Download Azospirillum VI and related microorganisms, genetics-physiology-ecology

Azospírillum VI
and Related Microorganisms
Genetics - Physiology - Ecology
Edited by
István Fendrik Maddalena del Gallo Jos Vanderleyden
Miklos de Zamaroczy
NATO ASI Series
Series G: Ecological Sciences, Vol. 37
Yoav Bashan", M. Esther Puente", M. Nieves Rodri
Toledo*, Ronald Ferrera-Cerrato*. and Sergio Pedrin
"Department of Microbiology, and ^Department of
The Center for Biological Research (CIB), A.P. 128
Abstract
The survival of Azospirillum brasilense strains
rhizosphere of wheat and tómalo plañís and in 23
from a wide range of environmental conditions in
brasilense was analyzed for 15 soil parameters
rhizospheres tested, regardless of soil type, bact
amount of rainfall each soil received prior to sam
survival characteristics of A. brasilense differed an
origin of the soil and not to prevailíng environm
brasilense is a rhizosphere colonizer which surv
periods. (ii) Some major physical and chemical s
bacteria in plantless soils.
Introduction
There is no doubt that Azospirillum species surviv
the rhizosphere of numerous plant species (Basha
in the soil per se is controversial. On one hand, se
Dobereiner and Baldani, 1979; Dobereiner et al. 1
but not exclusively (Germida, 1986) from tropical
found in nearly every sampling of soil, indicating
hand, studies mainly from températe and semi-a
(Nayak et al. 1986) found that Azospirillum surv
1983; Bashan, 1986 a; Bashan et al. 1987; De
Vandenhove et al. 1993) and hardly lasted from o
'*Institute of Natural Resources, Colegio de Po
56230, México.
Organisms
For all experiments we used Azospiríllum brasilense strains Cd (ATCC 29710) and Sp-24
(Baldani et al. 1986) with the plants wheat (Tríticum aestivum) cvs. "Deganit" (Israel) an
"Morelos" (Central México) and tomato (Lycopersicon esculentum) cv. "UC-82-L" (Baj
California Sur, México).
Bacterial inoculation
Bacteria were grown in one of the following growth media: Nutrient Broth (ín Baj
California, México and Israel) or N-free médium (NFb) (in mainland México) and prepare
for inoculation at various concentraüons as previously described (Bashan, 1986 b, Bashan e
al. 1993). The final bacterial concentrations were: IxlO 6 cfu/ml soil either in the presence o
absence of plants in Israel, 1.77xl07 cfu/ml soil for both soil or plant inoculation in Baj
California, México, and 4.2xl04 (to inocúlate plants) or 1.44xl08 cfu/ml (to inocúlate soil) i
Central México. Soil was directly inoculated by applying double washed bacterial suspensió
to each pot. Plants were inoculated at sowing as previously described (Puente and Bashan
1993).
hermetically sealed plástic containers at 4±1°
:s used in the studies. No two
commercial core samplers from the soil layer 20-3
:re not compared with those of
attempt was made to preserve the soil intact and
ntroversy in the soü survival of
disturbed. Several soils were from cultivated áre
¡süons of Azospiríllum research
land. Soils were collected from arid (< 200 mm r
p, 1991, Germany).
mm rainfall /year) (5 soils), mountains (500-800
>rrelating the soil parameters of
(1800+ mm rainfall /year) (one soil).
itain soils from Israel, semi-arid
The physical and chemical characteristics of eac
ya California, México) with the
standard methods: texture and organic matter (Ro
1 and Sp-245.
electric conducüvity (Chapman and Pratt, 1984
CaCo, content (Jackson, 1976).
Soil sterilization
To avoid competition with native microorganism
analysis, all soils were sterilized by a standard ty
autoclave. Later, the soil was incubated for 24
Cd (ATCC 29710) and Sp-245
Preliminary comparison between sterile and non-
ím) cvs. "Deganit" (Israel) and
in the level of survival for both strains Cd and Sp-
ilentum) cv. "UC-82-L" (Baja
Plant growth conditions and inoculation
Plants were grown in 500 mi plástic pots contain
with 10% NaOCl and thoroughly washed with s
:día: Nutrient Broth (in Baja
soils without plants was carried out in identical 2
nainland México) and prepared
chamber at 25±1°C, 100 mole/m2/sec and 60±2
>ed (Bashan, 1986 b, Bashan et
every week with 5-15 mi sterile, distilled wat
ni soil either in the presence or
different size of the growing plants. Plants w
il or plant inoculation in Baja
half-strength Hoagland's solution.
10* cfu/ml (to inocúlate soil) in
Sampling and bacterial countsfrom solí and root
ile washed bacterial suspensión
Samples (2 g soil or approx 500-1000 mg (fresh w
described (Puente and Bashan,
were taken at each sampling. Rhizosphere bac
colonize the roots and the adhering soil parücles
lightly sonicated al 25 W for 5 min (Colé Parm
procedure.
Resulte and discussion
The reléase of Azospiriüum and other PGPR into soils has a long history of unpredictable and
often disappointing results. One of the main obstacles has been the often poor establishmen
and survival of the introduced bacteria in the soil prior to root colonization (Bashan and
Levanony, 1990, Michiels et al. 1989, Jagnow, 1987). Survival of PGPR in the soil is
crucial issue for both ínoculant users and manufacturen, stemming from the commo
agrotechnical belief that seed inoculaüon is impracücal in many cases (perennial plants
vegetativo propagated plants, trees, or where more than one inoculaüon per season i
required, etc). When the PGPR is applied to the soil, it is hoped that it will survive lon
enough to fmd its target plant. Thus, before one considers an expensive field inoculaüon, th
prospect of bacterial survival in the soil must be considered.
The aim of this study was to address the soil phase survival of Azospirillum by creatin
sufficient data which will allow future modeling and prediction of bacterial behavior in an
gíven soil without laborious studies of bacterial survival in every field. To this end, w
collected 23 soil types representing different climatological conditions, from tropical to ari
ar (soils from Baja California,
available stains of A. brasilense.
>ils from Central México), by
One fact emerged immediately and was clear even
1) or by the time-limited liquid
A. brasilense proved to be a rhizosphere bacteria. I
bacteria fell below the level of
characteristics as long as plants were growing in
survival picture differed significantly. The g
geographical than climatological, i.e., in the arid
iL The rate of growth or death
while it proliferated in the arid soils of Baja Califo
;' Logistic Equation of Growth
in Fig. 1 in which the survival of A. brasile
representative soil types is demonstrated.
e soils were correlated with the
With the aid of several statistical analyses perfor
. type of soils using Linear and
the survival data from all the
Principie Componen! Analysis
soil parameters data, we were able to sort out the
A. brasilense in these soils. Two factors, the levéi
and significantly correlated with bacterial surviva
d c
s- ttí
ng history of unpredictable and
-O
"E
6
:n the often poor establishment
root colonization (Bashan and
•
O
•
n
»
ival of PGPR in the soil is a
stemming from the common
o Mexican soil
many cases (perennial plants,
me inoculation per season is
Israeli rhizosphere
Israeli soil
Baja California rh
Baja C a l i f o r n i a soi
Mexican rhizosphe
tjfl
O
loped that it will survive long
O
;xpensive fíeld inoculation, the
Days a f t e r in
il of Azospirillum by creating
Fig. 1. Survival of Azospirillum brasilense in the
from Israel, Baja California Sur, México a
Each point represents the mean of 3-5
conducted in triplicates. For simplicity, t
are: Israeli soil; rhizosphere (R) ±1.4851
±1.39024, S ±1.5149. Central México soil;
:m of bacterial behavior in any
every fíeld. To this end, we
Dnditions, from tropical to arid
10
20
T—•
1
—-1
1
T
y=-0.03644x-0.08098
r=0.86*
o -
y=-0.02354x-0.46746
r=0.82*
-0.5
-1.0
O
10
20
30
CaCO,, (%)
40
50
O
10
20
30
40
Rou»h Sand (%)
Fig. 2. Linear regression analyses between percentage of CaCO3 (A) and rough sand (B) and
the survival rate of A. brasilense in 23 and 13 soil types, respectively. Missing points
in each sub-figure are overlapped by other printed points. * - Significance of the
regression at P<0.05.
iüvely affected survival. On the
parameters may affect the survival of this bacteria in
level of fine sand in the soil had
:ts of single soil parameters on
L are known (Foster, 1988; van
Acknowledgements
y of this study is that only the
and positively determined the
This study was written for the memory of the l
ameters are unimportant when
encouraged agricultural research. We thank Ms. Lan
unknown are the proportional
for excellent technical assistance, Mr. Gustavo Pa
her manipulation of the soil
discussions on soil analyses and soil classificat
clarifying the English and Dr. J. Dübereiner, EMB
This study was partially supported by Conse
(CONACyT), México grant # 225130-5-3541 A.
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