia. Apparently, rhizobial
EPS absorbs water, because EPS layers are formed by the accumulation of
various types of polymeric substances of high viscosity around bacterial cell
walls (Potts 1994). Inoculation of plant roots with EPS-producing Rhizobium
sp. improved soil structure, probably, by increasing soil adhesion to roots
and/or by enhancing the stability of soil aggregates around roots (Alami et al.
2000). Thus, an explanation for the survival of A. senegal seedlings with glycine betaine under drought stress (III) could be that the increased rhizobial
population with enhanced EPS production and/or other activities colonises
roots and rhizospheres, subsequently protecting roots from drought.
4.5.3. Effects of exogenous compatible solutes on rhizobia
Both glycine betaine and trehalose functioned as osmoprotectants for the cells
of S. arboris strain 2180, when they occupied A. senegal soil exposed to
drought stress. Although the drought stress significantly reduced the numbers
of culturable cells from 107 - 106 cfu g-1, the presence of small amounts of
glycine betaine or trehalose (0.0001 and 0.0003 M) in the soil maintained
the cfu counts at the same level as detected in non-stressed soils (III: Table 1).
Based on previous data, (Sauvage et al. 1983, Bernard et al. 1986, Smith et al.
1988, Boncompagni et al. 1999), the roles of glycine betaine for sinorhizobia
might have varied during the course of the plant experiment. Under favourable
conditions and at the beginning of the drought experiments, the cells utilised
glycine betaine as an additional carbon and/or nitrogen source but when the
cells started to suffer from water deficit, glycine betaine was used as an osmoprotectant. Beneficial effect of trehalose might also contribute to its ability to
protect cells from desiccation (Bushby and Marshall 1977, Potts 1994).
4.6. RESPONSES OF S. ARBORIS POPULATIONS TO CHANGING
ENVIRONMENTAL CONDITIONS
Bacteria were earlier considered to exist as simple, solitary cells. This opinion
has changed and bacterial populations are viewed as colonial or multicellular organisms that use, for example, cell-to-cell communication to facilitate
their adaptation to environmental fluctuations (Whitehead et al. 2001). The
cell-to-cell communication at high population density (commonly known as
quorum sensing and mostly through N-acyl homoserine lactones in Gramnegative bacteria) prevents single bacteria from spending resources for a
certain function before the bacterial population is big enough. According
66
to recent data (e.g. Swift et al. 2001) rhizobia possess four quorum sensing
systems, which control several phenotypes: restriction of nodule number,
root nodulation, conjugation of plasmids, growth inhibition and stationary
phase adaptation.
To ensure survival in changing environments, bacteria have regulatory
elements that allow them to respond rapidly to stress conditions. The sigma
factors RpoS (ss) and RpoE (sss) belongs to these elements. RpoS plays an
important role when bacteria transform from the exponential growth phase
to the stationary phase, and during starvation process and under osmotic
stress (Kolter et al. 1993, Munro et al. 1995). In Pseudomonas fluorescens,
the sigma factor RpoE controled EPS production and tolerance of towards
desiccation and osmotic stress (Schnider-Keel et al. 2001).
Multiphasic alterations in the expression of bacterial phenotypes, the
phase variations, continuously generate cells with varied phenotypes leading
to a mixed population. This type of diversity ensures that a certain percentage
of cells in the population will express the phenotype needed for survival in
new environmental conditions. Phase variations are generally reversible and
random events occurring at high frequency (>10-5 per generations; Henderson et al. 1999). When Sinorhizobium cells were exposed to heat, drought,
salt and osmotic stresses in my thesis work, several extraordinary features
appeared.
4.6.1. Alterations in EPS production
Rhizobia, as other Gram-negative bacteria, produce variety of surface
polysaccharides, including EPS, LPS, CPS and periplasmic ß-glucans. EPS
and LPS are important when rhizobia infect root hairs and live as bacteroids
inside nodules. Periplasmic ß-glucans play an important role during osmotic
adaptation (Kijne 1992, Brewin 1998).
S. arboris strain 1552 as well as its gene-modified derivatives, formed
slimy colonies on agar, indicating that they were EPS producers (II, IV). If
the medium contained excess glucose or trehalose, it was apparently used
for both EPS production and cell growth (III). Generally, when strain 1552
was grown in liquid medium, the optical cell density determined with a
spectrophotometer increased in a proportion similar to the cell numbers (IV,
Fig. 1A). However, in thermally stressed S. arboris cultures, the final optical
density did not correlate with total cell counts or the numbers of culturable
cells (IV; Fig. 1B, Tables 1, 2). The reason for this phenomenon is unknown.
Strain 1552 also showed another peculiarity when grown under heat
stress. A small portion of the colonies changed their morphology from
mucous to dry when they were resuscitated from lyophilised cultures (L.
Räsänen unpblished), frozen cultures (IV) and thermally stressed A. senegal
rhizospheres (II) and liquid cultures (IV). The experiments performed with
67
the dry morphotype showed that the new colony morphology was a stable
property (IV: Fig. 2C, Tables 1, 2), and that the dry morphotype had retained
its nodulation ability (II). The change in EPS production seemed not to be
involved in improved heat tolerance (IV; Fig. 2). Instead, the cell morphology between the mucous and dry form differed to some degree when the
two morphotypes were grown as liquid cultures at 40ºC (IV: Fig. 4b-e, 5b,
c). This suggested that additional changes in cell morphology and physiology
were also associated with the altered EPS production.
In Gram-negative bacteria, phase variation particularly involves alterations
in expressions of cell surface structures (e.g. fimbria, flagella, outer membrane,
proteins, EPS, LPS), which often give rise to changes in observable phenotypes
(such as colonial morphology; Henderson et al. 1999). Thus, in the case of
S. arboris, the temperature shift, such as cold shock, might have induced a
change of the colony morphology through the phase variation mechanisms.
A halotolerant S. meliloti strain showed a decrease in mucoidy and produced
40% less EPSs when the medium was supplemented with NaCl (Lloret et al.
1998). In S. meliloti, EPS consists of EPS I (succinoglucan) and EPS II (galactoglucan). Later studies revealed that the changes in colony morphology in salt
were linked to EPS II. Regulation of EPS II appeared to be complex, varying
between strains. Possibly, regulation of EPS I and EPS II genes depends on the
environmental conditions (Lloret et al. 2002).
In R. galegae the tolerance of low pH was associated with the abundant
EPS production and long O-chain LPS (Räsänen and Lindström 1997). EPS
tends to absorb water (Potts 1994) and EPS is suggested to protect bacteria
from desiccation by altering their microenvironment (Roberson and Firestone 1992). The protective role of EPS against desiccation could explain
why the colony morphology of S. arboris 1552 cells remained unchanged or
became even more mucous when the bacteria were obtained from droughtaffected A. senegal soils (L. Räsänen, unpublished).
The complete sequence of the megaplasmid pSymB revealed that S. meliloti carries many more putative polysaccharide synthesis genes than was
previously known. From the 11 gene clusters (containing presumably 188
genes, 12% of the genes on pSymB), the existence of nine clusters was
unknown. The large number of surface polysaccharide genes was supposed
to reflect the very different conditions (e.g. desiccation and starvation) and
environments (e.g., soil, rhizosphere, and legume nodule) to which S. meliloti has had to adapt (Finan et al. 2001).
4.6.2. Changes in cell activity and culturability
Many studies have indicated that though bacteria rapidly lost culturability
after inoculation to soil, defined as visible colony formation on plates, the
cells were still metabolically active (Binnerup et al. 1993, Winding et al.
68
1994, Heijnen et al. 1995). This reproducible loss of culturability but maintenance of metabolic activity, studied especially in Vibrio sp., led to the
description of bacterial cells in this state as ”Viable But NonCulturable”
(VBNC; McDougald et al. 1998). Thus, natural bacterial population may
consist of i) culturable cells, which can be quantified as colony-forming units
(cfu) on solid media, ii) VBNC cells, and iii) dead cells. However, the exact
definitions are still under discussion (Kell et al. 1998).
During the routine growth tests with S. arboris strain 1552 in liquid
medium, it appeared that the strain grew oddly at 40ºC compared to at
28 and 37ºC (IV: Fig.1). Later studies revealed that in thermally stressed
S. arboris cultures the number of culturable cells (i.e. cfu counts) could
decrease considerably immediately after inoculation (IV: Fig.1B), but after
prolonged incubation, the cultures transiently regained culturability. To find
out whether this phenomenon was associated to the VBNC state, three fluorescent stains reflecting different physiological cell states were applied: i)
acridine orange – total cell numbers (IV; Fig. 4a-c, 5a, b), ii) Sytox Green
- dead or dying cells with permeable cell membrane (IV; Fig. 4f), and iii)
SFDA (fluorochromo 5- (and)-sulfofluorescein diacetate) - esterase active
cells (IV: Fig. 4d, e, 5c). Esterases were assumed to indicate basal enzyme
activity, being independent of the growth status and expressed by bacteria
during their entire lifetime. Thus, SFDA stained cells represented metabolically active cells. Moreover, strain 1552 was tagged with the luc gene
coding for the firefly (Photinus pyralis) luciferase (IV). The light output reaction (560 nm) of the firefly luciferase requires ATP, in addition to the presence of the D-luciferin substrate, O2, and Mg2+ (Bronstein et al. 1994).
Therefore, S. arboris cells tagged with the luc gene served as a sensitive
indicator of cellular energy reserves and growth status.
In heat-stressed S. arboris cultures, there were major changes in luciferase
activity and in the numbers of culturable cells but only minor changes in
the numbers of esterase active cells (IV: Fig. 2A, 3, Table 1). This suggested
that although the cells had low energy reserves and were no longer able to
be cultured on agar, they still maintained some basal enzyme activity. Thus,
these cells could be classified as VBNC cells by broad definition. Copper
in the growth medium, and starvation together with salinity have induced
VBNC state in rhizobia (Alexander et al. 1999, Lippi et al. 2000). The present
work (IV) indicated that a similar phenomenon may occur when rhizobia are
cultured under heat stress.
McDougald et al. (1998) proposed that there are subpopulations within
VBNC cultures, reflecting stages of VBNC formation. In the initial stage, cells
lose culturability but maintain the potential for resuscitation. In later stages,
VBNC cells will gradually degrade and lose the potential for resuscitation
(McDougald et al. 1998). Pseudomonas fluorescens tagged with the green
fluorescent protein (gfp) gene was observed to remain fluorescent following
69
starvation and entry into the VBNC state. Based on the GFP fluorescence and
the fluorescent dye propidium iodide (stains cells with permeable cell membranes), three cell categories were distinguished: i) dead cells, ii) damaged/
dying cells, and iii) viable cells (Lowder et al. 2000). Similarly, thermally
stressed S. arboris cell populations could be divided into three categories:
i) dead cells, stained with Sytox Green, ii) viable cells with energy reserves
(cells forming colonies on agar media and showing high luciferase activity),
and iii) VBNC cells, showing esterase activity but negligible luciferase activity and not able to form colonies on agar.
The extent to which VBNC cells containing resting forms can be resuscitated is not yet known. However, the sudden changes in luciferase activity,
both positive and negative (IV Fig. 2C, Tables 1 and 2), suggested that some
signalling occurred within S. arboris populations.
4.6.3. Moderately and highly stressed cultures
The studies with S. arboris strain 1552 tagged with the luc gene (= strain 2190),
revealed that the heat-stressed cultures did not grow in a logistic manner.
Based on cfu counts and luciferase activity, different growth phases of the cell
cycle could be distinguished (lag, exponential, stationary, and decline phases),
but the number and length of each phase varied considerably between replicate cultures (IV: Fig. 2B, C). Thermally stressed cultures could be separated
in moderately stressed and highly stressed cultures. In the former case, the
change in cfu counts and luciferase activity were small and gradual (IV: Fig.
2B, Tables 1 and 2) whereas in the latter case the two parameters changed
drastically (IV: Fig. 2C, Tables 1 and 2), and the cultures had a long lag phase
and/or a stationary phase was short or lacking (IV: Fig. 2C).
The rapid adaptation of E. coli strains to high growth temperature was
explained by equivalent useful mutations occurring at the same rate both
at 37°C and 42°C, but being lost in favourable growth conditions because
of a smaller selective advantage (Bennett et al. 1990). Multiphasic phase
variation, which ensures that a certain percentage of cells express properties needed for survival in new conditions (Henderson et al. 1999), together with other regulator mechanisms could explain why replicate S. arboris
cultures responded differently to thermal stress. In the beginning the cultures contained some cells that could tolerate heat or were capable of becoming heat tolerant. Other biological response systems could strengthen or
impair the development of heat tolerance. If the cells were heat-adapted,
they became only moderately stressed during heat treatment. On the other
hand, the cfu counts and luciferase activity of non-adapted cells were decreased considerably during the lag phase and the culture was regarded as
highly stressed. This phenomenon might explain why there was no definite
maximum growth temperature for liquid rhizobial cultures, above which
70
growth suddenly would cease (IV). Instead, the higher the growth temperature was, the fewer replicated cultures recovered, or they failed to recover.
4.6.4. Changes in cell morphology
In indeterminate nodules of many temperature legumes, differentiation of
vegetative rhizobial cells into N2-fixing bacteroids involves an increase in
size and pleomorphism (Fred et al. 1939, Vasse et al. 1990, Sprent 2001).
Some rhizobial species are known to be capable of changing cell morphology in particular conditions, such as in media containing some stress factor,
for example, at low or high pH, under osmotic stress, N deficiency and low
O2 concentration, and with the presence of bacteriophages or succinate (Fred
et al. 1939, Pankhurst and Craig 1978, Urban and Dazzo 1982).
In this work, the cell morphology changed remarkable when S. arboris
cells were exposed to heat stress. At 28 and 37 C S. arboris cells were typical
of rhizobia; small, spherical rods (IV, Fig. 4a), but after inoculation at 40 C,
they increased markedly in size and showed pleomorphism (IV: Fig 4b-e).
Surprisingly, the changes in cell morphology were related to the growth
phase. The cells sampled from slowly growing S. arboris cultures (i.e. lag
and early exponential phase) were approximately 2-4 times larger than normally, swollen and irregularly shaped (IV: Fig. 4b). In the exponential phase
the cell morphology had changed again, and the cells were large, elongated and slightly curved or wavy (IV: Fig. 4c, d, e). Especially elongated cells
demonstrated basal esterase activity (IV: Fig. 4d). Obviously, swollen cells
present in the lag phase were able to transform to elongated, culturable ones,
after the culture started to activate or grow. This study together with previous
studies suggests that the change in rhizobial cell morphology can be utilised
as an indicator of cell stress.
There is evidence that rhizobia are able to change cell morphology in the
soil. According to early study of Fred et al. (Fred et al. 1939), three forms
of rhizobia were always recovered from inoculated sterile soils: i) unbanded
rods, ii) banded or vacuolated rods, and iii) cocci. Proportions of each cell
form varied during the incubation, the rods predominating at the start and
the cocci after longer incubation. In a more recent study cells from several
rhizobial species underwent changes in the cell wall morphology after they
were transformed from broth cultures into peat. Obviously, these changes
were associated to enhanced survival of cells in peat (Feng et al. 2002).
4.7. IS IT POSSIBLE TO EXPLOIT SYMBIOTIC N2 FIXATION BY INOCULATION
OF ACACIA AND PROSOPIS SEEDLINGS?
Many studies have shown that inoculation of woody legume seedlings with
effective rhizobia has improved plant growth in greenhouse and nursery
71
conditions in the absence of fertiliser N (e.g. Odee et al. 2002b, I). However,
there are only few studies, in which effects of inoculation of tree seedlings
has been followed in the field (Lal and Khanna 1996, Requena et al. 2001).
Although these studies suggested that, inoculation is worthwhile in soils deficient in N, extensive field studies are still needed before the significance of
inoculation can be estimated. Perhaps, the major questions are: which role,
at which growth state and under which environmental conditions N2 fixation plays a role for tree seedlings. Furthermore, is the symbiotic N2 fixation
more important for young seedlings than for adult trees? Many of these questions can be solved only by using, in addition to traditional methods, modern
isotopic and/or molecular techniques and by studying both partners, host
plant and its symbiont, at the same time. However, because several factors
influece concurrently the organisms living in the field, laboratory and greenhouse experiments are also needed for more detailed studies, as well as to
make it easier to decide where the future studies should be directed in the
field.
4.7.1. Evaluation of the role of inoculation in the field
Tropical tree seedlings are likely to be planted to the field either by direct
seeding or after pre-germination in nurseries. Both techniques have both
good and weak points. The former method is economic but mortality of
seedlings can be high. The latter technique requires more work and rapidly
growing taproots of Acacia and Prosopis spp. may become disturbed if they
are not transplanted in time. However, presumably the establishment of
inoculant rhizobia on the root systems may be more pronounced in nurseries
than in the field.
In arid and semiarid regions, seeding or transplanting of seedlings occur
during the wet season. Then introduced rhizobia, applied together with seeds
or seedlings, will penetrate deeper to the soil with growing roots and rainwater. Liquid inoculant added around the root collar immediately after transplanting turned out to be suitable inoculation procedure for Calliandra
calothyrsys seedlings grown in local soils containing indigenous rhizobia
(Odee et al. 2002b).
Under favourable conditions, Acacia and Prosopis seedlings can nodulate rather fast, approximately within 10 days after sowing and inoculation of
germinated seedlings (size 5-10 mm; II). How fast nodulation of pre-germinated seeds occurs under field conditions is not known.
In general, Acacia and Prosopis seedlings grown under favourable conditions tended to have most N2-fixing nodules on roots located near the soil
surface (L. Räsänen, unpublished). Apparently, these nodules will decompose in the dry season due to drought and thermal stress (Monk et al. 1981,
II, III). It is unknown, at which extent young tree seedlings, aged between 0
72
and 5 years, have N2-fixing nodules in deeper soils, whether these nodules
are perennial or at which extent new nodules are formed during next wet
season. Furthermore, it is not known how long time the introduced rhizobia
will persist in arid and semiarid soils and at which extent it depends on
the properties of the strain. However, it can be possible that inoculant rhizobia will colonise soils for rather long time and survive trough unfavourable
growth periods by entering VBNC state.
Although there are numerous open questions to be research, the following aspects support application of inoculation on Acacia and Prosopis seedlings:
Assuring formation of N2-fixing nodules. Development both of effective
and ineffective nodules consumes plant resources but in the latter case the
host plant does not get any benefit (N). Inoculation could be especially
important when planting in to the field introduced tree species that have different rhizobial preferences compared to those of local ones, or when the
seedlings are grown in nurseries in local soils that may contain ineffective
indigenous rhizobia.
Improvement of N2 fixation under stress. Numerous studies on crop legumes have demonstrated that by application high amounts of rhizobia per
legume seed it is possible to improve nodulation and N2 fixation under
stress conditions (Lupwayi et al. 2000 and references therein). Large rhizobial population may protect plant roots due to their metabolic activity and
capacity to produce EPS (III).
Increased root growth. There are evidences that nodulated N2-fixing
legumes are capable of taking up more soil N compared to their non-nodulated counterparts, possibly because the fixed N supports increased root
growth (Unkovich and Pate 2000). Improved root growth would be important for Acacia and Prosopis seedlings, because their survival through dry
periods is often depending on that whether the roots reach deep water sources or not.
4.7.2. Inoculation as a procedure
Inoculation is beneficial for tree seedlings only, if inoculants of good quality
is available. I.e. it should be pure (no contaminants) and contain high
amount of viable, effective and tested rhizobia (107 - 109 cells g-1; Lupwayi et
al. 2000). In addition, the inoculant strain should be stable in its symbiotic
capacities, adequately competent against indigenous rhizobia and moderate
tolerant of environmental stresses. The widely accepted minimum quantitative per-seeds standards are 103, 104 and 105 per small, medium and big
seeds (Lupwayi et al. 2000). In some cases, most effective strain-tree provenance combinations could be proposed (Galiana et al. 1994), but testing of
different combinations is tedious. Presumably, the inoculant consisting of a
73
few effective rhizobial strains is more reliable in changeable soil conditions
than a single-strain inoculant (Sutherland et al. 2000).
Properties of the host tree play a big role. There are differences between
tree provenances with respect to growth, efficiency of the symbiosis and
resistance to various stresses (Aswathappa et al. 1987, Craig et al. 1991,
Kumar et al. 1999). The last-mentioned property is important because the
host tree determines growth ranges (Kumar et al. 1999).
Deficiency of P is quite common in African soils (Dakora and Keya 1997),
and low availability of P has limited symbiotic performance of Acacia seedlings in Australia (Hansen and Pate 1987). Colonisation of plant roots by
arbuscular mycorrhizal fungi (AM fungi) helps plants to take up P that is
immobile in the soil and may improve plant drought tolerance (Michelsen
and Rosendahl 1990, Ruiz-Lozano et al. 2001). Several studies have indicated that dual inoculation of Acacia seedlings with rhizobia and AM fungi
produced better results in plant growth and nutrition, and in soil fertility
and quality than inoculation with rhizobia or AM-fungi alone (Colonna et
al. 1991, Hatimi 1999, Requena et al. 2001). Dual inoculation has also increased the concentration of a compatible solute, proline in the leaf tissue of
Australian A. cyanophylla (Hatimi 1999).
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5. SUMMARY
In this thesis I first studied symbiotic properties and stress tolerance of
five putative inoculant strains belonging to the fast-growing Sinorhizobium
species, namely S. arboris, S. kostiense, S. saheli and S. terangae bv. acaciae.
The strains were previously isolated from the nodules of Acacia or Prosopis
trees growing in arid and semiarid in the Sudan and Senegal.
Host specificity. The above five strains induced N2-fixing nodules on
all African Acacia tested (A. mellifera, A. nilotica, A. oerfota (synonym A.
nubica), A. nilotica, A. senegal, A. seyal, A. sieberiana, A. tortilis subsp. raddiana) and on Latin American A. angustissima. In addition, the strains were
able to induce effective nodules on Afro-Asian P. cineraria and on several
Latin American Prosopis spp., both on native P. chilensis and P. pallida, and
on those that were introduced into Africa from Latin America (P. julifolora, P.
chilensis). The nodulation pattern of S. saheli confirmed the earlier view that
as in S. terangae, two biovars, bv. acaciae and bv. sesbanie, could be distinguished in S. saheli. The African Sinorhizobium strains induced ineffective
nodules on African P. africana and Australian A. holosericea.
Infection mode and nodule type. In general, all species had root hairs
on their roots but were particularly sparse in Acacia spp. The infection
mode resembled that of classical root hair infection. The infection threads,
along which the rhizobia penetrate the hair, were mostly formed on short
hairs in acacias and Afro-Asian P. cineraria and on longer ones in Latin American Prosopis spp. Elongation and ramification of the nodules indicated that
Acacia and Prosopis had indeterminate nodules although a persistent apical
meristem, characteristic feature of the indeterminate nodule, was not easily
detectable.
Stress tolerance of the two major effective Sinorhizobium strains. Two
major strains used in this study had competent and stable symbiotic properties. They also showed to be adapted to survive in arid and semiarid soils. S.
arboris strain 1552 was salt and osmotic tolerant strain whereas strain 1496
was sensitive to NaCl but tolerated higher growth temperatures.
Infection and nodulation under heat and drought stress. The unfavourably high root temperature and drought stress caused A. senegal hairs to be
partly abnormally deformed. Short and swollen bottle-like hairs were typical
under heat stress and very short or dwarfed, swollen hairs were characteristic
of water-stressed hairs. Under both stresses, infection threads were rare, and
they seemed to be either disintegrated or they had weak glucuronidase activity. Nodulation of A. senegal seedlings was impaired at 38°C, reduced to
50% at 40°C and completely inhibited at 42°C. The drought stress retarded
or stopped the normal nodule development so that the numbers of nodule
initials were higher on drought-stressed plants than on non-stressed ones. For
the numbers of true nodules the situation was reversed. The nodules formed
75
showed signs of premature senescence and displayed weak glucuronidase
activity, indicating low cell numbers and/or low activity of rhizobia occupying nodules.
The role of compatible solutes for plants and bacteria. Regarding endogenous glycine betaine, A. senegal was a non-accumulator plant. 60% of
the plants grown in drought-affected soils with 0.0003 M glycine betaine
were still alive 42 days after inoculation whereas the drought-stressed plants
grown without any increments were wilted 30 days after inoculation. The
treated soils tended to have more water left compared to that in untreated
soils. Possibly, glycine betaine enabled A. senegal seedlings to control water
uptake and transpiration more effectively. 0.01 or 0.05 M glycine betaine
and trehalose functioned as osmoprotectants for S. arboris strain 1552 and
S. saheli strain 1496 grown in liquid media with 9 and 17% PEG 6000
but in the case of salt stress (NaCl) only trehalose had a favourable effect.
In drought-affected A. senegal soils the number of culturable rhizobia decreased significantly but the addition of 0.0001 or 0.0003 M glycine betaine
and 0.0003 M trehalose maintained cfu counts of at the same level as in nonstressed soils (107 cfu g-1).
The heat induced peculiar changes in the numbers of culturable cells
and cell morphology when S. arboris 1552 cells were grown in batch cultures. Several complementary techniques (luciferase activity of S. arboris
strain marked with the luc gene; plate counts; optical cell density; two
fluorescent stains in order to microscopically identify metabolically active
and dead cells) were applied. It appeared that although the numbers of
cultural cells and cellular energy reserves decreased considerably during
heat stress, a majority of the cell population maintained basal enzymatic
activity. In other words, under adverse conditions, rhizobial cells did not
only die but also entered into a state, in which they were viable but nonculturable. Change in cell morphology, which was observed to be partly
related to the growth phase of the cell culture, can be utilised as an indicator of cell stress.
Heat experiments, both in batch cultures and in soil conditions, caused
changes in colony morphology in S. arboris strain 1552. Normally, this
strain produces mucous colonies but after the heat treatments, small portion of the colonies grew as small and compact ones. This dry colony morphology was a stable property for the bacterium, and it had retained its
symbiotic properties. Similar characteristic was observed, when strain 1552
was retrieved from lyophilised or frozen cultures. Change in colony morphology might be associated with the phase variation mechanisms, which
in Gram-negative bacteria involves alteration in expression of surface structures, such as EPS and LPS. The above mechanism ensures that a certain
percentage of cell population will express phenotype needed for a survival
in new conditions.
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6. TIIVISTELMÄ
Tropiikissa palkokasvit, kuten Acacia ja Prosopis -sukujen edustajat, ovat
pääasiassa puita ja pensaita. Nykyisin tunnetaan noin 1200 Acacia -lajia,
joista 850 lajia tavataan Australiassa ja 135 lajia Afrikassa. Sen sijaan Prosopis -sukuun kuuluvat lajit kasvavat lähinnä Amerikan mantereella (40/44).
Monet Acacia ja Prosopis –lajit ovat sopeutuneet kasvamaan tropiikin kuivilla ja puolikuivilla alueilla. Jotkut lajit voivat kasvattaa pääjuurta jopa
10-30 metrin syvyyteen vedensaannin turvaamiseksi. Akaasioita ja prosopiksia on perinteisesti hyödynnetty monipuolisesti mm. rehuna, polttopuuna,
puuhiilen ja lääkkeiden raaka-aineena, viherlannoitteena, rakennusaineena
ja ihmisravintona. A. senegal –puusta saadaan arabikumia, jota käytetään
mm. elintarvikkeiden ja lääkkeiden valmistuksessa. Kestävät ja monikäyttöiset akaasiat ja prosopikset sopivatkin erityisen hyvin karujen, eroosion vaivaamien alueiden metsittämiseen.
Ruohovartisten palkokasvien tavoin (esim. apila ja herne) suurin osa Acacia- ja Prosopis -lajeista kykenee symbioosissa juurinystyräbakteerien (ritsobit) kanssa sitomaan ilmakehän typpeä (N2) kasveille käyttökelpoiseen
muotoon. Monien viljeltävien, ruohovartisten palkokasvien ja niitä nystyröivien bakteerilajien väliset symbioosit tunnetaan varsin hyvin. Sen sijaan
palkokasvipuiden ja niiden ritsobeiden välisestä symbioosista ja sen merkityksestä taimille ja aikuisille puille tiedetään erittäin vähän.
Tässä työssä pyrittiin selvittämään sudanilaisten A. senegal – ja P. chilensis puiden nystyröistä eristettyjen, eri Sinorhizobium-sukuja edustavien kantojen symbioottisia ominaisuuksia, ts. mitä muita Acacia ja Prosopis -lajeja
kannat nystyröivät ja miten ritsobi pääsee puun taimen juureen. Tämän lisäksi
tarkasteltiin muodostuneiden nystyröiden rakennetta. Työssä tutkittiin myös
korkean lämpötilan ja kuivuuden vaikutuksia ritsobien ja A. senegal- taimien
kasvuun ja symbioosin kehittymiseen. Lisäksi kartoitettiin niitä tekijöitä, jotka
mahdollisesti parantaisivat ritsobien ja A. senegal -taimien osmoottisen ja
kuivuusstressin sietoa.
Sudanilaisten ritsobien isäntäspesifisyys oli laaja, eli ne muodostivat
typpeä sitovia nystyröitä sekä useisiin afrikkalaisiin Acacia-lajeihin että
Latinalaisesta Amerikasta kotoisiin oleviin Prosopis-lajeihin. Ritsobikannat
indusoivat tehottomia eli typen sidontaan kykenemättömiä, nystyröitä muistuttavia rakenteita australialaiseen A. holosericea -lajiin ja ainoaan kotoperäiseen afrikkalaiseen Prosopis-lajiin (P. africana). Ritsobit pääsevät Acacia
ja Prosopis -puun juureen juurikarvaan muodostuneen käytävän eli infektiolangan kautta. Muodostuneiden nystyröiden kärjessä on jatkuvasti kasvava
meristeemisolukko, mikä merkitsee sitä, että puiden nystyrät voisivat olla
monivuotisia.
Yleensä ritsobit sietivät korkean lämpötilan ja kuivuuden aiheuttamaa
stressiä paremmin kuin A. senegal –taimet. Työn kuluessa ilmeni, että
77
kasvatettaessa ritsobeja stressiolosuhteissa, joko ravintoliemessä tai/ja A.
senegal-taimien juuristossa niiden solu -ja pesäkemorphologia sekä kasvukäyrät poikkesivat suotuisissa oloissa kasvaneiden ritsobien vastaavista
ominaisuuksta. Viljeltäessä kuumuusstressistä kärsineitä ritsobeja maljoilla
osa pesäkkeistä oli muuttunut pintarakenteeltaan kannalle ominaisen limaisen muodon sijasta pieniksi, pinnaltaan kuiviksi pesäkkeiksi. Tämä ilmiö
liittyy Gram-negatiivisten ritsobeiden pintarakenteisiin kuuluvien pintasokereiden (eksopolysakkaridit eli EPS) tuotannon muuttumiseen. Stressatut ritsobit kykenivät menemään tilaan, jossa ne olivat eläviä, mutta eivät viljeltäviä.
Tämä muoto (VBNC, Viable-But-Not-Culturable) saattaisi olla jonkinlainen
säilymismuoto. Ilmeisesti samankin kannan ritsobipopulaatiossa muodostuu
jatkuvasti ominaisuuksiltaan erilaisia osapopulaatiota, mikä varmistaa sen,
että ympäristön muuttuessa äkillisesti ainakin osa bakteereista jää henkiin.
Glysiinibetaiini ja trehaloosi ovat osmolyyttisiä yhdisteitä, joita monet bakteerit voivat syntetisoida tai ottaa ympäristöstä suojautuakseen suolan ja
kuivuuden aiheuttamalta osmoottiselta stressiltä. Kummankin aineen lisäys
kasvualustaan tai A. senegal –taimien juuristoon lisäsi ritsobien kasvua
osmoottisen ja kuivuusstressin aikana.
Sekä A. senegal -taimien kasvualustan korkea lämpötila (38-40°C) että
kuivuus vähensivät nystyröiden määrää juurissa. Korkeammassa lämpötilassa (42°C) tai kuivuuden jatkuessa nystyröiden kehittyminen pysähtyi
kokonaan. Optimioloissa nuoret Acacia ja Prosopis -taimet pyrkivät kasvattamaan suurimmat typpeä sitovat nystyrät lähelle maan pintaa 1-5 cm
syvyyteen. Sen sijaan luonnossa maan korkea lämpötila estänee juurinystyröiden muodostumista 10 cm syvyyteen saakka, ja kuivana kautena
huomattavasti syvemmältäkin. Jotkut suolaa sietävät kasvit suojautuvat sen
aiheuttamilta haitoilta keräämällä soluihin glysiinibetaiinia. Glysiinibetaiinin lisäys A. senegal -tainten juuristoon alensi veden puutteesta kärsivien
kasvien kuolleisuutta selvästi. Oletettavasti glysiinibetaiini tehosti taimien
vedenkäyttöä. Trehaloosin lisäyksellä oli samantapainen, mutta lievempi
vaikutus. Trehaloosin tiedetään suojaavan biologisia rakenteita kuivuudelta.
Lisääntynyt ritsobipopulaatio sinänsä voi myös stimuloida juurten kasvua
ja suojata juuristoa.
Palkokasvipuiden ymppäys eli ritsobien lisäys kasvualustaan taimitarhoilla tai maahan siementen kylvön yhteydessä näyttää varmistavan sen, että
puulajille sopivaa ritsobilajia on saatavilla heti istutuksen jälkeen. Jos taimet/
siemenet istutetaan sadeajan alussa, jolloin maassa on riittävästi kosteutta,
ensimmäiset typpeä sitovat nystyrät kehittyvät parin viikon kuluessa. Typensidonta saattaa olla ratkaisevaa juuren kasvun kannalta. Selvitäkseen kuivasta kaudesta taimen on ehdittävä kasvattaa pääjuurta mahdollisimman
syvälle maahan ennen kauden alkamista. Kenttätutkimuksia kuitenkin tarvitaan, jotta ymmärrettäisiin paremmin missä vaiheessa ja minkälaisissa olosuhteissa Acacia- ja Prosopis -taimien ymppäyksestä olisi hyötyä.
78
7. ACKNOWLEDGEMENTS
T
his work was carried out during the
years 1994-2002 at Department of
Applied Chemistry and Microbiology, University of Helsinki. Work was
supported by funds and grants of The
Academy of Finland, EU contract No.
TS3-CT93-0232, The Kemira Oyj Foundation, Nordic Network, University of Helsinki and The Finnish Cultural Foundation,
which is gratefully acknowledged.
I’d like to express my warmest thanks
to my supervisor, docent Kristina Lindström, for the possibility to work in
her nice research group, N2-group, in
an inspiring, international and relaxed
atmosphere. I am especially grateful for
her giving me the opportunity to work
with the fascinating topic, to see the world
and for correcting my English writing.
I would like to thank Elena LapinaBalk for her nice company and help
during the experimental works. Sometimes the work was pleasant like working
on beautiful summer days under apple
trees, and sometimes we were as stressed as our research objects. I am grateful
to Salla Saijets, who promptly entered
into the difficult world of osmotic and
drought stresses, and Eva Tas for her patience when teaching me the use of different
computer programs.
I want to thank all present and former
members of the N2 group for their help
in the lab , and for the good atmosphere
and relaxing company during coffee and
lunch hours: Leena S, Gera, Zewdu,
Minna, Aneta, Leone, Jyrki, Kati, Petri,
Jenny, Inka, Endalka, Kaisa W, Seppo,
Aimo, Gise, Vesa, Kaisa H, Sirpa, Anne,
Gilles, Lena, Hannamari and all the
others not mentioned here.
I am grateful to Professor Mirja Salkinoja-Salonen for her kind support, and
to all the staff of the Department of Applied Chemistry and Microbiology for good
working facilities and for the friendly
attitude that I have always experienced.
I wish to thank co-authors, Prof. Janet
Jansson for giving me the opportunity to
visit Stockholm University and Annelie
Elväng for introducing the world of lucmarked bacteria. The other co-authors,
Prof. Janet Sprent (University of Dundee,
Scotland) and Dr. Kari Jokinen (Kemira
Oyj, Helsinki) are also deeply acknowledged for their expertise.
Prof. Ken Giller (Wageningen University, the Netherlands) and Dr. David Odee
(Kenya Research Forestry Institute) are
acknowledged for reviewing this manuscript and for their valuable comments that
significantly improved the text.
I like to thank my friends, Anne,
Hanna, Kirsi, Kirsti and Jukka for several
nice moments with good food and drinks,
and my godmother Aune for her kind
advises how to keep well.
Finally, my family deserves all my
thanks for their love and support. My
father Eino and my mother Anni, who
is not with us any more, have offered a
place where I could forget all my scientific
thoughts and worries. My brothers Pekka
and Tuomo have always been helpful in
transporting me or my things. Without
the practical help of my brother-in-law
and graphic designer Kari and my sister
Eeva-Liisa finishing this book would have
been a much more stressing experience.
As journalists they had the courage to ask
all the silly but important questions.
Helsinki, November 2002
79
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