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Comprehensive Summaries of Uppsala Dissertations
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Biological and Molecular
Characteristics of
Microorganism-Stimulated
Defence Response in
Lycopersicon esculentum –L.
BY
IDRESS H. ATTITALLA
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2004
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“To my marvellous mother for your support;
my sweet wife; wonderful daughters Sundos,
Ebthal, Estabrak; and my great brothers
Omar; Mustafa for every things”
http://islamicity.com/mosque/arabicscript/Ayat/1/1_1.htm
Translation:
1:1 In the name of Allah, Most Gracious, Most Merciful.
1:2 Praise be to Allah, the Cherisher and Sustainer of the worlds;
1:3 Most Gracious, Most Merciful;
1:4 Master of the Day of Judgment.
1:5 Thee do we worship, and Thine aid we seek.
1:6 Show us the straight way,
1:7 The way of those on whom Thou hast bestowed Thy Grace,
those whose (portion) is not wrath, and who go not astray.
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Paper III: Copyright 2001. Firenze University Press.
Paper IV: Copyright 2001. Blackwell Wissenschafts-Verlag GmbH.
Contents
Introduction.................................................................................................................1
Microbe-related plant defence ....................................................................................4
Localized plant defence.........................................................................................4
Plant immunization ...............................................................................................6
Immunization as a mode of action...................................................................6
Systemic induced resistance (SIR) .......................................................................8
Single gene and multiple gene resistance ........................................................9
Stimulating SIR expression (signals) ............................................................11
Cross-talk between microbes and plants .............................................................11
Molecular plant response...............................................................................12
Cross-talk complexity....................................................................................16
Phytophthora cryptogea in SIR ................................................................................18
Fusarium oxysporum complex .................................................................................19
Life history..........................................................................................................20
Vegetative compatability groups (VCGs) ...........................................................21
Species status ......................................................................................................22
Biology of Fusarium oxysporum f. sp. lycopersici (Fol).....................................23
Modes of existence..............................................................................................23
Restriction fragment length polymorphism (RFLP) .................................................23
The phylogenetics of Fo-(IMI 386351)...............................................................24
Distinguishing between Fol and Forl ..................................................................24
Possible origins of Fol and Forl ..........................................................................29
Fusarium oxysporum and Lycopersicon esculentum –L. (an extensive relation)......31
Details of the relation ..........................................................................................31
Evidence for SIR in plants ..................................................................................34
Ways of demonstrating microbe-elicited SIR................................................36
Experiment 1 (III) .........................................................................................38
Experiment 2 (IV) .........................................................................................40
Experiment 3 (II) ..........................................................................................44
Summary........................................................................................................47
Searching for biocontrol agnets (Bs) ........................................................................48
Obtaining microbial isolates ..........................................................................48
Ultimate possibilities for the MF30 strain .....................................................48
Concluding remarks..................................................................................................51
Appendix...................................................................................................................53
Summary in Swedish ................................................................................................54
Acknowledgements...................................................................................................56
References.................................................................................................................61
Abbreviations
Fusarium oxysporum
F. oxysporum f. sp. lycoperici
F. oxysporum f. sp. radicis-lycopersici
F. oxysporum f. sp. melonis
Phytophthora cryptogea
Restriction fragment length
polymorphism
Elongation factor
Mitochondrial small subunit
Systemic induced resistance
Biological control agents
Disease incidence
formae speciales
Vegetative compatibility groups
Salicylic acid
Programmed cell death
Hypersensitive response
Peroxidases
Fo
Fol
Forl
Fom
Pc
RFLP
EF
mtSSU
SIR
Bs
DI
f. sp., singular, and f. spp., plural
VCGs
SA
PCD
HR
POXs
Introduction
Plants have developed effective mechanisms for avoiding or mitigating the
detrimental effects of infectious agents in their environments. These
mechanisms exist because they help plant genotypes survive the selection
pressures of evolution (Kuü 1987). To defend themselves from aggressive
parasite attack, plants have several means, which have evolved in the course
of interaction with microbes over geological time. Like animals, plants are
able to perceive and respond to changes in their environments (Schaller and
Ryan 1996). To understand these appropriate and adaptive responses (which
are often biochemical), knowledge about how plants perceive external
stimuli has increased substantially within recent years (Jones 1994).
In the course of evolution, many plants have developed distinct strategies
to resist and protect themselves against the attack of viruses, bacteria, fungi,
insects, and other biotic stress factors. Heath (1997) has summarized details
about the probable course that evolution has taken in plant resistance, which
has led to a variety of chemical defence mechanisms that plants use to
protect themselves against a broad spectrum of pathogens. The deterrent
effects of the chemicals produced, ranging from small organic molecules to
large proteins and enzymes, affect in various ways the attacking pathogens
that consume them.
As early as the beginning of the past century, Beauverie (1901) and Ray
(1901) realised that, upon infection by a pathogen, plants may develop an
enhanced resistance to further pathogen attack. Later Chester (1933)
provided a descriptive study in which he summarised various phenomena of
pathogen-induced disease resistance of plants. One of these phenomena is
induced systemic resistance (ISR; Kuü 1995a, b; Heil and Bostock 2002) or,
synonymously, systemic acquired resistance (SAR; reviewed by Ryals et al.
1996, Sticher et al. 1997).
Plant immunity (against various pathogens) induced by agents (biotic or
abiotic) has been reported since the 1930s, when Chester (1933) proposed
the term “acquired physiological immunity”. Since then several terms have
been used to describe the phenomenon of induced resistance, including
“systemic acquired resistance” (Ross 1961a, b), “translocated resistance”
(Hurbert and Helton 1967), and “plant immunization” (Kuü 1982a, b; Tuzun
and Kuü 1991; Tuzun 2001). Induced resistance is thus defined as an
enhancement acquired after appropriate stimulation of a plant’s defensive
1
capacity against a broad spectrum of pathogens and pests (Knoester et al.
1999; Ramamoorthy et al. 2001).
For a plant, surviving a pathogen attack depends both on performed
barriers, such as constructed mechanical barriers, and on induced active
defence mechanisms (Nandakumar et al. 2001). In the latter case, biotic and
abiotic elicitors induce a plant’s defence mechanisms against various
pathogens (Baker et al. 1997). Biotic inducers include pathogenic
microoganisms (Dalisay and Kuü 1995), non-pathogenic microorganisms
(Lemanceau et al. 1993; Duijff et al. 1998; Larkin and Fravel 1999; Olivain
and Alabouvette 1999; Benhamou and Garand 2001), plant-growthpromotion rhizobacteria “PGPR” (Leeman et al. 1995; Liu et al. 1995a, b),
plant products (Singh et al. 1990), and tissue damage caused by organisms.
Abiotic factors include man-made chemicals (Lawton et al. 1994) and
factors such as UV radiation or mechanical damage to plants (van Loon
1997; Porat et al. 1999). These elicitors can induce a plant’s defence
mechanisms by activating genes that invoke induced systemic resistance
(SIR). The invoked resistance can be a specific defence response or a
complex unspecific response. The origin of a biotic elicitor derived from a
pathogen may be exogenous (e.g., fungal cell wall elements such as ȕ-1,3glucans or chitin) or endogenous if released from the plant cell wall during
infection (Ebel and Scheel 1997; Hutcheson 1998).
Plant defence mechanisms can be divided according to their function into
structural and biochemical mechanisms. Structural mechanisms restrict the
developing population of infecting pathogen cells by the construction of
mechanical barriers; biochemical mechanisms pathogen inhibitory activity
that involves substances that participate in biochemical actions and reactions
between plant and pathogen (Kombrink and Somssich 1995; Lebeda et al.
2001).
This research project has focused overall on clarifying the phenomenon of
SIR, and in particular, that of tomato plants. The aim has been to provide
facts and understanding that will contribute to our ability to take greater
advantage of biological means (as opposed to chemical means) for reducing
the ability of the fungus Fusarium oxysporum f. sp. lycopersici to cause
tomato wilt, a disease that can devastate tomato plants and commercial
production of tomato. Biological control of tomato wilt can be achieved by
applying certain microorganisms to the soil around a plant’s root or to parts
of the plant (seed, root or green parts).
The research work is comprised of studies of the response of tomato
plants to several microorganisms that have potential as biocontrol agents,
and studies of those microorganisms. One in particular is a non-pathogenic
Fusarium oxysporum strain IMI 386351 (Fo-(IMI 386351)). Study of the
interaction of Fo-(IMI 386351) with tomato plants led to examination of
some of the physiological responses of the host plant to Fo-(IMI 386351),
and to examination of the biology of Fo-(IMI 386351), as an endophyte of
2
tomato. The examination of the biology of Fo-(IMI 386351) included a study
of its molecular phylogenetics, with a particular interest in establishing the
taxonomic relation of Fo-(IMI 386351) to the species Fo. Morphological
properties were also examined confirm or disaffirm its taxonomic position
within that species. As part of the phylogenetic study, the research project
was also concerned with developing an efficient molecular technique for
distinguishing two morphologically indistinguishable formae speciales (to be
abbreviated f. sp., singular, and f. spp., plural) within the Fo complex,
namely, F. oxysporum f. sp. lycopersici (Sacc.) Snyder and Hansen (Fol),
which causes wilt, and F. oxysporum f. sp. radicis-lycopersici Jarvis and
Shoemaker (Forl), which causes crown rot and root rot.
3
Microbe-related plant defence
Plants constitute the largest and most important group of autotrophic lifeforms on earth. Their abundant organic material serves as a nutritional
source for all hetertrophic organisms, including animals, insects, and
microbes. Despite the large number of fungi and bacteria in the
decomposition of dead plant material, only very few of these potential
pathogens have acquired, through evolution, the ability to colonize living
plants: that is, few have evolved from saprophytic organisms to necrotrophic
or biotrophic pathogens. Most plants exhibit natural defence to microbial
attack (i.e., non-host resistance, basic incompatibly), and therefore, disease is
the exception rather than the rule.
Localized plant defence
When a pathogen attacks a plant, the biochemical responses observed are
usually cell-wall modifications, such as the synthesis and deposition of
phenolic compounds and proteins in the cell wall, and rapid cell collapse and
death, known as the hypersensitive response or reaction “HR” (Slusarenko et
al. 1991). When the host plant cells of hypersensitive tissue come in contact
with the pathogen, the plant inhibits pathogen development by forming
substances such as hydrolytic enzymes, and inhibitors. In local plant
defence, only plant cells within and around the neighbourhood of cells
attacked or damaged by a pathogen need respond. By localizing its
interaction with the pathogen, the plant can continue to grow and flourish.
Local plant defence includes the classical HR, which can be defined as
rapid local cell death occurring in response to pathogen attack. Such
programmed cell death (PCD), which serves to remove redundant,
misplaced, or damaged cells, is essential to the development and
maintenance of multi-cellular organisms. As Hoeberichts and Woltering
(2003) point out, plant PCD involves plant-specific mechanisms, as well as
mechanisms (conserved for both plants and animals) that have
morphological and biochemical similarities to the relatively well described
PCD for animals, called apoptosis (e.g., DNA laddering, caspase-like
proteolytic activity, and cytochrome c release from mitochondria). In
addition, several plant hormones may exert their respective effects on plant
PCD through the regulation of reactive oxygen species (ROS) accumulation,
4
which is an important signal, along with nitric oxide, in the activation of
plant PCD (de Pinto et al. 2002). Likewise, the work by Irving and Kuü
(1990) demonstrates both local and systemic defence in cucumber plants
(Cucumis sativas L.). In this model plant, SIR has been extensively studied
(Kuü 1982c).
The role of accumulation of anti-microbial phytoalexins on hypersensitive
tissue is not yet clear (see reviews by Kuü 1984, 1995b; Lamb and Dixon
1997). Of the induced defences, phytoalexin production has received much
attention since the phytoalexin concept was introduced over 60 years ago.
However, the specific role of phytoalexins in disease resistance is unclear for
the majority of host-parasite systems (Hammerschmidt 1999). Much of the
research on phytoalexins has relied on the identification of induced antifungal compounds, and on correlating their presence with resistance.
Although this is an important first step, more studies are needed, especially
definitive ones. Approaches that use in situ localization and quantification
have provided good evidence that phytoalexins can accumulate at the right
time, concentration, and location to be effective in resistance, as
Hammerschmidt points out. In addition, studies on phytoalexin tolerance in
pathogenic fungi have also shown a relationship between virulence and the
ability of fungi to detoxify phytoalexins.
Brishammar’s (1987) proposes, however, that at least in potatoes,
phytoalexins might not help plants resist attack by pathogens, for several
reasons. His results showed that although those compounds did accumulate
in both healthy and infected tubers, no significant difference between them
could be detected, nor could the synthesis of pathogenesis related proteins
(PR proteins) comprising a wide range of different defence proteins,
including ȕ-1,3-glucanase. Phytoalalexins, which appeared for compatible as
well as incompatible interactions, only accumulate in the tubers, in amounts
that depended on the tuber’s physiological condition. Young tubers, even
though they synthesized extremely small amounts of phytoalexins, were not
easily infected with Phytophthora infestans. Because markedly different
types of pathogens induce synthesis of the same substances in host cells,
perhaps all the phytoalexins are synthesized in response to stimulation by an
endogenous elicitor. When potato tubers were inoculated with the late blight
fungus, bacteria that appeared secondarily were not retarded, despite the
presence of phytoalexins, which were restricted to the attacked parts of the
tubers.
Little knowledge is available regarding the biosynthesis of these
sesquiterpenes, and many previous determinations have presumably been
erroneous. There is no generally accepted hypothesis describing the
mechanism by which phytoalexins inhibit pathogens, and the distinction
between the effects on necrotrophs and biotrophs has yet to be made. Thus
far, no convincing inhibitory effects have been reported, partly because
adequate bioassays capable of measuring the effects of inhibition have yet to
5
be developed. During purification of the phytoalexins, the risk of artifacts
forming is high, implying that specific compounds cannot be detected with
certainty. Moreover, present analytical methods must be improved before we
can determine how phytoalexins act in vivo. Phytoalexins are probably
synthesized at a stage in the infection too late to be able to restrict its
expansion within the tissues of the host. There is no evidence indicating that
these compounds pose health risks when present in potatoes used for
consumption.
Plant immunization
Being able to immunize plants of concern has long been a dream, whether
house plants, plants in one’s garden or yard, or in the field. After all, we
protect ourselves against many diseases this way, and observations over the
last 100 years suggest that plants also have that possiblity. For example,
prior infection of plants with a pathogen may result in increased resistance of
that plant to the same or other pathogens. Such protection does seem to be
analogous to the immunity we acquire after successfully fighting off certain
diseases.
Starting about 25 years ago, plant pathologists began to examine seriously
whether or not plants can be "immunized" by prior infections. Studies
demonstrated that by causing a local infection in a plant (such as cucumber
and green bean) with one pathogen would result in the entire plant becoming
resistant to disease. This "immunization" phenomenon—an induced
physiological change in the plant that allows it to resist infection more
effectively—is known as "induced" or "acquired resistance".
Immunization as a mode of action
Scientists have sought chemicals (both natural and synthetic) that can
activate disease resistance in plants, as substitutes for pathogen infection.
These compounds, which are not directly toxic to pathogens, cause
expression of the genes that plants naturally use for defence against
infection. For example, benzo (1,2,3) thiadiazole-7-carbothioic acid Smethyl ester (BTH) induced acquired resistance in wheat against powdery
mildew (Gorlach et al. 1996). BTH protected wheat from infection
systemically by affecting multiple steps in the life cycle of the pathogen.
Later, resistance against scab (caused by Cladosporium cucumerinum)
accompanied by the accumulation of chitinase was rapidly induced in
cucumber plants after treatment with acibenzolar-S-methyl (a BTH), and in
soybean against white mold (Narusaka et al. 1999).
Researchers studying traditional biological control have taken advantage
of recent research on induced resistance; and consequently, the mode of
6
action of certain biocontrol agents has been re-evaluated. Several root
colonizing bacteria (rhizobacteria), particularly Pseudomonas, strains,
thought to control disease through competition with pathogens, have been
found to reduce infection, at least in part, by inducing disease resistance
(Achuo and Hofte 2001). Other beneficial microbes, such as soil borne
mycorrhizae, are also being tested for their ability to induce resistance (Pozo
et al. 2002).
The majority of inducible defence responses are the result of
transcriptional activation of specific genes, the regulation of which in plants
has been studied extensively in both intact plant-pathogen interactions and in
model systems consisting of cultured plant cells and elicitors (inducers).
Kombrink and Somssich (1995) suggest three classes of defence responses:
x Immediate, early plant-defence responses—these constitute the
first line of defence towards pathogen ingress, and are considered
to be involved in recognition and initial signalling events
x Locally activated defence mechanisms—these are thought to have
direct adverse effects on the pathogens
x Induced systemic responses (SIR)—papers II and III focus on
these responses.
Kombrink and Somssich’s base their classification on the temporal and
spatial patterns of expression of defence responses, as observed in several
systems (Fig. 1). However, because certain responses have a dual function,
or can exhibit variable expression patterns in various systems or under
various conditions, the classification is somewhat arbitrary. In fact, because
so few model systems have been studied in sufficient detail with respect to
the expression of the many defence responses, no conclusion on causal links
or successive signalling mechanisms can be inferred from Kombrink and
Somssich’s classification.
7
Fig. 1: Schematic representation and summary of mechanisms involved in plantpathogen interactions. Abbreviations: En, endogenous elicitor, E×, exogenous
elicitor. From Kombrink and Somssich (1995).
“Reprinted from publication title: Advance in Botanical Research Incorporating Advances in Plant
Pathology, 21, Chapter 1., In: J.H. Andrews and I.C. Tommerup, Disease Responses of Plant Pathogens.
Edited by Erich Kombrink and Imre E. Somssich, p. 4.Copyright (1995), with permission from Elsevier”.
Plant defence responses involve the activation of multiple, coordinated, and
apparently complimentary defence responses, such as the formation of
physical barriers through increased cross-linking, elicitation of HR, or the
elevated expression of pathogenesis related proteins, production of
phytoalexins, or other anti-microbial compounds (Tuzun 2001). Enhanced
activity of defence enzymes might contribute, such as phenylalanine
ammonia-lyase (PAL), peroxidase (POX), chitinase, ȕ-1,3-glucanase and
accumulation of phenolics (van Peer et al. 1991; M’Piga et al. 1997).
Systemic induced resistance (SIR)
Although there is no consensus on a definition for SIR (synonymously ISR
or SAR, see Conrath et al. 2001), Kloepper et al. (1992) have referred to it as
the state of enhanced resistance to pathogen infection that is activated by
biotic or abiotic agents. By this phenomenon, a plant, upon appropriate
stimulation, acquires an enhanced level of resistance against a broad
8
spectrum of pathogens Knoester et al. (1999). The signal originates at the
site of infection and moves throughout the plant Shulaev et al. (1995).
Single gene and multiple gene resistance
A pathogen must possess particular properties to invade successfully a host
plant, to survive in the host, and to secure sufficient nutrients to grow and
reproduce. Such properties can exist as a result of coevolution with plants
(Kombrink and Somssich 1995). The microorganism acquires pathogenicity
factors enabling individuals to overcome host defences and to establish a
state of basic compatibility with the plant sufficient to survive in the host and
to secure sufficient nutrients to grow and reproduce. During subsequent
coevolution, genetic variants of the susceptible host emerge that are able to
recognize the pathogen and that resist infection by activating host defence
systems. During further evolution, pathogen variants able to avoid or counter
the recognition process eventually evolve. Such a coevolutionary dynamic is
equivalent to a type of ‘arms race’ between the plant and the pathogen (Keen
1990; Briggs and Johal 1994; Beckman and Roberts 1995).
According to this gene-for-gene theory, host-pathogen specificity must
include genetic factors conferring not only basic compatibility, but also
physiological interactions involving single complementary genes in the host
and pathogen. Such specificity and gene-for-gene complementarity can quite
clearly be an artifact of plant breeding, where a single resistance gene is
introduced into crop cultivars, and then a matching virulence gene is
eventually selected in the pathogen population (Kombrink and Somssich
1995). However, recent analysis of host-pathogen relationships in wild
plants, such as Arabidopsis, has shown that gene-for-gene interactions can,
and in some cases do, regulate the host range of bacterial and fungal
pathogens in natural systems. Hence, single gene resistance as described by
the gene-for-gene model seems to be a fundamental potential evolutionary
route that plant-pathogen relationships can take, either by means of plant
breeding programs or, in nature, by means of coevolution.
Single gene resistance.—Gene regulation is likely to be the determinant
of disease resistance in plants. A gene-for-gene relation could evidently exist
(Kroon and Elgersma 1991) for tomato wilt caused by Fol. Evidence for
race-specific avirulence factors in Fo (Huertas-Conzález et al. 1999) suggest
the presence of gene-for-gene interaction between Fol-tomato and Fommuskmelon. In that interaction, cross protection (another form of SIR against
a virulent race) is mediated by recognition of a specific elicitor from the
avirulent race by the plant resistance gene product and by subsequent
induction of the plant defence reaction.
In a gene-for-gene system, pathogen-specific recognition occurs when a
resistance (R) gene in the host plant is matched by a corresponding
avirulence (avr) gene in the pathogen (Kombrink and Somssich 1995). The
9
simplest explanation for the fact that plant resistance genes and pathogen
avirulence genes usually segregate as single dominant characters is that the
product of the avr gene interacts directly with the product of the R gene.
Based on this interpretation, models predict that the R gene product is likely
to be a receptor located on the host cell membrane, and the avr gene is likely
to encode a secreted, or surface-located, signal molecule that binds to the
receptor (de Wit 1992; Pryor and Ellis 1993). This molecular binding then
triggers a complex cascade of molecular and physiological responses
involved in active plant defence. Buell (1999), who developed this “Signal
gene” disease-immunity hypothesis, explains that if the R gene of a plant
does not match at least one of the avr genes possessed by an invading
pathogen, the plant’s R gene will be unable to detect and stop the pathogen
(Tuzun 2001). The expression of genes for the gene products contributing to
disease resistance is regulated by compounds of plant and microbial origin
(West et al. 1985; Sharp et al. 1984a, b; Madamanchi and Kuü 1991).
Multiple gene resistance.—Multigenic (i.e., horizontal, quantitative, or
polygenic) resistance is another type of resistance, which almost all plants
possess, and which depends upon multiple genes in the plant host.
Pretreatment of plants with a variety of organisms or compounds can induce
the activity of those genes. This general phenomenon, induced systemic
resistance (SIR), can depend on the timely accumulation of multiple gene
products related to plant defence (Tuzun 2001). In other words, as Tuzun
states, pretreating plants with an inducing organism or compound appears to
incite an effective plant defence response upon subsequent encounters with
pathogens. He goes on to state that for subsequent encounters with
pathogens, plant defence, at least in some cases, can be viewed as a process
of converting what would have been a compatible interaction to an
incompatible one. Thus, as Tuzun states, plants in general, by virtue of past
coevolutions with pathogens, have evolved two fundamental forms of
defence against pathogens, the single-gene form and the multiple-gene form.
In the wild, plants have numerous occasions to need at least single gene
defence, which is pathogen-specific in several wild plants. Some plants have
multiple gene defence, which is less pathogen-specific and which is systemic
rather than local. Multiple gene defence is invoked only under certain
conditions for plants in the wild populations, that is, under conditions that
tend to be appropriate for invoking the far more general single-gene defence.
Tuzun (2001) documented that multigenic-resistant plants “constitutively
express specific isozymes of hydrolytic enzymes that release cell wall
elicitors, which in turn may activate other defence mechanisms”. SIR
induces accumulation of these and other gene products prior to challenge.
Known to function against multiple organisms when induced, SIR has,
however, no specificity in the accumulation patterns of defence-related gene
products. Thus, the constitutive accumulation of specific isozymes of
hydrolytic enzymes, or other defence related gene products, is hypothesized
10
to be an integral part of multigenic resistance and of the phenomenon of SIR.
Plants that have activated SIR seem to move from latent resistance to a form
of active resistance that is multigenic and non-specific.
Stimulating SIR expression (signals)
The induction of plant defence responses, such as enhanced resistance to
pathogens, seems to involve several signal transduction cascades (Karban
and Kuü 1999). Dean and Kuü (1986a, b) have shown that an infected leaf
can produce a signal invoking SIR, and that detachment of the leaf before
the development of the HR blocks SIR induction. The SIR signal can move
in the phloem. This is known through grafting experiments and stem girding
experiments with cucumber and tobacco (Guedes et al. 1980; Tuzun and Kuü
1985). Plants can acquire SIR after treatment with necrotizing attackers,
non-pathogenic root-colonizing pseudomonads, salicylic acid, ȕaminobutyric acid, and many other natural or synthetic compounds (Conrath
et al. 2002).
Salicylic acid (SA) has recently been qualified as a plant hormone
(Mishra and Choudhuri 1999) because of its physiological and biochemical
roles in plants (Enyedi et al. 1992; Malamy and Klessig 1992; Dempsey et
al. 1999). Evidence now indicates that SA does play an important role in
plant defence against pathogen attack, and that it is essential for the
development of SIR, but is not the mobile translocated signal responsible for
that phenomenon (Vernooij et al. 1994; Ryals et al. 1995). Yalpani and
Raskin (1993) conclusively demonstrated the biosynthetic pathway of SA in
tobacco plants by 14C-tracer studies with cell suspensions, Lee et al. (1995)
reviewed the biosynthetic and metabolic pathways of SA and the key
enzymes for its biosynthesis and catabolism. Malamy et al. (1990) and
Métraux et al. (1990) discovered that SA accumulates in infected leaves of
tobacco plants, and to a low level, in uninfected leaves.
Induced resistance is often associated with a process called “priming”, an
enhanced capacity to mobilize infection-induced cellular defence responses
(Conrath et al. 2002). Although priming has been known for years, the
understanding of the phenomenon is only recent. The studies by Conrath et
al. (2002) show that priming often depends on NPR1 gene (also known as
NIM1 or SAI1), which is the key regulator of SIR, and which has a major
effect on the regulation of cellular plant defence responses.
Cross-talk between plants and microbes
Immunization, an effective deterrent against diseases caused by fungi,
bacteria, and viruses, has been successfully tested in laboratory and field
(Kuü 1990). Immunity (i.e., modification of susceptibility) to fungal plant
11
pathogens induced by earlier or simultaneous inoculation with a strain of the
same organism, or another, is commonly described as cross protection,
systemic resistance, or local resistance. The phenomenon immunity,
described above as SIR, has been demonstrated in various host plant species
against several pathogens. Heil and Bostock (2002) regard resistance elicited
by one group of enemies and active (also) against another as cross-resistance
(e.g. resistance against pathogens induced by herbivores and vice versa).
Cross-talk phenomenon has been found in several systems, as for example,
feeding by thrips and aphids reduced infection of watermelon by the fungus
Colletotrichum orbiculare (Russo et al. 1997).
Molecular plant response
Plants are under constant threat of infection by pathogens that have evolved
a diverse array of effector molecules used to initiate plant colonization. In
turn, plants have evolved sophisticated detection mechanisms and response
systems that decipher pathogen signals and then induce appropriate defence
responses. Genetic analysis of plant mutants with impaired mechanisms for
mounting a resistance response to invading pathogens has revealed several
distinct, but interconnecting, signalling networks that are under positive and
negative control (Feys and Parker 2000). These pathways operate, at least
partly, through the action of small signalling molecules, such as salicylate,
jasmonate, and ethylene. The interplay of signals probably allows the plant
to fine-tune local and systemic defence responses.
Excellent and comprehensive reviews have been published on the various
biochemical and molecular responses of plants to pathogens, as well as on
important related aspects such as recognition, specificity, and signal
transduction (Bowles 1990; Dixon and Harrison 1990; Cutt and Klessig
1992; Keen 1992). Through evolution, disease resistance is multi-factorial,
because plants are continually exposed to various pathogens, and therefore
have developed numerous physiological mechanisms for self-defence that
incorporate both physical and chemical factors. The chemical factors include
the production of secondary metabolites and defence-related peptides.
Kombrink and Somssich (1995) propose that a plant’s response to a
pathogen includes (a) synthesis of phtoalexins (i.e., low molecular weight
anti-microbial compounds that accumulate at site of infection), (b)
systemically produced enzymes that degrade the pathogen (e.g., chitinases,
ȕ-1,3-glucanases, and proteases), (c) systemically produced enzymes that
generate anti-microbial compounds and protective biopolymers (e.g., POX
and phenoloxidases), (d) biopolymers that restrict the spread of pathogens
(e.g., hydroxyproline-rich glycproteins, lignin, callose), and (e) compounds
that regulate the induction or the activity of the defence compounds (e.g.,
elicitors of plant and microbial origin, immunity signals from immunized
plants, and compounds that release immunity signals).
12
PR proteins.—First identified in tobacco infected with tobacco mosaic
virus (TMV) (van Loon and van Kammen 1970), pathogenesis-related
proteins (PRs) are defined as plant proteins that are synthesized specifically
when plants come into contact with pathogens (van Kan et al. 1992). PR
proteins are grouped into two major classes: (i) acidic, which are mostly
found in the apoplast, and (ii) basic, mostly found in vacuoles. PR protein
gene expression or accumulation is commonly used as a measure of the state
of SIR under inducing or under non-inducing conditions (see reviews by Bol
et al. 1990; Linthorst 1991). Different PR protein families are recognized
(van Loon and van Strien 1999).
For the majority of the PR families, activities are known or can be
inferred. Putative anti-fungal, anti-bacterial, anti-viral, and insecticide
activity may explain why PRs accumulate during plant-pathogen
interactions, in stress conditions and upon wounding (either by insects and
nematodes or mechanically). The only PR family for which no biochemical
function is known consists of the PR-1 proteins; they are anti-fungal proteins
of unknown function. Other families include PR-2, a group of ȕ-1,3glucanases with anti-fungal activity; PR-3, a group of anti-fungal chitinases
and anti-bacterial lysozymes; PR-5, an osmotin similar to thaumatin;
proteinases; a-amylases; POXs; and cysteine- and glycine-rich proteins. PR1 can have activity against pathogens. The constitutive expression of a PR-1
transgene in tobacco resulted in enhanced resistance to the oomycetes
Peronospora tabacina and Phytophthora parasitica (Alexander et al. 1993).
Furthermore, Niderman et al. (1995) showed that PR-1 proteins purified
from tomato can inhibit germination of Phytophthora infestans zoospores in
vitro.
In relation to these investigations, we attempted to find relationships
between the accumulation of POXs, total phenols, and proteins, and also
polygalacturanase activity (unpublished results)—upon stimulating plants
with Fo-(IMI 386351). Differences occurred between treated and non treated
plants in terms of accumulation of those enzymes. There was no clear
evidence for those enzymes being key factors in SIR expression. Perhaps
pathogenesis related proteins are only expressed for certain plant species
being attacked by certain pathogens under certain conditions. For example,
in tomato SIR induced by Penicillium oxalicum against Fol was not
associated with accumulation of pathogenesis-related proteins (Garcia-Lepe
et al. 1998). The same was true of SIR obtained by Pseudomonas
fluorescens strain WCS417r (Duijff et al. 1998). However, SIR obtained
non- pathogenic Fo strain Fo47, well known for its capacity to suppress
tomato wilt in vivo (Alabouvette et al. 1993), might be related to
accumulation of PR-1 and chitinases (Duijff et al. 1998). Hoffland et al.
(1995) also demonstrated in their study that SIR expression obtained in
radish is not associated with accumulation of PR proeins. However, there are
several reports relating SIR with concomitant accumulation of PR proteins
13
(Hammerschmidt and Kuü 1995). Wubben et al. (1993) observed that upon
infection by Cladosporium fulvum, tomato plants start to produce PR
proteins (glucanse, chitinase, PR-1b), near the stomata in C. fulvuminoculated tomato leaves. van den Elzen et al. (1993) also found that tomato
plant constitutively expressing both class I ȕ-1,3-glucanse, and that class I
chitinase genes exhibit enhanced immunity against Fol.
Phenolics compounds.—The phenolics (or polyphenols) are a large
chemically diverse family of compounds ranging from simple phenolic acid
to very large and complex polymers such as tannins and lignin (Hopkins
1999). The flavonoids and other secondary phenolic products appear to be
involved in plant-herbivore-interactions. Some are important structural
components, such as lignin, whereas others seem to be simply metabolic end
products with no obvious function (Hopkins 1999). The phenolics are almost
universally present in plants, and are known to accumulate in all plant parts
(root, stem, leaves, flower, and fruits). Although they represent the most
studied secondary metabolites, the function of many phenolic compounds is
still unknown (Raven et al. 1999).
Accumulation of phenolic compounds in plant tissues in response to
infection has been widely reported (e.g., Dixon and Paiva 1995; El Hadrami
et al. 1997; Mohammadi and Kazemi 2002; Dihazi et al. 2003). Such
accumulation facilitates plant immunity (Chet et al. 1972; Retig and Chet
1974). After polymerization or oxidation, some phenols act as inhibitors of
pathogens (Kuü 1968). Winstead and Walker (1954) showed that the
pectolytic enzyme extract from cultures of several Fo f. spp. caused vascular
browning in non-susceptible as well as susceptible plants, presumably due to
the breakdown of cell wall materials, which in some way released phenols.
Polyphenol oxidase of the host converted those phenols to melanins.
An initial increase in phenols after infection with Fol was very
pronounced in resistant tomato cultivars, but small, and probably
insignificant in susceptible ones (Matta et al. 1967). Previous inoculation
with non-pathogenic organisms increased resistance of susceptible plants in
some studies (Chakraborty and Sen Gupta 1995; Blok et al. 1997; Fuchs et
al. 1997; Olivain and Alabouvette 1997; Benhamou et al. 2002). In the plant
Lycopersicon esculentum –L., increased immunity induced by a nonpathogenic fungus was followed by an increase in phenolic compounds
(Matta et al. 1969). Our results reported in (Paper II) show clear variation in
levels of phenolic compounds between plant treated with Fo-(IMI 386351)
and disease-control plants, suggesting that phenols might be involved in SIR
expression.
Peroxidases (POXs).—The peroxidases are oxido-reductive enzyme that
participate in the cell wall-building processes such as oxidation of phenols,
suberization, and lignification of host plant cell during defence reaction
against pathogenic agents (Kolattukudy et al. 1992; Chittoor et al. 1999).
14
Lignin.—Participation of precursor biosynthesis and polymerisation in
SIR has often been ignored. Yet, in most tissues undergoing resistance
responses, phenyl ammonia lyase (PAL) is induced, and provides the
precursors for lignin synthesis, but also for the synthesis of phenolics,
isoflavonoid phytoalexins, coumarins, and salicylic acid (Sticher et al. 1997).
Lignin precursors and other phenolics are directly toxic to pathogens
(Hammerschmidt and Kuü 1982); their polymerisation makes cell walls
more difficult to penetrate and degrade (Ride and Barber 1987). In SIR
tissues of cucumber, the HR is repressed, and instead of necrosis upon
pathogen challenge, the cell wall under the germ tube of the pathogen
becomes heavily lignified, preventing its ingress (Richmond et al. 1979).
Besides being involved in lignification, some PR proteins are components
of cell walls (e.g., glycine-rich glycoproteins). POX cause them to undergo
oxidative cross-linking, which increases further the cell wall's strength
(Sticher et al. 1997). In rice leaves, increase in biosynthesis of lignin occurs
due to Pseudomonas fluorescens treatment, which increases levels of POX,
PAL, and 4-coumarate: those enzymes are considered to be fundamentally
involved in lignin biosynthesis (Smith et al. 1994, Vidhyasekaran et al.
2001), as a consequence of treatment with an endophyte, for example. Crosslinking due to the action of POX and deposition of lignin during secondary
wall development may limit Fol (pathogen) ingress and spread in the host
tissue. Also, reactive compounds associate with a signal released by Fo-(IMI
386351), or by the Fo-(IMI 386351)-Fol complex (see Fig. 8 in Paper II).
Those compounds may also inhibit Fol growth. As suggested in (Paper II),
the endophytic Fusairum strain Fo-(IMI 386351) may release a signal that,
alone or in combination with a signal released by the pathogen, might
enhance the lignification process related to SIR expression.
Accumulation of lignin and of phenolic compounds has been correlated
with disease resistance in many plant–pathogen interactions (Mohammadi
and Kazemi 2002). These include wheat, Puccinia graminis f. sp. tritici
(Beardmore 1983); tomato, Verticillium albo-atrum (Robb et al. 1987); and
rice, Xanthomonas oryzae pv. oryzae (Reimers and Leach 1991). Enhanced
POX activity has been correlated with resistance in rice (Reimers et al. 1992;
Young et al. 1995), wheat (Flott et al. 1989), barley (Kerby and Somerville
1989), and sugarcane (McGhie et al. 1997) following the inoculation with
phytopathogens. POX activity was delayed (or remained unchanged) during
the compatible interaction with susceptible plants. Polyphenol oxidases are
involved in the oxidation of polyphenols into quinones (anti-microbial
compounds) and lignification of plant cells during the microbial invasion.
Phenol oxidases generally catalyze the oxidation of phenolic compounds to
quinones using molecular oxygen as an electron acceptor (Mayer 1987;
Sommer et al. 1994).
15
Cross-talk complexity
Some bacterial strains might well reduce DI by invoking SIR, and in some
cases eliminate it (Konnova et al. 1999). Such induction might vary due to
differences in receptors of the tomato plants. If so, the effectiveness of SIR
might depend on how it is induced. None of the selected bacteria have
inhibitory effects on Fol in culture media (Paper IV).
Table 1: Eight bacterial strains tested in (Paper IV) have a potential capacity to
suppress Fol in vivo, along with codes and the scientific names of the plant species
from which they were isolated.
Bacteria code reference
MF7
MF9
MF11
MF20
MF27
MF29
MF30
MF44
Scientific name
Viola biflora
Viola biflora
Carex sp
Fabaceae sp
Rumex acetocella
Rumex acetocella
Rumex acetocella
Equisetum sylvaticum
Such a dependence of effectiveness of SIR on type of induction might reflect
strain-specific plant-defence against bacteria (See Table above), which might
include complex coevolving systems, such as several strains of bacteria
coevolving with a single plant species, such as Rumex acetocella with three
strains of Pseudomonas sp. (MF27,MF29,MF30) (See Table 1. above).
Other bacterial strains were isolated from plant species other than Rumex
acetocella, as indicated in the table. Some bacterial strains (Pseudomonas
sp.), although acclimatized to the relatively low temperatures of northern
Sweden, grew well in vitro at rather high temperatures, such as 25°C and 28°
± 2°C.
Dependence of SIR on strength of induction.—Biochemical changes in
the host plant always accompany pathogenic infections. Those changes can
be different in susceptible and resistant reactions of the host to the pathogen
(Chakraborty and Sen Gupta 2001). However, a multitude of changes in
biochemistry or physiology of a plant can occur, not as parts of a susceptible
or resistance reaction, but as consequences or by-products of such reaction,
such as physiological adjustments or compensations that automatically occur
as part of homeostasis.
Our results seem to suggest that SIR induction had different strengths in
the experiments reported in (Papers II and III), and that different degrees of
SIR occurred in response to the different induction strengths, which
increased with increase in Fo-(IMI 386351) dosage (Paper II). Moreover,
there appears to have been a threshold strength for induction, which, when
passed, caused a qualitative shift in plant protection, which could well have
been due to one or more plant genes being turned on (or off) when the
16
threshold was passed (Paper III). Such a threshold shift would explain the
fact that the pathogen Fol shifted from a virulent and undetectable (but
apparently low) density within the stem of the host plant (when induction
strength was below the threshold) to an apparently benign high density
(when the induction strength was above the threshold). Correspondingly,
SIR shifted from a lesser to a distinctly higher level and effectiveness.
Fo-(IMI 386351) has yet to exhibit, however, pathogenicity (in the true
sense) to any plant, although it can readily gain access to a tomato plant, and
does cause phytotoxicity in some of the lower leaves. But Fo-(IMI 386351)
does not seem to functionally “attack” the plant. The result is plant
protection and plant growth promotion. Fo-(IMI 386351) also may well lack
clear host-specificity, and any specificity that exists may well be highly
changeable, for reasons discussed below.
Complementary modes of action.—A microbe, as an agent for mitigating
disease for a plant caused by another microbe that is pathogenic to the plant,
can directly antagonize the pathogen, by parasitism, antibiosis, or
competition, or can indirectly antagonize it by induced resistance carried out
systemically by the host plant (SIR). Since these several modes of action do
not exclude one another, and can instead complement one another, several
can be occurring simultaneously, or at different times. Hence, the cross-talk
triangle between plant, pathogenic microbe, and SIR-inducing microbe can
be dynamically a symphony of action played by the biochemistries of the
plant’s cells, in concert with the population dynamics and cell biochemistries
of the population of pathogen cells infecting the plant, and of the population
of cells of the SIR inducing microbe within and around the plant. Since the
symphony’s composer is evolution, it is played in the same way whenever
the given triangle of organisms convenes for plants of a given species.
Cross-talk triangles can include Fusariam oxysporum strains as inducers of
SIR, as for example, the Fusariam oxysporum strain Fo47 involves
competition with other microbes for nutrients in soil, for root colonization
sites, and for opportunities to induce systemic resistance (Fravel et al. 2003).
Likewise for the non-pathogenic Fusariam oxysporum isolates C5 and C14
isolated by Mandeel and Baker (1991).
17
Phytophthora cryptogea in SIR
Phytophthora species belong to the order Peronosporales, class Oomycetes
(Pseudofungi), which includes some of the world’s most destructive plant
pathogens. The Pseudofungi, sometimes also referred to as the water molds,
have a phylogenetic relationship with some algae, and therefore are no
longer classified as “ true fungi” in the kingdom Myceteae, but belong now
to the new kingdom Chromista (Cavalier-Smith 1989; Judelson et al. 1992;
van de Peer and Wachter 1997). Pseudofungi differ from other “fungal
groups” in many genetic characters (Quintanilla 2002).
One of the Phytophthora spp., P. cryptogea (Pc), was characterized as a
non-pathogen in potato (Strömberg and Brishammar 1991). In our previous
study, we found that Pc was not useful in acting as an inducer when applied
to the root system (Attitalla et al. 1998), but was able to induce SIR in
tomato plants by using an application described by Quintanilla and
Brishammar (1998). Upon developing this application, we sprayed a
zoospore suspension of P. cryptogea on the green parts of tomato plants,
instead of applying, as is customary, the suspension to roots of plants (Paper
III). Cryptogein, a genus-specific elicitor that purportedly elicits necrosis
only on tobacco and certain radish cultivars (Kamoun et al. 1993); but they
did not include tomato in their tests. Pernollet et al. (1993) reported that
cryptogein is a non-specific elicitor in Solanaceae in that it induces necrosis
on potato cuttings (Solanum tuberosum -L.), tomato seedlings (Lycopersicon
esculentum – L.) and on leaves of pepper (Capsicum annum –L.) (Strömberg
1994).
Elicitin proteins are secreted by all species and most isolates of
Phythopthora (Kamoun et al. 1994; Huet et al. 1995). In tobacco, they
induce a response phenotypically indistinguishable from HR observed when
challenged by avirulent races or species of Phytophthora (Ricci et al. 1989).
Elicitins also can induce SIR (Keller et al. 1996). However, in the
combination between the tobacco plant and the pathogen P. parasitica pv.
nicotiana, the elicitins may act as avirulent factors in tobacco (Ricci et al.
1992). On the other hand, most plant species do not respond to elicitins
(Keizer et al. 1998). In our study (Paper III), Pc enhanced necrosis on the
susceptible tomato cultivar Danish Export, but not in the moderate resistant
one Elin F1, that might be related to one of the above elicitors.
18
Fusarium oxysporum complex
The genus Fusarium contains a number of soilborne species with worldwide
distribution and known to be important plant pathogens (Moss and Smith
1984; see review by Roncero et al. 2003). Recently, Fusarium sp. have been
reported as an emerging human pathogen in immunocompromised patients
(Vartivarian et al. 1993). The anamorph Fo species belongs to the order
Moniliales, and is placed in the family Tuberculariaceae; they include some
of the world’s most destructive plant pathogens, and differ from other
“fungal groups” in many genetic characters. Although most of members of
the Fo-complex are anamorphs, some produce telemorphic stages, as for
example, each of the Gibberella. fujikuroi and Nectria haematococca species
complexes, once considered by Snyder and Hansen (1940) to be represented
by F. moniliforme and F. solani, respectively, are now known to harbor over
40 phylogenetically distinct species with a hypothesized Gondwanic
biogeographical history (Xu and Leslie 1996; O’Donnell 2000; Jurgenson et
al. 2002).
All strains of Fo exist saprophytically, but some are well known for
inducing wilt or root rots on plants, whereas others are considered nonpathogenic (Fravel et al. 2003). Soil-borne populations of Fo are diverse. In
suppressive soils, pathogenic and non-pathogenic strains interact in such a
way that the observed result is often that of disease control. Non-pathogenic
strains are used therefore as biocontrol agents. As Fravel et al. (2003) point
out, non-pathogenic Fo strains can contribute to biological control through
several modes of action: for example, competition for nutrients in the soil,
competition for infection sites on the root, reduction in the rate of
chlamydospore germination of the pathogen, triggering of local plant
defence reactions, and induction of systemic resistance. Which mechanisms
are used or emphasized depends on the strain. The non-pathogenic Fo are
easy to mass-produce and formulate, but economically feasible and effective
application conditions for biocontrol under field conditions have yet to be
determined.
Numerous studies clearly identify a role for non-pathogenic Fusarium
spp. in suppressive soils from different areas in the world (Schneider 1984;
Paulitz et al. 1987; Larkin et al. 1993; Larkin et al. 1996). For biocontrol of
Fusarium diseases, most research has focused on the modes of action of the
non-pathogenic strains of Fo. Strains of Fo and F. solani were more efficient
in establishing suppressiveness in soil than were other Fusarium sp. Several
19
Fo isolated from the stems of healthy plants have been found to be effective
biocontrol strains (Ogawa and Komada 1984; Postma and Rattink 1992).
Life history
The view that Fo is an asexual fungus with ascomycete affinities is widely
accepted (Gordon 1993). In culture, this fungus produces three kinds of
asexual spores: microconidia, macroconidia, and chlamydospores. The
typical spores, microconidia, which are 1-to-2-celled, are produced most
frequently and abundantly under all conditions, including inside the vessels
of infected host plants, macroconidia, which are 3-to-5-celled, they have
gradually pointed and curved ends, and appear in sporodochia-like groups on
the surfaces of plants killed by the fungus.
Chlamydospores are viable, asexually produced accessory spores
resulting from the structural modification of vegetative hyphal segment (s)
or from conidial cells possessing a thick wall mainly consisting of newly
synthesised cell wall material (Griffths 1973; Schippers and van Eck 1981).
The primary function of chlamydospores is survival in soil. They are 1- or 2celled, thick-walled, and round, and produced within old mycelia or the third
kind of spore, macroconidia.
All three types of spores are produced in cultures of the fungus, and
probably in the soil, although only chlamydopsores can survive long in the
soil (Beckman 1987; Agrios 1997). For Fusarium solani f. sp. cucurbitae
and Fusarium sulphureum, the chemical composition of the cell walls of
macroconidia and chlamydopsores mainly consists of glucosamine, glucose,
mannose, galactose, amino acids, and glucronic acids; but the proportions of
these compounds are different in the two spores types (van Eck 1978a, b;
Schneider et al. 1977). The mycelium, colorless at first, becomes
successively darker with age—cream-colored, pale yellow, pale pink, or
somewhat purplish. Fig. 2 shows several conidia produced by Fo-(IMI
386351).
20
Fig. 2A,B,C,D,E,F,G,H: Scanning electron microscope (SEM) monographs illustrate
Fo-(IMI 386351) conidia and myclial structure under SEM. A through C show
macro and microconidia, D show the ariel mycelium, E and F shows
chlamydospores in two resolutions, and G and H shows germination conidium.
These pathogenic strains, which express a high level of host specificity, are
classified into more than 120 f. spp. and races (Armstrong and Armstrong
1981). After gaining entrance into the host plant through the root system,
mycelial cells become established (Beckman 1987). In the xylem, they
produce toxic materials that pass throughout the plant bringing about
yellowing and wilting of leaves and stem, and eventually resulting in the
plant’s death (Beckman and Roberts 1995).
Vegetative compatibility groups (VCGs)
Recent studies with molecular markers reveal that Fusarium oxysporum (Fo)
has extensive subspecies diversity. Whether or not sexual reproduction must
be invoked to account for this diversity might therefore be an appropriate
consideration. No sexual stage of Fo occurs in the field or in the laboratory.
Thus, there is no direct evidence that Fo spp. reproduce sexually. This might
be why most researchers accept that they are asexual. An argument that
sexual reproduction occurs in Fo spp. might develop from studies of the life
history of this species.
Fo is a taxon comprised principally of non-pathogenic strains that are
saprophytic, root-colonizing, and weakly parasitic (Gordon et al. 1989). One
might argue that sexual reproduction occurs among these non-pathogenic
strains but that progeny (descent lines) that become virulent on a particular
host species also become committed to clonal propagation. The abundance
21
of phenotypes, based on VCGs found among non-pathogenic strains, might
be taken as evidence in favour of this interpretation. For example, from 300
Fo isolates, 60 VCG phenotypes were identified (Gordon and Okamoto
1992a). Data from population studies, including frequency distributions for
virulence and for VCG phenotypes, and non-random associations between
nuclear and mitochondrial (mt) genotypes of Fo, also provide strong support
for the dominance of asexual reproduction in this species. However, this
level of VCG phenotype diversity does not make compelling the argument
for sexual reproduction in Fo (Gordon 1993).
Further evidence for clonality in Fo can be gleaned from recent data on
mtDNA in this species (Paper I and II). In both pathogenic and nonpathogenic strains, strong correlation between some VCG phenotypes and
particular mtDNA haplotypes has been observed. For example, in F.
oxysporum f. sp. melonis, a single VCG (0130) was consistently associated
with the same mtDNA haplotype (Jacobson and Gordon 1990). Also, among
non-pathogenic strains, 13 isolates sharing a common VCG phenotype all
had the same mtDNA haplotype (Gordon and Okamoto 1992b). Such nonrandom association between nuclear and extranuclear genetic markers
indicates clonally propagating populations (Tibayrenc et al. 1991).
Species status
The concept of species generally includes the concept of sexual fertility, but
there are some semantic problems in applying to as asexual forms. An
“Asexual species”, as a term used by Gordon (1993), is “a biologically
meaningful grouping as distinct from an assemblage of strains unified by a suite of
morphological characters”. When a species lacks a teleomorph, mating
compatibility cannot be used to confirm the taxonomic identity of individual
strains. Thus, Fo has been extensively studied to examine the variability in
morphological characters which serve to define it as a taxon (Nelson et al.
1983). Critical evaluation of these characters provides support for Fo being a
phenetic species (Sokal and Crovello 1984). Two lines of molecular
evidence also support the view of Fo as a species. First, sequence
comparisons (of the large nuclear rRNA gene) among Fusarium spp.
indicate a close relationship between the three strains of Fo that were
included in (Paper I). The result of mtDNA analysis by the restriction
digests of total DNA confirmed that all strains of Fo are closely related.
Examination of mtDNA for over 300 isolates of Fo, pathogenic and nonpathogenic, indicates that all variants can be related to a single reference
strain by relatively simple changes (Gordon and Okamoto 1992a, b; Gordon
1993).
22
Biology of Fusarium oxysporum f. sp. lycopersici (Fol)
Modes of existence
An analysis of the behaviour of species and infra-species populations of
Fusarium spp. indicates that they have three basic modes of existence. Two
of these can be designated traditionally as soil-borne and air-borne, whereas
a third applies to those Fusarium spp. that are common in soil and have
efficient mechanisms for air dispersal. Survey data and routine isolations do
indicate, however, that many Fusarium spp. can occur outside their common
habitats.
Fol has a relatively high requirement for micronutrients. Deficiencies of
copper, iron, manganese, molybdenum, and zinc reduce growth and
sporulation. Tomato wilt incited by Fol was first described by Masee in
1895. The disease is of world-wide importance, having been reported in
several locations—to our knowledge, in 32 countries (Jones and Woltz
1981). Generally, tomato wilt is a warm-weather disease, most prevalent in
soils that are acid and sandy, and where soil and air temperatures approach
the optimum of 28 qC for disease development. Soil temperatures that are
too warm (34qC) or too cool (17í20qC) retard wilt development (Clayton
1923; Walker 1971; Agrios 1997).
Some wilt diseases have been considered “low-sugar diseases”, and
others, “high-sugar diseases” (Horsfall and Dimond 1957). Tomato wilt is
classified as a low-sugar disease: low- and high-sugar-content tissues favour
susceptibility (Cohen et al. 1996). Low light intensity or short photoperiod
markedly decreased resistance in tomato, whereas root damage, which
causes sugar levels to rise in the plant’s stem, increased resistance (Bell and
Mace 1981).
Restriction fragment length polymorphism (RFLP)
RFLP analyses uses restriction enzymes to cut DNA at specific 4-6 bp
recognition sites (Dowling et al. 1990). Sample DNA is cut (digested) with
one or more restriction enzymes. Fragments that result from base
substitutions, additions, deletions, or sequence rearrangements within
restriction-enzyme recognition sequences are separated according to
molecular size using gel electrophoresis (Avise 1994). RFLP is most suited
to studies at the intraspecific level or among closely related taxa. Presence
and absence of fragments resulting from changes in recognition sites are
used to identify species or populations. In Fusarium spp. as well as in other
fungal species, variation in rDNA has been estimated by RFLP analysis
(Kistler et al. 1987; Nicholson et al. 1993). Intra- and inter-specific
relatedness has also been estimated using the RFLP-analysis of
23
mitochondrial and genomic DNA, or using analysis of rDNA (Förster et al.
1988; Förster et al. 1990).
The phylogenetics of Fo-(IMI 386351)
Using sequencing to study the phylogenetics of the isolate Fo-(IMI 386351)
= 29868 biocontrol (see Fig. 2 in Paper II), we found that Fo-(IMI 386351)
has a typical mtDNA profile similar to Fol (Paper I). Fo-(IMI 386351) was
located in the same clade with other Fol strains (based on sequencing of two
genes a mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and the
nuclear translation elongation factor 1Į (EF-1Į)). Three trees (Paper II)
show that some clades contain both Fol and Fusarium oxysporum f. sp.
melonis (Fom), and that Forl was located in a clade containing f. spp. other
than Forl. This is an example in which a PCR-based DNA-sequencing
method, although a good way to study infraspecific complexity and
diversity, worked insufficiently for clear-cut, accurate, and objective
differentiation between f. spp. This was one reason for seeking some other
method for distinguishing between the two f. spp., Fol and Forl. Another
reason was the fact that laboratories used for fieldwork are far less likely to
be equipped with means of mtDNA RFLP profiling, and that sequencing is
far more time consuming.
Distinguishing between Fol and Forl
To distinguish between the two f. spp. Fol and Forl, we sought a tool that
would provide a simple “yes” or “no”, an unambiguous accurate decision as
to which of the two f. spp. a given isolate belongs. Such a screening tool
would make field studies feasible for examining the ecological and
population dynamics of the two f. spp.
We feel that a PCR based DNA-sequencing method, although often
employed to differentiate between species, is not a useful means of
differentiating between infraspecific taxa, such as f. sp., as clearly expressed
by Edel et al. (1996). To illustrate this point further, we used O’Donnell’s
sequencing method on two isolates, Fol-(CBS 165.85) and Forl-SW, using
two genes, mtSSU and ITS (Unpublished) and using the gene bank to
determine similarity. Fol-(CBS 165.85) had about 99.6% similarity with Fol,
but Forl-SW, although distinctly Forl (based on pathogenicity), did not
indicate similarity with any f. sp. in the Fo complex. In (Paper II) we show
that one cannot distinguish between Fol and Forl for either of the genes, nor
when one combines them. Sequencing of other genomic regions may help to
separate these two f. spp. of F. oxysporum. (Perhaps a gene can be found that
keeps Fol and Forl in separate clades). To demonstrate the phylogenetic
24
breadth of the tool’s applicability, we specified in (Paper I) the VCGs to
which Fol and Forl isolates belong.
The telemorphic stage of most Fusarium spp. is unknown, and therefore
Fusarium taxonomy depends on morphological criteria of the anamorph: for
example, the size of macroconidia, the presence or absence of microconidia
and chlaymydospore, color of the colony, and conidiophore structure
(Windels 1992). Delineating species based on these features has problems,
however (Booth 1971; Nelson et al, 1983). Many taxonomic schemes group
the species into sections. Based on morphological characters, distinguishing
Fo from many other species belonging to the sections Elegans and Liseola
can be difficult. Plant pathogenic, saprophytic, and biocontrol strains of Fo
complicate matters because many are morphologically indistinguishable.
Pathogenic Fo are commonly host specific, attacking only one or a few
species of plants, and in some cases attacking only certain cultivars of a
plant species. The specificity for a given host species or given cultivar of
that host species is designated, respectively, as a f. sp. or race of the
pathogen.
Because many pathogenic fungi are morphologically indistinguishable
from each other (e.g., Fol and Forl), and because the same is true of many
non-pathogenic fungi (Fravel et al. 2003), in 1985, Puhalla proposed a
system of classification for strains of Fo—a system based on their vegetative
compatibility. For determining the VCG of each strain, he described a
method that is based on pairing nitrate non-utilizing mutants. Kistler et al.
(1998), in recognizing the usefulness of VCGs in classifying Fo strains,
proposed a system for standardizing their numbering. Although some f. spp.
correspond to a single VCG, others include several VCGs. In a recent
review, Katan (1999) recognized 59 VCGs for 38 f. spp. Many isolates in
non-pathogenic populations are single member VCGs, and some are even
self-incompatible (Gordon and Okamoto 1992b; Kondo et al. 1997;
Steinberg et al. 1997). For this reason and others, the determination of
VCGs, although useful, cannot be used as a universal tool to identify f. sp.,
or to identify non-pathogenic isolates.
A rapid molecular method (Paper I) was developed to differentatiate
between two morphologically identical Fo f. spp., Fol and Forl, since both f.
spp. share the same host Lycopersicon sp. but cause different disease
symptoms, wilt rot and foot root, respectively. The result led to a tool that
distinguishes between them, and does so easier and faster than possible with
in vivo tests, and at a lower cost than other biochemical and physiological
methods—and that requires equipment that is available in all ordinary
laboratories. Some scientists have failed to distinguished between these two
f. spp. For example, Benhamou et al. (1998) considered a strain of Forl to
cause symptoms of wilt disease (i.e., symptoms known to be caused by Fol,
not Forl). Recorbet et al. (2003) emphasizes the urgent need for powerful
molecular markers, for distinguishing between pathogenic and non25
pathogenic Fusarium. Molecular approaches, such as the polymerase chain
reaction (PCR), which has been developed for fungal systematic studies, are
often based on the analysis of ribosomal DNA (rDNA) sequences that are
universal and that contain both conserved and variable regions, allowing
therefore discrimination at several taxonomic levels (Edel et al. 1996). The
development of the PCR combined with the selection for fungi of universal
oligonucleotide primers (White et al. 1990) has provided a tool for easily
accessing rDNA sequences. That tool is suitable for rapid taxonomic studies
of fungi (Vilgalys and Hester 1990; Cubeta et al. 1991).
At the species level, non-coding rDNA, such as the internal transcribed
spacer (ITS) region, has been a good taxonomic character for many fungi,
such as Pythium spp. (Chen 1992), Phythopthora spp. (Lee and Taylor
1992), and Verticillium spp. (Morton et al. 1995). For many fungi, and
certainly Fusarium spp., nucleotide sequence comparisons after direct rRNA
sequencing has been used to estimate variation in rDNA (Logrieco et al.
1991). Also, polymorphism in the ITS region has been shown for a few
Fusarium spp. (O’Donnell 1992; Donaldson et al. 1995). That was also the
case when we sequenced that region (unpublished) for Fol and Forl in our
attempt to distinguish between them. Unfortunately, we failed to obtain
relevant results: in fact, even the Forl-SW sequence appeared to be not well
identified after comparing our sequence with the reference sequences in the
GenBank data set: Forl-SW is classified as a Fusarium sp., which is another
reason for seeking a better method to differentiate between Fol and Forl
(Paper I).
In coding rDNA regions, some domains are variable between fungal
species (Hibbett and Vilgalys 1993; Sherriff et al. 1994). PCR-based
methods can be powerful tools for characterizing Fusarium spp. strains at
the species level. Edel et al. (1995) compared three molecular methods: (1)
RFLP, (2) analyses of total DNA after hybridization with a random DNA
probe, and (3) PCR-based fingerprinting with primers that match
enterobacterial palindromic (REF) elements and with restriction fragment
analysis of PCR-amplified ribosomal intergenic spacers (IGS). All three
methods yielded interspecific polymorphisms, but levels of discrimination
were different. Edel et al. (1995) also found high correlation between the
groupings by these three methods.
Sequences of the EF-1Į and the mtSSU rDNA genes have been valuable
in distinguishing between Fusarium spp. and their origins (Baayen et al.
2000; O’Donnell et al. 2000; Baayen et al. 2001; Skovgaard et al., 2001).
DNA sequences of UTP-ammonia ligase; trichothecene 3-Oacetyltransferase, a putative reductase (O’Donnell et al. 2000); nitrate
reductase; and phosphate permease (Skovgaard et al. 2001) have also been
used to distinguish between Fusarium spp. successfully. Sequences of the ȕtubulin region have been useful to distinguish between some Fusarium spp.
(O’Donnell et al. 2000), but not others (O’Donnell et al. 1998). Although
26
DNA sequences of the ITS regions are highly successful in distinguishing
species for many eukaryotic taxa, they have not been so for Fusarium spp.
(O’Donnell and Cigelnik 1997).
Need for an efficient diagnostic tool.—Adaptive variation can quickly
arise within any pathogenic fungal species when host-related ecological
circumstances change, as when new or modified agricultural methods of
disease control change. Natural selection can use such variation to create
new adaptive intraspecific forms, which can split and diverge further into
more forms. Adaptive breakthroughs can be the result, along with new
adaptive pathways that complicate the infraspecific taxonomy of the species.
Such complexity can be difficult to sort out and to keep track of it. Yet
knowledge and means of monitoring the infraspecific f. spp. and races of a
pathogenic fungal species can be critical to studying the ecology and
population-genetics associated with host-pathogen interactions. Being able
to distinguish accurately and routinely between f. spp. (such as Fol and Forl)
can thus be important in successful disease control for a plant species (such
as tomato).
Finding a diagnostic tool.—Although many physiological, biochemical,
or genetic differences between Fol and Forl have been found, none thus far
has provided a convenient, efficient diagnostic tool. The osmotic method,
complemented by high performance liquid chromatography “HPLC”
(Knudsen 1982; Joelsson 1983; and Engström et al. 1993), has yet to reveal
differences between Fol and Forl. In this study, the fungal exudates from the
Fol-(CBS 165.85) and Forl-SW isolates produced no distinguishing
differences, indicating that the metabolisms of Fol and Forl are perhaps
basically the same.
In addition, no genetic differences between the Fo-(IMI 386351) strain
and other Fol isolates were found (i.e., they produced the same Fol mtDNA
pattern), with the exception of a minor but distinct difference found for Fol(CBS 165.85). However, the Fo IMI 386351 endophytic strain (Attitalla et
al. 1998) did produce a HPLC-definable biochemical feature that separated it
from the other Fol isolates. This feature may be the consequence of a
compound that causes special effects when the isolate interacts nonpathogenically with the host plant.
Isozyme analysis.—This procedure is commonly used in population
genetics studies of fish (Allendorf et al. 1977; May et al. 1979), mammals
(Wheeler 1972), insects (Yang et al. 1972), nematodes (Huettel et al. 1983),
and plants (Cinkle et al. 1981)—as well as in fungi (Vogler et al. 1996;
Banniza and Rutherford 2001). Isozyme analysis, conducted with starch gel
electrophoresis, can be used to study fungal taxonomy and genetics (Micales
1986; Zervakis et al. 2001).
Electrophoresis banding patterns reflect the genetic and nuclear condition
of the organism. The ambiguous results of early isozyme analysis of fungi
(Shecter 1973) originally discouraged the application of this technique. But
27
since then, it has been used to quantify genetic variation in Agaricus (Royse
and May 1982), Entomophora (May et al. 1979), Neurospora (Spieth 1975),
Perononsclerospora (Bonde et al. 1984), Phytophthora (Tooley et al. 1985)
Puccinia (Burdon and Roelfs 1985) Fusarium spp. (Patel and Anahosour
2001). Recently Aly et al. (2003) used multi-locus enzyme and protein gel
electrophoresis to discriminate five Fusarium spp. Their data revealed that
sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) and
estrase isozymes are more efficient in grouping isolates, whereas POX and
malate dehydrogenase isozymes have more limited resolution in organizing
isolates into their respective species-specific clusters.
Isozyme analysis can be used in at least three major areas: the
clarification and delineation of fungal taxa, the identification of fungal
cultures to the species, and the study of genetics, including population
genetics, of specific fungi. Clarification and delineation of taxa—the
interpretation of banding patterns in term of specific alleles—allows the
determination of ratios of alleles expressed in common among fungal
isolates. These ratios are excellent means of determining phylogenetic
relationships among organisms.
As a means of distinguishing between “electrophoretic types”, isozyme
analysis has been employed as an important method for studies of fungal
taxonomy, especially when morphological characters are insufficient to
distinguish between samples (Micales 1986). However, below the species
level of fungi, isozyme analysis seems to lack sensitivity, except in few
cases, such as Phytophthora spp. (Oudemans and Coffey 1991) and
Aphanomyces spp. (Larsson 1994).
Taxonomic clarifications corroborate this limitation of isozyme analysis.
For example, the two enzyme systems, G6PDH and MDH (used in this
study), yielded different banding patterns (Rataj-Guranowska and Wolko
1991) when tested on strains that were thought to be different infraspecific
forms of Fo, F. oxysporum var. redolens and F. oxysporum f. sp. lupini.
However, F. redolens (now given species status) is apparently not a member
of the Fo complex, based on the ribosomal DNA (rDNA) internal transcribed
spacer region and gene sequence analyses. In fact, gene genealogies indicate
that F. redolens is not even within a sister group of the Fo complex
(O’Donnell et al. 1998; Baayen et al. 2000).
Such facts seem to indicate that enzyme-based banding-pattern
differences in fungi may generally be a product of evolutionary divergence
that results in speciation. Any divergence that stops short of speciation, such
as divergence leading to stable f. spp., can often be insufficient for the
development of variation in enzyme mechanisms that produce significant
differences in banding-patterns. In other words, the process of speciation (as
opposed to that for creation of f. spp.) may commonly involve substantive
divergence of enzyme systems that ecologically have resource-specific
28
consequences, and that diverge enough to produce clear differences in
isozyme banding patterns.
Should this be true, then rather than seek differences in isozyme banding
patterns between Fol and Forl, one should perhaps seek conserved genetic
differences between the two f. spp. Such difference can be discrete without
there being recognizable differences in isozyme banding patterns. However,
although PCR/(genomic RFLP) methods as described by Edel et al. (1996)
provide a simple and rapid procedure for differentiation of Fusarium spp.
strains at the species level, they have failed so far to provide those features
below that level.
Anomalies.—The mtDNA profile (Paper I) for the seven Forl isolates
(including Forl-SW) was clearly distinguishable from the profile of the five
Fol isolates including Fo-(IMI 386351). Nevertheless, the banding pattern of
one of the Fol-(CBS 165.85) was peculiar in that, just as Forl-12 and Forl-13
had one or two distinct differences from the basic Forl pattern (as detailed
above), the banding pattern of Fol-(CBS 165.85) likewise had only a couple
of distinct differences from the Forl pattern.
We have no simple explanation for why this was true only for Fol-(CBS
165.85) and untrue for all the other Fol isolates, whose patterns were
fundamentally different from that of all Forl isolates, assuming that the Fol(CBS 165.85) isolate is, by evolution, a Fol instead of Forl isolate.
Sequencing of other genomic regions of Fol-(CBS 165.85), and detailed
examination of the pathogenic mechanisms and properties of Fol-(CBS
165.85), will probably resolve this seeming paradox of evolution.
The mtDNA profile for the non-pathogenic isolate Fo-(IMI 396351) was
also interesting. It had the typical features of the Fol profile, with the
exception of Fol-(CBS 165.85). This result for Fo-(IMI 386351) is consistent
with its identification by O’Donnell (self communication), based on the
sequencing of several genomic regions.
Possible origins of Fol and Forl
Our results (Paper I) provide some clues as to the evolutionary origin of Fol
and Forl, as f spp. of Fo, and the maintenance of their genetic and
phenotypic differences. Questions, therefore, about the origin, evolution, and
ecology of Fol and Forl, and about the functional significance of the single
mtDNA band difference between Fol and Forl, deserve brief mention.
Perhaps all Forl isolates share the mtDNA band not present for the Fol
isolates in present study. If so, perhaps Fol and Forl arose through a single
evolutionary subdivision, and since that time (starting from the initial
subdivided population) both have spread worldwide. Alternatively, Fol and
Forl may have arisen during several independent evolutionary events; but
why (or how) then have all of these independent within-population
divergence processes yielded the same band difference between Fol and
29
Forl? Presumably, geographically widespread locations have significantly
different selection pressures.
The third possibility is that Fol and Forl are genetically the result of two
sets of co-adaptive alleles, with each set having an environment-specific
selection advantage over the other. For any given locality, as the numbers
and frequencies of alleles of the favoured set increase, the selection
advantage for the other alleles within that set might increase. The increase
may be due to a local selection advantage facilitated by competition arising
from some niche overlap of the two f. spp. If this is the case, then finding Fol
and Forl in the same locality could be unlikely. Thus, starting from an almost
undetectable founding population within a given locality, natural selection
might so rapidly eliminate one or the other set of morph-determining alleles
that, by the time the population reaches a noticeable size, only one or the
other set (Fol or Forl) is present.
30
Fusarium oxysporum and Lycopersicon
esculentum –L. (an extensive relation)
Details of the relation
Defence reactions observed in Lycopersicon esculentum –L., when colonized
by non-pathogenic strains of Fo, appear to be essentially those of other plant
species infected by compatible or incompatible f. spp. of Fo (Charest et al.
1984; Brammall and Higgins 1988; Tessier et al. 1990; Pharand et al. 2002).
Using histochemistry and immunocytochemistry to determine the
chemical nature of the substances that accumulated in the cells and the
intercellular spaces of infected tomato plants, callose and phenolic
compounds are the most frequently cited substances. These two compounds
amass in the cells and the intercellular spaces of infected tomato plants
(Fravel et al. 2003). In response to plant infection, these compounds appear
in both susceptible and resistant varieties of tomato plants. Usually they
accumulate more, and more rapidly, in resistant than in susceptible varieties
(Jordan et al. 1988; Benhamou et al. 1991; Shi et al. 1992). Colonization of
resistant cultivars by fungi is apparently limited by such defence responses,
which generally disallow invasion of the vessels.
Perhaps then the defence response in tomato plants (of susceptible
varieties) infected by a nonpathogenic strain of Fo are liken to those in
tomato plants of resistant varieties colonized by a pathogenic strain of Fo
(Olivain and Alabouvette 1997). They state that an intense colonization of
the root surface by a nonpathogenic strain of Fo can quickly occur after
inoculation, and may produce a physical barrier that prevents direct contact
of the pathogenic strain of Fo with the root surface. Such colonization by a
nonpathogenic strain probably entails intense competition for root exudates.
Upon being denied access to such nutrients, propagule germination for the
pathogenic strain might be curtailed. But because colonization of the root
surface is never complete, some noncolonized areas of the root surface might
allow the pathogen to reach the root in any case.
In the case of nonpathogen strains of Fo, a plant defends itself from
invasion more by the thickening of cell walls and by intracellular plugging.
A similar defence response was observed in transformed pea root, when
confronted with a nonpathogenic Fo strain (Benhamou and Garand 2001).
31
That response prevented the nonpathogen from reaching the stele, but the
pathogenic strain grew quickly towards the vessels, which were invaded.
Antagonism by a fungus or a fungal strain (e.g., a biological control
agent) can reduce disease symptoms for plants when caused by a pathogenic
fungus or strain. Such as mechanisms of direct antagonism include
parasitism, antibiosis, and competition. Attitalla et al. (1998), by means of in
vitro and in vivo experiments, showed that a Fo strain Fo IMI 386351, has
the potential to antagonize Fol in selected culture media, such as PDA (see
Fig. 3), and can suppress Fol when applied to soil around the root system. In
our previous study, we speculate that SIR was the fundamental mode of
action for disease suppression, however, other mechanisms may well have
been at work also. By definition, endophytic fungi, living asymptomatically
in plant tissues, constitute a valuable source of bioactive secondary
metabolites for potential therapeutic use (Lingham et al. 1993; Dreyfuss and
Chapela 1994; Bills et al. 1994). Fo IMI 386351, which was detected in the
green part after treatment (Paper II), presumably acts as an exogenous
endophyte.
Inhibition zone
Fol
Fo-(IMI 386351)
Fig. 3: Illustrate antagnostic activity by Fo IMI 386351 in culture media.
Fo IMI 38635 definitely produces secondary metabolites, which are
oligosaccarides, preliminarily identified by nuclear magnetic resonance
(NMR) spectra. Briefly, osmotic methods were applied (Paper I) to find a
rapid method for differentiation between Fol and Forl. We included Fo IMI
38635 in that study, because the PCR based method described in (Paper II)
suggest that Fo IMI 38635 might be a member of Fol group, as confirmed
using a mtDNA RFLP method (Paper I). Using the osmotic method, we also
observed that all Fusarium spp. including Fo strain produced a typical peak
which can be helpful in distinguishing between Fo IMI 38635 and other
fungal species (unpublished). In that study, we included several Fo strains
and three methods, described in detail in (Paper I). We observed no
differences between those strains, by isozyme analysis or by the osmotic
method (revealed by HPLC) methods. We then decided to exclude those
strains from the (Paper I) and to focused only on Fol-(CBS 165.85) and
32
Forl-SW, and to include more Fol and Forl in the third method analysis,
mtDNA RFLP. Two fractions of Fo IMI 38635 obtained by HPLC were
investigated by means of NMR spectroscopy, as will now be explained.
Signal transduction.—NMR proceeded as follows. Material of both Fo(IMI 386351) fractions (less than 0.1 mg) was dissolved in 140 µl MeOD
and 1H NMR on a Bruker 600 MHz. The signals from the protons in the
sample indicated that the compound was an oligosaccharide, but due to the
small amount of the compound used, the structure could not be determined
by NMR, so we repeated the methods after obtaining more material.
Interestingly, the signal was probably that of an oligosaccharide because
plants have the ability (a) to recognize some oligosaccharides or their
derivatives as a potent biological signal and (b) to initiate various cellular
responses that lead to important biological phenomena such as defence
reactions or differentiation (Ryan and Farmer 1991; Ebel and Scheel 1997).
Albersheim et al. (1987) proposed the name "oligosaccharins" for such
biologically active oligosaccharides. It seems reasonable such
oligosaccharides, produced by a fungus, serve as signal molecules by which
plants are distinguish their tissue from the fungus, and to initiate defence
responses. These polymers, which do not exist in plants, are widely
distributed among various fungi, including pathogens. Various plant cells
can respond to fragments of chitin, chitosan, or ȕ-1,3- glucan, ȕ-1,6-glucan
and thus can initiate defence reactions. Research is still needed to explore the
molecule under investigation and to demonstrate the role of that fraction in
SIR expression (Shibuya–self communication). The molecular machinery
involved in the perception and transduction of oligosaccharide signals in
these systems has been studied (Ryan and Farmer 1991; Ebel and Scheel
1997).
Oligosaccharides (Bishop et al. 1981) and oligogalacturonides (Doares et
al. 1995; Norman et al. 1999) released from damaged cell walls might play a
role in the elicitation of the general wound response (Heil and Bostock
2002). In some cases, the receptor molecules for the oligosaccharides have
been purified, and their genes cloned. Fragments of pectin
(oligogalacturonides, OGA) can also induce defence reactions in plants
(Cote and Hahn 1994). In this case, the degradation of the cell surface of the
host plant by invading pathogens can be interpreted as generating the
warning signal (Ryan and Farmer 1991). Some plants have been shown to
produce a polygalacturonase-inhibiting protein, which may serve to prevent
further degradation of OGA by pathogenic polygalacturonase to maintain the
suitable size of OGA as the warning signal (Shibuya-self communication).
Plants provide interesting and excellent model systems for the study of
glycobiology, especially because simple oligosaccharides rather than
glycoconjugates function as a signal molecule in these systems. These
studies also raise interesting questions regarding the relationships between
defence responses and development/differentiation, as seen in the case of
33
OGA as well as chitin/lipochitin oligosaccharides (Darvill and Albersheim
1984; Dixon et al. 1994).
Evidence for SIR in plants
Inoculating the lower leaves of a tomato plant with Phytophthora infestans,
the causal agent of late blight, increased the immunity to this pathogen
(Heller and Gessler 1986). The result was resistance expressed as smaller
and fewer necrotic lesions, reduced sporulation, and delayed or inhibited
penetration into the epidermis. Yan et al. (2002) applied two plant-growth
promoting rhizobacteria (Bacillus pumilus SE-34 and Pseudomonas
fluorescens 89B-61) and one chemical inducer to tomato plants. SIR was
obtained in various ways. Several other elicitors were used to enhance SIR in
tomato against Fo, and to some degree, SIR occurred. Recently, Siddiqui and
Shaukat (2004) found that fluorescent pseudomonads stimulate SIR against
root-knot nematode via a signal transduction pathway, which is independent
of SA accumulation in roots.
SIR is one of the most important mechanisms for protecting plants (see
review by Cao Ke-qiang and Forrer 2001; Kuü 2001). In case of tomato wilt,
SIR can be an important mechanism for disease control. The recent key idea
is to elicit SIR in tomato plants by various systems of in vivo application.
The goal is to find a promising in vivo system of application that merits
proceeding with the next step—field trials. So far, few field trials have been
run to test the application of SIR as a means of disease control in agriculture.
One example, however, has already led to commercial application in Japan.
In these field trials, stem cuttings of sweet potato (Ipomoea batatas) were
challenged with a high-level of inoculum of a non-pathogenic Fusarium sp.,
and then planted at field sites, where they showed increased resistance to
infection by the vascular wilt disease Fusarium oxysporum f. sp. batatas
(Komada 1990). The method is practical because high agricultural subsidies
in Japan make it economic to transplant sweet potato cuttings. Immunization
has been successful in both the laboratory and field (Caruso and Kuü 1977;
Tuzun and Kloepper 1995).
Others have also tried to make the SIR system applicable. Kuü and his coauthors did an experiment in the field to obtain SIR in cucumber against
Colletotrichum legumenarium, the causal agent for Anthracnose in
cucumber. To some extent, the plant was immunized when the same fungus
was used as an elicitor. The experiment, as far as I know, was never
completed, but the idea of applying SIR in the field makes sense. I feel that a
wide range of specific conditions are needed to establish the SIR as a
significant component of plant defence in many plant-pathogen-systems.
Descalzo et al. (1990) pointed out that considerable skill, time, and labor are
34
required in field applications of plant immunization, making such
applications expensive.
In tomato plants, many compounds have been described that can or might
be responsible for enhanced resistance to Fol: for example, POXs and
polyphenoloxidases (Abbattista et al. 1988) and E-1,3-glucanases and
chitinases (Retig 1974; Carrasco et al. 1978; Ferraris et al. 1987; Tamietti et
al. 1993). The development of the disease caused by Fol was noticeably
limited in tomato plants inoculated with the mycorrhiza-producing fungus
Glomus mosseae (Dehne and Schönbeck 1975). Chlorosis was less
pronounced on the leaves of mycorrhizal plants. Additionally, the rate of ion
leakage from the stem and leaf cells of induced plants was less than that
observed in controls after challenge inoculation with Fol. This report also
noted that phenol metabolism had changed, which was reflected in enhanced
deposition of lignin in the cell walls of the induced host. The induction
causes physiological conditions that activate or enhance natural resistance
mechanism; such conditions constitute the establishment of SIR in the plant,
a physiological activity in the plant discussed earlier and extensively
reviewed by (Agrawal and Karban 1999; Karban et al. 2000, Montesinos,
2000; Gatehouse 2002).
Although the degree of protection is not absolute, in tobacco (Nicotiana
tabacum –L.), however, the plant for which induced resistance has been
most studied, disease reduction can reach 90% in field tests and can last for a
growing season (Tuzun et al. 1986).
The systemic development of resistance after induction implies that a
signal (or signals) in the plant leads to the activation and coordination of
resistance mechanisms (Dean and Kuü 1986a, b; Malamay and Klessig
1992). Endogenous signals associated with SIR include a peptide termed
systamin (Pearce et al. 1991), a fatty acid termed jasmonic acid (Farmer et
al. 1992 and Cohen et al. 1993), electrical potential (Scott 1967), and
salicylic acid (Gaffney et al. 1993). How the signal(s) are recognized,
mediated, and transported in the plant is not understood, however. Our
research confirmed that SIR play important role in protecting tomato plants
from the Fol (Paper II, III, IV). Elicitation of SIR commercially in
Solanaceae plants is unlikely to replace pesticide application and artificial
selection of major genes for resistance, but such elicitation will reduce the
detrimental side effects of pesticides and of manipulated genes and genomes
(Strömberg 1994).
As a phenomenon that utilizes natural resistance mechanisms involving
the plant as a whole, SIR has only been partially explored. The goal of
scientific application of SIR is responsible combinations of the old and new
techniques for plant disease control, techniques that will ensure that the
necessary conditions for activation of natural defence systems in plants are
present. Thus, SIR provides an additional technique to be used in
35
combination with other control measures. SIR might become one factor to be
considered in the development of future integrated control programs.
Ways of demonstrating microbe-elicited SIR
The invocation of SIR in plants is not determined by the presence or absence
of genes for resistance mechanisms (Quintanilla and Brishammar 1998);
rather, it determined by the speed and degree of gene expression and the
activity of the gene products. All plants likely have the genetic potential for
induced resistance. Plant’s can express such potential (Paper II and III) as
immunization—after restricted inoculation with pathogens, attenuated
pathogens, inoculation with a selected non-pathogen, treatment with
chemical substances that are produced by immunized plants, or chemicals
that release such signals. To provide a strong argument that an occurrence of
significant reduction in disease symptoms is the result of SIR induced by an
eliciting agent such as a microorganism, one can proceed experimentally in
several ways.
The basic experimental means of providing a strong argument for SIR as
the cause of reduced disease symptoms is to divide a set of plants (seeds or
seedlings) processed under the same conditions into four treatments (one
primary treatment and three controls). The primary treatment is exposure of
plants to the biological control agent (B), and then some time later, exposure
of the plants to the pathogen (P). The control treatments are as follows: Pcontrol plants (pathogen-control plants) are only exposed to the P. The
same holds for the B-control plants; plants are only exposed to the B. For
the healthy-control plants, the same procedure is carried out except for
leaving out exposure to the B and to the P.
If disease symptoms (caused by a P) are significantly fewer or of lower
degree for the B+P- plants than for the P-control plants, then (assuming the
experimental procedure is properly carried out) one can draw the following
conclusion.
x Conclusion 1. B is ultimately responsible for the reduction in
disease symptoms.
One then has the possibility of showing that the disease-symptom
reduction is due to SIR elicited by the B and not to something else, such as
direct interaction between the B and the P. That is, the question arises then
as to whether the B causes a systemic physiological response in the plant,
and that response is responsible for the reduced disease symptoms in B+P
plants relative to the P-control plants. If one can show experimentally that
the reduced disease symptoms for B+P plants cannot be the result of direct
interaction between the B and the P, then one can draw a penultimate
conclusion.
36
x Conclusion 2. B causes a physiological response in the plant and
that response is directly responsible for observed reduction in
disease symptoms.
The induced resistance obtained can be local and not necessarily systemic. If
one then can show, however, that the plant’s response to B is systemic, one
then one can draw the ultimate conclusion constituting proof of SIR.
x Conclusion 3: SIR is responsible for the observed reduction in
disease symptoms.
One way of constructing an experiment that makes drawing all three
conclusions possible is that of a split-root experiment. One physically splits
the root of all experimental plants and applies B to one split and P to the
other, so that direct interaction between B and P is physically impossible for
those plants. If then the B+P plants have demonstrably lower disease
symptoms than P-control plants, conclusion 1 can be drawn. And so can
conclusions 2 and 3, if the splitting of the root did not itself induce the SIR.
But the latter possibility can be ruled out if disease symptoms of P-control
plants with split roots and P-control plants whose roots are not split are not
significantly different.
One way of constructing an experiment that makes drawing all three
conclusions possible might be a split-root experiment. One physically splits
the root of all experimental plants and applies B to one split and P to the
other, so that direct interaction between B and P is physically impossible for
those plants. If then the B+P plants have demonstrably lower disease
symptoms than P-control plants, conclusion 1 can be drawn. And so can
conclusions 2 and 3, if the splitting of the root did not itself induce the SIR.
But the latter possibility can be ruled out if disease symptoms of P-control
plants with split roots and P-control plants whose roots are not split are not
significantly different.
However, for a given experimental procedure and its reproducible results,
many events and circumstances can point to SIR as the mechanism for
reduction in disease symptoms in B+P plants (relative to P-control plants).
We ran three experiments in which experimental procedure combined with
experimental results indicate that SIR was an important, if not the major
contributor, to reduced DI. The basic arguments for SIR in those three
experiments will now be presented.
For each experiment, after mentioning the particular B and P used in the
experiment, the abbreviations B and P will be used to present the arguments,
instead of using the names of the actual biological control agent and
pathogen. We do this with the idea of focusing on the argument, and
accordingly on types of experiments and corresponding experimental results
that indicate SIR as the mechanism of plant defence. Note though that for all
three experiments, the plant was tomato and the pathogen was Fol; only the
biological control agent was different for each experiment, along with some
important details of the experimental procedure. For several reasons,
37
including explanatory reasons, each experimental tomato plant was thought
of as having four spatially distinguishable regions of tissue: the roots, the
stem, the petioles, and the leaves.
Experiment 1 (Paper III)
In this experiment, B is Phytophthora cryptogea (Pc).
The basic rationale for SIR.—B+P plants have significantly less disease
symptoms than P-control plants, so conclusion 1 (see above) could be
drawn. The petioles of B+P plants never had detectable amounts of B or P,
and yet in P-control plants, that region did eventually have detectable
amounts of P; and similarly, in B-control plants, that region had detectable
amounts of B. B was apparently the ultimate cause of the absence of P in the
petioles of those plants. The absence of P was either due to direct interaction
with P in the stems of plants, or to SIR triggered by exposure to B. If direct
interaction were the case, one would expect that B+P plants would have
detectable amounts of B in the petioles, as did B-control plants, unless B
and P, in direct interaction in the stems, mutually denied each other access to
the petioles in B+P plants. But that seems improbable because the B was
applied to the leaves of the plants, and found there for the B-control plants.
These results strongly support conclusion 2. The fact that the B was applied
to the leaves of plants, but P was applied to the soil around the roots of B+P
plants, and yet tomato wilt was observed to be significantly suppressed
compared to P-control plants, indicates that active and effective plant
defence activity occurred in the stems and probably the roots of B+P plants.
Exposure to B in the leaves of the plants apparently caused or triggered the
plant defence: that is, such defence was not observed in P-control plants.
These results provide strong support for validly drawing conclusion 3.
The details of the results, as given more precisely below, suggest a two
step process of SIR in the tomato plants, as follows. Exposure to B triggered
SIR: that is, triggered a systemic response in the form of preparedness in
B+P plants and B-control plants. But in B+P plants, when they were
exposed to P at the region of the root, after exposure to B on leaf surfaces,
the B-induced SIR in the plant was converted from a state of preparedness to
one of active defence. The plant’s active defence not only denied P access to
the petioles, but also reduced B in the leaves to an undetected level. This
“SIR explanation” is corroborated by delayed colonization of P in the stems
of B+P plants, relative to P-control plants, even though amounts of B in
the stem were not detectable during the occurrence of the delay.
The experiment, the results, and the argument for SIR in more detail.—
The experimental procedure and results are as follows:
x The green parts (stem and leaves) of B-control plants and B+P
plants are exposed to B.
38
x Later, the roots of P-control plants and B+P plants are exposed
to P.
x B+P plants have significantly less disease symptoms than Pcontrol plants.
x P was never found in detectable amounts anywhere in B+P plants.
x At time t, P is first found in the stems of P-control plants.
x Then, somewhat later, at time t+x, P and B are found in the stems
of B+P plants, but not in the petioles.
This indicates that colonization of stems by P was delayed in B+P plants
by an interval x (relative to t in the P-control plants), and that somehow,
exposure of stems, leaves, and petioles to B in B+P plants was responsible.
Because B was not found in the stems of B+P plants during time interval t
to t+x, either B induced SIR, which was responsible for delaying stemcolonization by P, or undetectable amounts of B in the stem interacted
directly with P in the stem, causing the delay.
x However, B was found in the petioles of B-control plants
beginning at time t+x+y, but was never found in the petioles of
B+P plants.
x P was found in the petioles of P-control plants by time t (the
same time as it was found in the stem), but P was never found in
the petioles of B+P plants.
Apparently, exposure of B+P plants to B was ultimately responsible for
P never being found in the petioles of those plants, and because exposure of
B to those plants apparently triggered a physiological response in B+P
plants, such that exposure of those plants to P caused a further physiological
response that did not occur for B-control plants. That physiological
response of the plant, first to B-exposure to its leaves, was apparently
systemic in that it caused P, when exposed to the roots, to set off a more
active physiological response that not only resulted in reduction of disease
symptoms, but also reduction in level of B in the leaves, which again
indicated that the response was systemic. That is, the shift to a more active
response disallowed B to accumulate in the petioles of B+P plants in
detectable amounts). In short, this means that for B+P plants, there were two
phases of SIR. Exposure to B near the time of B-application triggered a
response that established a state of systemic plant-resistance activity in the
form of preparedness for active defence, which was presumably present in
B-control plants as well. Then, at least two days later, exposure of the
plants to P triggered a conversion of the first and current SIR activity (or
state) to a second, which took the form active plant defence, a form that did
not apparently occur for B-control plants, nor for P-control plants. That is,
P was found in the petioles of P-control plants by time t—at the same time
as found in the stems of those plants—but P was found nowhere in B+P
plants.
39
In other words, reduction in disease symptoms in B+P plants for a
colonization experiment of the sort defined above demonstrates a two-step
process for SIR. B-exposure triggers a first SIR state, that of preparedness
for active plant defence, and later, that first SIR state entails physiological
conditions for that plant that cause P to trigger a second SIR state in B+P
plants, when the roots of those plants are exposed to P. The second SIR
state takes the form of active defence.
Experiment 2 (Paper IV)
In this experiment, B is Pseudomonas sp. (strain MF30) bacterium strain.
The basic rationale for SIR.—B is applied to the soil around the roots of
the plant, and thus is able to make contact with the plant’s root surfaces.
However, B never enters the plant, based on colonization observations. Since
disease incidence is significantly reduced for B+P plants relative to Pcontrol plants, conclusion 1 can be drawn: that is, in one way or another,
exposure of the soil around the roots of plants to B was the ultimate cause.
Shoot dry weight of B-control plants is reduced relative to healthy-control
plants, meaning that exposure of root surfaces to B for B-control plants and
B+P plants did trigger a systemic response of some sort in those plants,
which may or may not have been responsible for the reduction of disease
symptoms of B+P plants relative to P-control plants.
If the disease-causing features of the phenotype of P cells in B+P plants
is different from those in the P-control plants, then that difference might be
responsible for the difference in reduction of disease symptoms in the B+P
plants relative to P-control plants. Any such phenotypic difference would
have to be under genetic control, because otherwise, the difference would
disappear almost immediately (i.e., such a difference would be absent for
generations of pathogen cells in the plant beyond that of the cells that
entered the plant). But genetically controlled virulence features between the
P cells in B+P plants and the P cells in the P-control plants would have to
have been established outside the plant (in the soil), because the B cells did
not enter the plants, or if some did, the level was undetectable. But
establishment of genetic differences between P cells in B+P plants and the
P-control plants due to direct or indirect interaction between P cells and Bcells outside the plant, in the soil, seems highly unlikely. Somehow, the B
cells in the soil would have to create environmental conditions (through
production of anti-microbial compounds, changes in availabilities of
resources, or other environmental factors). Those conditions would have to
(1) increase rates of mutation for specific mutant virulence-related genes,
and (2) provide those genes with a sufficient selection advantage, within the
population of P cells within soil, to quickly spread throughout that
population. From the standpoint of population genetics, that is an enormous
requirement for an organism to make specific changes in soil conditions for
40
other resident organism, regardless of the fact that microbes in the soil can
have fast generations times relative to that of most other organisms.
Imagining possible selection pressures required for creating the needed
mutator alleles and for providing the appropriate virulence-related mutations
with a sufficient selection advantage—that is, high selection pressures
specific to a given soil environment—is indeed difficult, especially
considering requirements for overcoming overwhelming hurdles of
population genetic time tables (See Appendix).
In other words, the possibility of the P cells that first enter the plant (i.e.,
those P cells that effectively end up colonizing the plant due to their
numerical dominance in the plant) being mostly mutants due to conditions
(outside the plant) created by the B cells in the soil seems remote. A postrecolonization experiment to see whether P cells of the two sets of plants (Pcontrol plants and B+P plants) do in fact behave differently, based on
genetic differences, was not done because the results of experiment 2 (Paper
IV) were not anticipated.
Taking account of these matters seems to leave only one other possibility
for not drawing conclusion 2, as follows. At least one of the P cells that
entered the plant early acquired a mutation due to a brief exposure to
environmental conditions outside the plant caused by the presence of B cells
in the soil. That mutant, once in the plant at a low frequency, happened not
only to produce a non-virulent phenotype, but also a phenotype that provided
a high selection advantage over the other genetically unperturbed virulent P
cells that first entered the plant, and which were in the process of colonizing
the plant. Although such a scenario is possible, theoretically, it seems to be
far-fetched and unlikely, for many reasons, but especially the fact that the
same unlikely mutation of an early-entering P cell, or similarly unlikely
mutation, would have had to have occurred for all the B+P plants in the
experiment, a statistically unlikely coincidence.
Having now essentially eliminated the only apparent possibilities for not
drawing conclusion 2, that conclusion seems to be valid. If conclusion 2 is
drawn, then conclusion 3 follows, since B apparently did not enter and
colonize the plant, and therefore was unable to retard the development of
disease within the plant by direct (or indirect) interaction. Hence, only the
plant—by means of SIR induced by B—was apparently responsible for
defence against P. The B cells (in the soil) presumably made contact with
the root surfaces of the plants, and that contact induced SIR.
There is, oddly, yet another possibility, but that possibility leads again to
conclusion 3. Perhaps interaction of P cells with B cells outside the B+P
plants results in a reduction in rate of entry of P cells into the plant—enough
reduction for the plants to have had time to develop sufficiently a systemic
physiological response to early contact with the P cells at the plant’s root
surfaces. That time, due to reduced rate of P-cell entry, was not available to
P-control plants. Such delay in P-cell entry for B+P plants might have
41
allowed early contact with P cells not only to have triggered SIR in B+P
plants, but also to have allowed the SIR to develop sufficiently to be able to
act effectively in defence against P. As mentioned, this scenario also leads to
conclusion 3, but in this case, the plant’s contact with P, instead of with B,
triggered SIR—although B is still ultimately responsible for the SIR.
This possibility for induction of SIR, due to the reduced rate of entry and
colonization by P, due to agonistic interaction of B with the P in the soil, or
on the root surfaces of the tomato plants, deserves further consideration.
Reducing the rate of entry by one half will delay colonization by about one
doubling time, on average. Presumably, the doubling time was at least 5 h,
and more likely 10 h or more, in that doubling times in the soil are generally
very slow relative to in the laboratory. Reducing the rate by three-fourths
would delay colonization by about two doubling times, and so forth, which
could easily be a day or more. But even a delay of a few hours could have
been sufficient to allow the B+P plants to establish a temporal edge over P,
sufficient to allow full development of SIR, and consequently, suppression
of the ability of P to cause observable disease symptoms.
In fact, plant genotypes that have a superior capacity to take advantage of
reduced rates of P-entry, to cause P cells to be responsible for triggering SIR
against themselves, have a selection advantage, all else being equal. Hence,
plants may well have genetically determined thresholds for rate of pathogen
entry required to cause disease. Any rate below the threshold allows the
plant to make use of initial contact with P in triggering SIR, and thereby
avoiding disease. Indeed, many interpretations of experimental results that
clearly provide evidence that direct interaction between B-cells and P-cells
in the soil was responsible for reduction in disease symptoms may well
overlook the fact that in many, and perhaps even most of those cases, SIR
rather than direct interaction was the immediate cause of the reduction in
disease symptoms. Hence, the role of SIR in plant protection against disease
may well be considerably underestimated. That is, although direct interaction
between B-cells and P-cells within the soil is observed, in many of those
cases, and perhaps in most, such interaction is a means of allowing the plant
to take advantage of initial contact with P-cells to trigger SIR against those
cells. In other words, in such cases, B, instead of itself triggering SIR, it does
so indirectly, by causing P to do the triggering—by delaying P’s rate of
entry.
In other words, below a genetically determined threshold for rate of entry
of P-cells, P ends up triggering SIR, which then suppresses or eliminated its
own ability to cause significant disease symptoms. Should this be true, then
non-pathogenic microorganisms in the soil, by sufficiently reducing rates of
pathogen entry into plants, can cause a soil to be suppressive—but
mechanistically, the direct cause of the suppression is SIR, and not direct
interaction with B, which nonetheless is ultimately the ultimate cause.
Should this be true, then suppressive soils include an intrinsic stability for
42
suppressiveness: that is, pathogen genotypes are not entirely denied means of
living and survival by means of entering plants, and therefore are able to
persist in suppressive soils, even though plants exhibit resistance.
Consequently, selection pressures on pathogens to create mutants that reduce
soil suppressiveness are diminished, and soil suppressiveness is intrinsically
self-perpetuating. In this respect, the soil in the pots of B+P plants in
experiment 2 (Paper IV) might represent a microcosm of the basic
mechanism of soil suppressiveness, and experimentally an ideal case in the
sense of being simple and experimentally tractable.
The experiment, the results, and the argument for SIR in more detail.—In
our in vitro experiment, there was no effect on the radial growth of Fol by
the eight selected bacteria when grown in dual cultures for different media
(Wei et al. 1991; van Loon et al. 1998). Although such results lend some
support for SIR in the climate chamber experiment, as van Loon et al.
explain, even when inhibition is not observed in culture, direct inhibition of
the pathogen in the rhizophere cannot be excluded: conditions in the
rhizoshere differ from those on artificial media, and an antibiotic produced
exclusively in planta may go undetected.
But the argument presented above for the likelihood of SIR being
responsible for suppression of disease symptoms entails the in vivo
experiment and not the in vitro experiment. Here now in outline is the in vivo
experiment.
x B is applied to the soil around the roots of plants.
x Later, P is also applied to the soil.
x B+P plants have significantly less disease symptoms than Pcontrol plants.
x Shoot dry weight of B-control plants is reduced relative to
healthy-control plants.
x No B is found within B-control plants and B+P plants.
x P-colonization and vertical spread within the stem is substantially
reduced in B+P plants relative to P-control plants.
The P cells that colonize the B+P plants were likely unchanged
genetically (and phenotypically) because the first P cells entering the plant
were those that upon inoculation were immediately in contact with the roots.
The early-contact P cells probably entered plants almost immediately, with
little or no chance of being significantly exposed to B cells in the soil or
around the root surface, and were therefore unlikely to be genetically
modified by changes in soil environment caused by B cells. One can show
with simple population genetics that in general the genotypes of the first P
cells entering a plant will generally be able to double several times before
most other cells have entered, and therefore can be expected to be dominant
numerically within the plant from start to finish. This statement holds insofar
as doubling times within a plant are essentially the same for all cell descent
lines within a plant, allowing then the decent lines of those P cells that first
43
enter the plant to maintain their initial numerical dominance. Hence, the
descent lines of P cells that have spent enough time in the soil to be
genetically modified by B can be expected to be at a strong numerical
disadvantage to P cells that entered almost immediately. That is, in general,
the descent lines of mutated P-cells will in general gain access to the plant
long after non-mutated P-cells have acquired a sustainable numerical edge
for colonizing of the plant. Therefore, any mutated P cells that are avirulent
due to selection pressures caused by B cells must have a considerable
selection advantage over the non-mutated virulent P-cells. Otherwise they
will have no possibility of overriding the expected development of disease
initiated and driven by the colonizing non-mutated P-cells that will continue
to colonizing the plant at a rate normally sufficient to overpower any plant
defence that the plant is able to muster without SIR. The odds seem to be
stacked against such a scenario. Thus conclusion 2 seems to be valid, and
conclusion 3 then follows because B-cells are presumably not in the plant to
interact with the B-cells, or so few in number as to be undetectable, leaving
the plant’s own systemic defence as the only apparent credible cause of the
observed reduced disease symptoms of B+P plants relative to P-control
plants.
The argument presented for drawing conclusion 3 (i.e., for SIR being
responsible in experiment 2 for the observed reduction in disease symptoms
of B+P plants relative to P-control plants) hinges on two apparent facts. (1)
Significant interaction between B and P apparently could only have occurred
on or near the surface of the plants’ roots, since B-cells were detected neither
in B+P plants nor in B-control plants. (2) Any effects of those interactions
that might have been retained by normally virulent, non-mutated P-cells
entering the plant would have been lost in their progeny within the plant
(i.e., first generation offspring within the plant).
Note that Konnova et al. (1999), in another set of experiments, but using
the same B and P, proposed that SIR induced by B might be an important
means for controlling P. They point out that SIR, activated by tomato-plantassociated bacteria, can involve a number of pathways (Pieterse et al. 1996;
van Loon et al. 1998), and that different pathways can have synergistic
effects. Unlike our study, instead of pouring a P suspension on the soil
surface around the root system of tomato seedlings, they injected
lipopolysaccharides extracted from B into the plant’s cotyledons, which
resulted in significant suppression of wilt development.
Experiment 3 (Paper II)
In this experiment, B is Fusarium oxysporum Fo-(IMI 386351), an
endophytic fungus for tomato.
The basic rationale for SIR.—The experiment includes several seed
treatments, representing several doses of B applied to the seeds of plants. B
44
and P are in contact with one another within B+P plants. The experimental
results clearly show that plants to which B was applied (B-control plants
and B+P plants) experienced two distinctly different physiological
responses to B: a low-dose response and a high-dose response. A threshold
dose apparently subdivided the two responses: that is, doses below the
threshold distinctly resulted in the low-dose response; and doses above, in
the high-dose response. Hence, the two response types can be considered
separately.
For the low-dose response of B+P plants, as the B dose increased,
disease symptoms were significantly reduced compared to P-control plants.
Hence, conclusion 1 can be drawn. From the results of the experiment,
conclusion 2 cannot be drawn because B was found everywhere that P was
found in plants. Nonetheless, SIR may well have occurred in the low-dose
response in that shoot fresh weight for B-control plants increased, and shoot
dry weight decreased, indicating that there was a systemic response to Bapplication. There was also a clear decrease in POX activity within Bcontrol plants. The reduction in disease symptoms for B+P plants relative
to P-control plants may well have included both SIR and direct interaction
between the B cells and the P cells, which may have competed for sites
within the stems and petioles of plants.
For the high-dose response of B+P plants, conclusion 1 can be drawn
because there were no disease symptoms for B+P plants, whereas all Pcontrol plants died as a result of disease. Conclusion 2 can be drawn
insofar as in B+P plants, P is found in the stems of plants, but not B, which
was found in the petioles; but B was found in the stems and petioles of Bcontrol plants. Apparently, the elimination of disease symptoms in the B+P
plants (high B dose) was essentially the result of the plant’s own defence
activity. Conclusion 3 then follows, because the B-control plants and the
B+P plants did respond systemically, at least to the application of B in that
shoot fresh weight seems to have consistently increased with increase in B
dose, as did shoot dry weight for B+P plants.
The experiment, the results, and the argument for SIR in more detail.—
x B is applied to the seeds of plants at several doses, which cover a
sufficient range of reduced disease symptoms to make dosage a
useful parameter for evaluating the response of plants to both B
and P.
x Upon planting the seeds, P is applied to the soil around the root
systems of plants.
x For B-control plants and B+P plants, shoot fresh weight
consistently increased with increase in B dose.
These results indicate that B induced systemic physiological activity, and
did so dose-dependently under the given experimental procedure.
45
x For B-control plants, shoot dry weight decreased with increase in
B dose for the two lowest B doses, but increased for the three
higher doses.
x For B-control plants (10 d and 20 d after planting), phytotoxicity
(apparently caused by B) increased with increase in B dose for the
three lower doses, but decreased with increase in dose for the last
two higher doses.
This last result for dose-dependent phytotoxicity supports the hypothesis
that B induced two systemic physiological activities under the given
experimental conditions, at least with respect to effect on plant properties,
that of low-dose activity and that of high-dose activity. Moreover, this result
suggests that the low-dose and high-dose activities are related to plant
defence, and thus, at least the high-dose activity includes a form of SIR
(SIR-H), since for B-control plants, phytotoxicity was reduced with
increase in B dose for the higher doses. This reduction in phytotoxicity may
have been related in some way to the corresponding increase in shoot dry
weight with increase in dose.
x For B-control plants, POX activity decreased (with increasing B
dose) for the first two lower doses, but then increased for the next
three higher doses.
This further confirms the hypothesis of dose-dependent shift in
physiological activity, from a low-dose activity to a high-dose activity.
x For B+P plants also, POX activity was B dose dependent: that
activity increased as dose increased.
This suggests that for the low-dose B-control plants, POX activity served
a function that is at least somewhat different from that which it served for
low-dose B+P plants, and therefore, POX activity apparently tended to
descend with low-dose increase in B-control plants, but tended to ascend
with low-dose increase in B+P plants. In other words, it seems that P
induced a modification in the function of POX activity relative to its
function induced by B. Apparently, P converted the B-induced systemic
physiological activity to an activity that is physiologically qualitatively
different, inasmuch as it changes the B-dose-dependent direction for POX
activity.
x Disease in disease incidence is distinctly B-dose dependent, with
incidence decreasing sharply with increase in dose.
This suggests that the plants responded physiologically to being exposed
to B. Because the systemic response depended on B dose, the observed
physiological activity, associated with POX activity, was induced by B. The
associated systemic physiological activity may well have included SIR as
one of its component activities, insofar as reduction in disease incidence
increased with increasing POX activity.
x For B+P plants (10 d after planting), phytotoxicity (caused by B
and perhaps by P also) increased with dose until a threshold
46
consistent with that for B-control plants was reached, with
increasing phytotoxicity as dose increased, until reaching a
threshold dose at seemingly the same dose level as for B-control
plants.
x For the B+P plants, it seems that beyond that threshold B dose,
phytoxicity was first diminished, but then increased with
increasing dose, suggesting that a SIR response to B doses above
the threshold was different from, and stronger than, the SIR
response to B doses below the threshold dose.
An experiment with such results indicate, apparently, that the SIR
response was not only quantitatively dose-dependent, but also qualitatively
dose-dependent. Olivain and Alabouvette (1997) note that a strain belonging
to a given f. sp. can be used to protect an incompatible plant against its
specific pathogen (Matta 1989), and that many attempts have been made to
utilize strains of different f. spp. to control tomato wilts (caused by Fusarium
spp.) through induced resistance (Biles and Martyn 1989).
Summary
These three experiments support and emphasize the idea that many plantpathogen scenarios can indicate SIR, and that SIR as a complex adaptive
trait, allows plants to take advantage of environmental signals of pending
circumstances by becoming prepared physiologically for those
circumstances. The experiments also suggest that a part of such signalinduced plant states can entail preparedness for having certain other
environmental signals trigger, at appropriate times, rapid systemic changes
and activities as suitable responses to circumstances that are encountered.
One might conceive of these complex adaptive plant activities as being plant
interpretations of environmental circumstances, which the plant translates
into expectations, for which the plant then initiates appropriate physiological
and behavioural preparations. The experiments also indicate that plants can
configure physiological and behavioural responses in terms of genetically
determined propensities for certain successions of physiological states, such
as successive states of SIR, and with each state setting up physiological
preparedness for triggering the next state, if needed. In other words, we may
find that, as organisms, plants are far more capable of sensing and
responding to environmental change than we thought was possible, and in a
concatenated fashion, almost as if they have minds of a sort, of the most
rudimentary sort perhaps. Almost certainly species of plants and species (or
strains) of pathogens are, in final analysis, evolutionary complements to each
other, as are predator and prey in animals, each being important for
ecological balance, and therefore complementing each other, as allies in
macro-evolutionary success, that is, long-term evolutionary trends viewed
from the perspective of geological time.
47
Searching for biocontrol agents (Bs)
Searching for Bs generally involves some sort of isolation and selection
method, even before the screening process, in order to obtain a set of
microbial isolates to be screened. One wants, of course, to select a set of
microorganisms with high potential for becoming effective Bs.
Obtaining microbial isolates
For this first step, taking into account natural environmental conditions
(physical and biological) within which the pathogen that needs to be
controlled is found, is of crucial importance. Such conditions are
fundamentally involved in the evolutionary success of a pathogenic
microorganism, since those conditions are the physical and biological
medium to which the microorganism has become adapted and is able to
thrive, by utilizing metabolic products from the host plant or from other
endogenous or commonly found plants. Hence, in planning a program for
screening in search of suitable Bs, taking into account, first, optimal physical
conditions for the pathogen’s growth, and then second, optimal biological
conditions, is important. Given that a field (or other place under cultivation)
with suitable conditions is found, then obtaining suitable endogenous
microbial isolates is made more likely if isolation is done after one or two
crop rotations, because then the designated pathogen (target organism) is
more likely to be aggressive. Finally, to obtain an optimal set of microbial
isolates for screening (bacterial or fungal), a comprehensive sampling of the
area at different time points can be important. That is, if one only samples at
a single location and at a single time point, the sample may miss the best
isolates, which might happen to exist at other places in the given field or
fields, or at other time points.
Ultimate possibilities for the MF30 strain
Progress is being made in searching for effective Bs. Streptomyces spp. have
been shown to suppress tomato wilt (Turhan 1981; El-Abyad et al. 1993),
without use of herbicides, but also with herbicides (El-Shansoury et al.,
1996). Several Pseudomonas spp. are recognized as potential Bs against
48
many soil-borne diseases, including tomato wilt (Fuchs and Défago 1991;
Lemanceau and Albouvette 1991). Combinations of Pseudomonas spp. with
non-pathogenic Fusarium strains (Biles and Martyn 1989; Larkin et al. 1996;
Benhamou et al. 2002) are recognized as potential Bs against tomato wilt.
If a microbial strain B triggers SIR for a given plant cultivar against a
given pathogen P, and does so under all or most environmental conditions,
then the strain’s biological control capability might have the following
potential, if one considers two extremes at two ends of a spectrum. At one
extreme, B+P plants allow uninhibited colonization by P so that
colonization of B+P plants and P-control plants is the same. Mutant B
genotypes that benefit the growth and vigour of plants, and their disease
resistance to other pathogens, will then have a selection advantage, because
the plants that those genotypes colonize will be more likely to grow well and
avoid disease. With time, as such B mutants accumulate within the genome,
either by natural selection or by artificial selection, B will become
endophytic to the tomato cultivar. At the other extreme, P is unable to
colonize plants of the cultivar after B application. If all cultivars of the plant
species, except for the given cultivar, were destroyed to the point of
extinction, and if all plants of the given cultivar are immunized against P by
application of B, the plant species will no longer serve as a host for P. Then
P will cease to exist, unless it acquires hosts other than the given plant
species. A similar sort of scenario became reality for the smallpox virus,
which is now essentially extinct, after the smallpox vaccine was
administered worldwide (Fenner 2000; Berche 2001; Jodar et al. 2001).
The isolate MF30 (closest but not identical to Pseudomonas orientalis;
unpublished data) has suppressed disease development of Fol (tomato wilt)
in tomato (Paper IV), likewise it suppressed F. culmorum (seedling blight)
and Microdochium nivale (snow mold) in wheat (Johansson et al. 2003), as
well as Drechslera teres (net blotch) in barley (Konnova et al. 1999).
However, being isolated from an area, such as northern Sweden, which is
extremely cold during the winter, some of those isolates (MF1 to MF50)
might be difficult to test because those strains are adapted to cold
environments. Nevertheless, for in vivo experiments, some of those isolates
revealed a capability of adjusting to environmental temperatures, given that
other conditions were optimal, such as pH, soil moisture level, humidity, and
soil or air temperature.
49
Interestingly, Konnova et al. (2003) tested the isolate MF30 for
production of biologically active metabolites. Their investigation, using
HPLC, H+ NMR and MS in order to characterized active compounds,
revealed four peaks with retention times 6.6, 6.8, 7.3, and 8.0 minutes,
whose UV spectra were highly correlated (0.999-1) to massetolide type A
(1) (Fig. 4), a low molecular weight lipopeptide (Gerard et al. 1997). This
compound, as well as several of its derivatives (massetolides types B-H),
were recently isolated from two Pseudomonas spp. isolates of a marine alga
and a marine tube worm origin (Gerard 1997). Moreover, the isolates
produced viscosin (2) (Fig. 4) (Burke et al. 1989; Moore 1999). No
suppressive effect of this compound on in vitro growth of Fol was detected
(Konnova et al. 2003). However, lipopeptides increase attachment of
bacteria to hydrophobic surfaces, such as root or shoot surfaces. This might
be considered as an important factor of effectiveness of B (Konnova and
Hedman unpublished). To date, there are only two reports of in vitro antituberculosis activity expressed by massetolide and viscosin (El Sayed et al.
2000). When tested against Mycobacterium tuberculosis, both massetolide A
and viscosin inhibited M. tuberculosis H37Rv at a concentration of 12.5
mg/mL for 97 and 66%, respectively, without showing significant
cytotoxicity (Rodriguez et al. 1999).
Fig. 4: Chemical structure of Massetolide and Viscosin.
“Reprinted from Publication title: Tetrahedron, 56, El Sayed, K.A., Bartyzel, P., Shen, X., Perry, T.L.,
Zjawiony, J.K. and Hamann, M.T., Marine natural products as antituberculosis agents.949-95. Copyright
(2000), with permission from Elsevier”.
50
Concluding remarks
Immunity in plants, such as induction of resistance by microbes, may well be
more developed than we earlier thought, and may approach a level of
sophistication on par with processes of immunity in animals. Numerous
biological and chemical products that control disease by induced resistance
will likely be developed over the next few years. These advances should be
useful in developing new, environmentally friendly means of disease control.
We found that the direct RFLP mtDNA technique consistently produced
clear banding-pattern differences between Fol and Forl isolates, regardless of
the geographic origin of our isolates. We also found that the technique was
rapid enough for routine differentiation, suggesting that the technique might
be useful worldwide as a routine diagnostic tool for differentiating between
Fol and Forl. Studies of the ecology, the evolution, and the population
dynamics of Fol and Forl might now be readily conducted. In addition, as a
diagnostic tool, the technique might prove an efficient means of monitoring
the infraspecific structure of Fo populations, and thereby facilitate control of
the tomato diseases, wilt, root rot, and crown rot.
Some of the conclusions below concern the potential of nonpathogenic
strains of Fo to control tomato wilt. But nonpathogenic strains of Fo have
been used for many crops, including banana (Gerlach et al. 1999), basil
(Fravel and Larkin 2002), carnation (Garibaldi et al. 1986), cucumber
(Mandeel and Baker 1991), cyclamen (Minuto et al. 1995), flax (Alabouvette
et al. 1993), gladiolus (Magie 1980), melon (Rouxel et al. 1979), tomato
(Fuchs et al. 1999), spinach (Katzube et al. 1994), strawberry (Tezuka and
Makino 1991), and watermelon (Larkin et al. 1996). Few of them have been
applied on a commercial scale.
The MF30 bacterial strain belongs to the genus Pseudomonas, which is
well known for its B capacities, and is probably a species closely related to
biovars of Pseudomonas fluorescens, which have natural suppressiveness to
Fusarium wilts, along with nonpathogenic Fo (Alabouvette and Lemanceau
1996). Three properties make MF30 a promising candidate for managing
tomato wilt caused by Fol: (1) MF30 has a capability, when inoculated into
the soil around the roots of tomato plants, of eliminating DI caused by Fol;
(2) our experiment strongly suggest that this capability is the direct result of
SIR, triggered directly or indirectly by MF30; and (3) the biological process
of elimination of DI appears to involve no discernable hazardous effects
51
(e.g., no necrotic spots or phytotoxic signs observed on MF30 plants and
MF30+Fol plants.
The performance of biological control based on the applications of strains
of nonpathogenic Fo has lacked consistency, which is the main reason that
biological control has not been adopted. But field studies that integrate
biological control into commercial production systems, and studies that
provide us a more thorough understanding of the genetics, biology, and
ecology of Bs, and their modes of action, will eventually enable optimal
exploitation of nonpathogenic Fo for disease control. Molecular techniques
can provide insight in each of these interrelated areas of research. Molecular
genetics, for example, offers new tools for unravelling mechanisms and for
understanding genetic relationships among Fusarium spp. and strains. For
example, an approach using transposon mutagenesis has recently been used
to produce mutants of F. oxysporum f. sp. melonis that affect their
pathogenicities (Migheli et al. 1999), and to produce mutants of Fo47 that
show either an increased or a decreased biocontrol capacity (Trouvelot et al.
2002). Research is needed in several areas, including field studies and
studies of how to optimally integrate Bs into production systems, and studies
of risk assessment, mechanisms of action, and means of genetically
improving Bs.
Here are some comprehensive remarks that derive from this study:
x Two fungi, Phythopthora cryptogea (Pc) and Fusarium oxysporum
strain Fo IMI 386351, and one bacterium, Pesudomonas sp. (strain
MF30), apparently induced SIR in Lycopersicon esculentum –L.
under certain experimental conditions in different ways.
x Fo IMI 38635, an endophytic fungus, was confirmed to belong to
the Fol family.
x Fo IMI 38635 invoked physiological and biochemical changes,
which were probably involved in SIR expression.
x Activation of plant defence mechanisms can be accomplished
either by the presence of the eliciting microbe or by one of its
products.
x Based on the phylogenetic trees, Fo IMI 386351 might be a
member of Fol or Fom, but Fol and Fom have polyphyletic
evolutionary origins.
x mtDNA RFLP analysis supports the sequencing data and suggests
that this strain probably belongs to the Fol family.
52
Appendix
In a study of fitness distributions and fitness effects of advantageous
mutations in evolving Escherichia coli populations, Imhof and Schlötterer
(2001) found that Malthusian fitness parameters, commonly used for
bacteria (Pringle and Taylor 2002), ranged from 0.006 to 0.059. They found,
as suggested by population genetic theory (Fisher 1930), the most
advantageous mutations had a small effect. Only a small fraction had large
selection coefficients. In their most striking example, an allele markedly
increased in frequency after 270 generations.
Because stress can cause the frequency of mutator alleles to increase
(Taddei et al. 1997; Sniegowski et al. 1997), high stress on Fol cells entering
the soil might increase the probability of occurrence of a certain mutation.
Suppose then that circumstances are the most favourable—that is, doubling
times in the soil are extremely short, say 5 hours, and an appropriate
mutation occurs almost immediately after soil inoculation of Fol cells. Even
then, about 60 days (270 generations, each of a duration of about one
doubling time) would be required for a marked increase in frequency of the
mutant allele. For more realistic doubling times of 10 or more hours, a
comparable increase in frequency would take about four to six months.
Although the doubling time of colonizing Fol cells within soil seems to be
unknown, a Fusarium graminearum sp. grown in a glucose-limited
chemostat at a dilution rate of 0.05 h-1 had a doubling of 13.9 h (Wiebe et al.
1994).
53
Summary in Swedish
Attitalla, I.H. 2004. Biologisk och Molekylär Karakterisering av Inducerat
Försvar i Tomat (Lycopersicon esculentum –L.)
Två svampar Phytophthora cryptogea (Pc) och Fusarium oxysporum stam
Fo-(IMI 386351) samt en bakterie Pseudomonas sp. (stam MF30) testades
avseende deras förmåga att stimulera till försvarsreaktion i tomat
(Lycopersicon esculentum –L.). Det har visat sig att växter som vanligtvis är
mottagliga för sjukdomsalstrande mikroorganismer kan bringas till att inta
ett tillstånd av beredskap genom påverkan av icke-patogena
mikroorganismer. Detta tillstånd benämns inducerad resistens och kan vara
lokal eller fördelad i hela plantan, s.k. systemiskt inducerad resistens (SIR). I
föreliggande avhandling har tomatplantan använts som modellväxt vid
undersökningarna av de mekanismer som är aktiva vid interaktionerna då
växter induceras av mikroorganismer. Därmed får man ett biologiskt system,
som tillåter uppföljandet av en evolutionsprocess, där särdragen och
komplexiteten i denna nära samverkan studerats och delvis klargjorts.
Det förfarande som undersökningen baseras på har varit inokuleringen av
plantorna eller den omgivande jorden med någon av de inducerande
mikroorganismer som förutsättes ha en biologisk kontrollverkan (B). Denna
behandling uppföljes av en s.k. ”utmaning” varvid plantan inokuleras med
den patogena svampen Fusarium oxysporum f. sp. lycopersici (Fol) som
orsakar vissnesjuka i tomat. Vid användningen av de inducerande
mikroorganismerna visade resultaten ifrån vart och ett av dessa
undersökningar att en påtaglig interaktion uppstod mellan de olika B och
växten, varigenom en systemisk inducerad resistens kunde erhållas – d.v.s.
en fundamental stimulerad växtförsvarsmekanism blev tillgänglig.
Antagonismen mellan den patogena svampen och den biologiskt
aktiverande organismen har studerats. Två av dessa - Pc och Fo-(IMI
386351) – stimulerade till bildning av systemisk inducerad resistens i växten,
varvid två olika uttryck av SIR kunde iakttagas, vilket antyder att två former
av svar kan vara involverade i den observerade, stimulerade resistensen.
Plantor som stimulerades av Fo-(IMI 386351) svarade med vissa
fysiologiska och biokemiska förändringar, som troligen är delar av den
systemiskt inducerade resistensen.
Genom att använda molekylära metoder kunde vi bekräfta den
taxonomiska placeringen av Fo-(IMI 386351). Denna mikroorganism, med
54
biologisk kontrollaktivitet, kunde åtminstone under vissa förhållanden visa
sig vara, en s.k. endofyt – d.v.s. en organism som lever i en växt utan att
orsaka menliga symtom. Utförandet av fylogenetisk analys av partiella
sekvenser i två gener: en mindre ribosomal DNA-subenhet – (mtSSU rDNA)
– ifrån mitokondrier och en s.k. ”translation elongation factor” – (EF-1D) –
ifrån kärnan, gav ett fylogenetiskt träd som antydde att Fo-(IMI 386351)
skulle kunna vara en typ av Fol eller en typ av Fusarium oxysporum f. sp.
melonis (Fom), som har s.k. polyfyletiskt evolutionärt ursprung.
Medelst s.k. RFLP-analys av mtDNA erhölls stöd för sekvenseringsdata,
vilka angav att Fo-(IMI 386351) troligen tillhör Fol. Molekylära metoder
utvecklades likaså för att få en differentiering mellan två formæ speciales (f.
spp.), som morfologiskt inte är möjliga att skilja ifrån varandra, nämligen
Fusarium oxysporum f. sp. lycopersici och Fusarium oxysporum f. sp.
radicis-lycopersici, som i tomat orsakar sjukdomen rot- och stambasröta. Vi
jämförde resultaten från denna metod med de från två andra biokemiska och
molekylära metoder (osmosmetod i kombination med HPLC och isozymanalys).
Aktivering av plantans försvarsmekanismer kan åstadkommas antingen
vid närvaro av en stimulerande mikrob eller genom verkan av någon produkt
från denna. Vid de HPLC-spektra som erhölls vid separation av
preparationer från Fo-(IMI 386351) uppvisades närvaron av en extra topp
vid test av två fraktioner. När dessa fraktioner analyserades med
kärnspinnresonans (NMR), framgår det av de preliminära resultaten att
extratoppen kan motsvaras av en oligosackarid. Denna oligosackarid anser
jag kan vara en signalmolekyl som utlöser försvarsmekanismer i plantan.
Den huvudsakliga slutsatsen från studien av de tre mikroorganismerna
med B-verkan är att plantan genom framkallandet av systemisk inducerad
resistens kan utnyttja sina egna resurser för försvar och immunitet gentemot
patogener.
De interaktioner som uppstår mellan mikrober och växter, när plantans
försvar igångsättes, kan till viss grad studeras i laboratoriet även om
reaktionsmönstret är komplext. Molekylära verktyg kan vara kraftfulla
hjälpmedel vid identifieringen av svampstammar och vid klarläggandet av
deras taxonomiska släktskap.
55
Acknowledgments
First I would like to thank ϰϟΎόΗϭ ϪϧΎΤΒγ Ϳ΍ [ALLAH – S.W.] (GOD) for huge
help in different ways and support during my study, as well as for health and
keeping me well. I’m thankful to SWEDEN, which I spent part of my life,
and had really a nice time, also when consider me as a PhD candidate and as
I expect its excellent school to everybody to do high style work. My country
LIBYA for supporting me during long destination study.
AN PROVERB FROM ARABIC SOCIETY WHICH I ADOPT IT HERE THAT
“IF YOU DO NOT THANK PEOPLES YOU NEVER THANK GOD”
I would like to start with peoples who without them I will never think even
how I could finish my PhD study: my supervisor Professor Sture
Brishammar: who not just only scientific adviser for me, he became so
closed than anything else, thanks Sture for everything you did with me,
thanks for accepted me to study in your laboratory, when the chances was so
limited, when the time is gone, when I was going to lose my scholarship
opportunity, when I get no single response from other places around the
world, all of them asking so many questions and nobody feel what my
situation is, you was the right person and I will always think for sure that
you are a Great Person. I wish you a wonderful life. Again thank you Sture
for unfailing support of my work, for always being a listening ear, ready for
discussions and for introducing me to an independent view on scientific
matter. Thanks you also for your powerful help and your encouragements
and believing in me, thanks for all ideas and all contribution to solve my
scientific matters.
Dr. Paul Johnson: you are the one which I will never forget, I learned a
lot from you, and you showed me how reactions should be, you improved
me a lot in my scientific style and thinking and I belive that without you this
thesis will never get to this form. I understand several things I never have
dreamed to learn without you, you was so close all the time. I appreciate that
and I feel in fact so happy when I pick up the phone or of course meeting
you personly, I do not know why (?) but this is absoultely truth.
Dr. Jamshid Fatehi: you are one of the best scientist I have met in my
live, thanks for introducing me in something I always have struggled to
learn, you helped me a lot and the words thanks I belive is not enough but
God know how much respect I have for you.
56
Professor Berndt Gerhardsson: for your kind suggestion, thanks for
beliving in me and your good deal of advice makes me think in different
mannar.
Professor Siv Andersson: thanks for believing in me and your
acceptance to be one of your department members, that I really had wished a
long time ago to be and I am happy I could finish my PhD in your
department, this is really the best present in my life.
Associate professor (my co-supervisor) Paloma Melgarejo (INIA)
Madrid, Spain: huge thanks for you, your ideas have always helped me,
your long term comments make my paper acceptable and I am so happy that
you are a part of my PhD-contribution.
Associate professor Lennart Johnsson: without your brain my data will
be in trouble, thanks so much for given me times, for your kind help and
always patience with my inquiry.
Associate professor Sandra Wright: your encouragement is always on
my mind, thanks for your comments on my papers and your pieces of advice,
and your nice deal.
Dr. Jens Levenfors: It was a great time to share knwledge with you, and
to do lab-work togther, and Dr. Jolanta Levenfors: for excelent job we do
togther concerning MF30 and PCR, thanks for your comments in my thesis.
Assoictae professor Kerry O’Donnell (NCAUR) in the USA: my best
regard and respect for someone who always is positive and helping me,
never ever get disappointed when I disturb you by many e-mail and phone
calls, and of your critical review of (Paper II). you are the one I will always
remember, and you can see an evidence for that, my thesis reference list
contains several papers of yours in the high rank scientific journal.
Professor Mark J Bailey: Director CEH-Oxford Virology and
Environmental Microbiology, Oxford, UK, thanks for reading my second
paper.
Dr. Stefan Gunnarsson, Mr. Gary Wife and Ms Annette Axén at
Microscopy and Imaging Unit, EBC for your help with SEM and TEM and
Images of my Fusarium strain.
57
I WISH TO THANK EVERY BODY, WHO IN ONE WAY OR ANOTHER
CONTRIBUTED TO THIS THESIS
MY GREAT MOTHER MABRUKA ABDU SALAM SALEIM
Life without you is nothing, for this please accept this dissertation as present
for your scarify (?) since I grew up and did not see my father, you was my
mother and my father as well, you was always helpful, since I moved from
Faculty of medicine and I was depressed, I remember all your
encouragements all your word, IDRESS be a real man and show and prove
that you are able to be a doctor, this is I learned only from you, I wish God
always keep you in a better health and make life for us so sweet, thanks for
your wishes when you pray, without your wishes “DOAA” (ARABIC
WORD) I can not struggle for this long time.
MY FAMILY IN SWEDEN
My sweet wife Mabruka Abdali: with nick name Ebreka, my daughter
Sundos, Ebthal, and Estabrak thanks for be patient with me those last
years, for giving inspiration in my work. My wife’s family, for
understanding my situation and pushing things going well. I express my
deepest appreciation to all of you, without your love, patience and support
this thesis could never have been done.
MY RELATIVES IN MY HOME LAND (LIBYA)
My brothers Omar and Mustafa: for several common interest and your
helps in many many several ways, let me feel stable and strong to go on with
my study. My brother’s wife: Yasmen, her family, and her brothers
especially Mr. Moftah who was so close to me and supporting me in my
different ways until I got the chance to be back in Sweden, and of course I
will never forget Samie, Arwa and Tkwa.
FRIENDS IN SWEDEN AND LIBYA
My best friend Dr. Mikaeel Faturi: from my country he is absent now,
after he did his PhD in Sweden as well, just to let you know what we discuss
earlier some thing special for me. I consider you a gentle man, with huge
ethical opinion I never seen. Thanks also for helping me to interpret my data
after statically analysis.
Dear brother Emad Abd-Elrady: and his family particularly his wife
and his sweet daughter Hadel, thanks for your time spending solving
problems with us, excellent social relationship.
Brother Usama Hegazy: and his family, Brother Mohamed Issa: and
his family, we became so closed. Brother Mohamed Abdel-Aziz El Sayed:
and your family so nice to know you brother and to be friends in very short
time.
Football (Sudan football group) was great time with you all. My friend
Brother Moawiea and his kind wife Sana, I consider time with you like
58
Gold. Mr Ali El Kaelanye: for given me the last min chance to be back to
Sweden.
My friend Jörgen Sjögren: Department of Chemistry at SLU, for
helping me to run the NMR for my sample; Dr. Farhan Rizvi: EBC,
Department of Development and Genetics: Thanks for your reading and your
effective comments in the last version of my thesis.
Professor M’Barek Fatmi and Professor Mohamed Achouri: Institut
Agronomique Et Veterinaire Hassan II Complexe Horticole D’Agadir
Agadir, Morocco for your suggestions and friendship. Sisters Dr. Olga
Vinnere and Ms Inga Morocko: for every nice time we had together,
sharing problems and solving problems together by hard discussion!
BIOCONTROL GROUP
Ph. Lic. Ola Arwidsson: I must say you was so helpful, even in the last
minute, I was on your mind, I avoided so many problems by taking good
pieces of advice from you about using new types of software, and managing
programs, to be sure that all will be under control.
Dr. Janne Lager: I’m grateful to that time we spent together discussing
many troubles concerning social and scientific matter and I remember your
test you did for me, I found you extraordinary and by time we became more
closer, thanks for helping me fixing my data analysis in good table format.
Secretaries: Mrs Ewon Enqvist, and Catarina Falgin: your support
was something special for me, I do really mean it, thanks so much for
understanding other peoples culture, I knew its hard, but I believe you did.
Dr. Pablo Quintanilla: I want to say SIR is completely right?
Dr. Riccardo Tombolini: you are the first who let me go to do something
I like in research; I appreciate your first lecture and the working under your
supervision that time.
Associate professor Mauritz Ramstedt: for good times to find solutions.
Dr. Tahsein Amein and Dr. Ibrahim AlWindi: nice to know both of
you, we have great discussion about different things, and I feel really happy
when I share ideas with both of you, thanks for your encouragements. Also, I
thank my colleagues at Biocontrol Unit for your friendship.
MOLECULAR EVOLUTION GROUP
Associate professor Robert fast: for your patience with my questions
concerning Uppdok. Professor Otto Berg, Associate professor Rolf
Bernander and Asssociate professor Mikael Thollesson: for introducing
me through seminar presentations and accepted me to be one of your
department member during that official meeting. Karin Olsson: thanks for
the time you gave to me. Ola Lundström: in simple word and frankly, you
are one of the best I met in Sweden, thanks for helping me with figures; Dr.
Karin Hjort and Hillevi Lindroos: your positive reaction when I started at
59
MolEvol Dept. and showing me the procedure about and role at Uppsala
University; Dear Håkan Svensson and Stefan Ås: thanks so much for
solving problem in my computer. Davids Wagied: I am happy to know you.
Thanks for your comments in my thesis and my manuscripts. Brothers and
sister at Department of Molecular Evolution I appreciate so much the time I
spent with you which really was so nice and a nice deal as well. I got an
impression that all of you really are true scientists and I enjoyed so much
listening to your great presentations. All of you are high quality peoples.
Mats: thanks for refining one of my manuscript as well, Hans-Henrik: was
always helpful. MolEvol group, I really enjoyed you, I wish to have longer
time, to have common interest in research too, I would like to say even more
but I prefer to only keep that on mind! I like you all and I wish you all will
be successful, under the perfect leader (Siv).
Omar Al Mukhtar University, El-Beida, Libya http://www.omulibya.org/
for given me an opportunity to complete my study in Sweden by granting me
a scholarship.
The General Secretariat of Education and Scientific Research, Ministry of
Education (Libya); Libyska Folksocialistiska Arab Jamahiriya as
Folkkontor, Stockholm (Sweden)
For finical support during all my study period.
Especial thanks for The finance director Mr. Alsedeiq Alshaibi
Being so close friend and for his encouragement and his believe in what I am
doing!
The Culture Attaché Mr. Hakoma Alnaqib
Always been so kind with me.
60
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phenoloxidase activities as a consequence of stress that induce resistance to
Fusarium wilt of tomato. Journal of Phytopathology 122: 45–53.
2. Achuo, A.E. and Hofte, M. (2001) Potential of induced resistance to control
Oidium lycopersici on tomato and tobacco. Mededelingen - Faculteit
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3. Agrawal, A.A. and Karban R. (1999) Why induced defenses may be
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University Press, Princeton, N.J. Pp. 45–61.
4. Agrios, G.N. (1997) Plant Pathology. 4th ed. Academic Press. NY. Pp.16–
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5. Alabouvette, C., Lemanceau, P. and Steinberg, C. (1993) Recent advances
in biological control of fusarium wilts. Pesticide Sciences 37: 365–373.
6. Alabouvetter, C. and Lemanceau, P. (1996) Natural suppressiveness of soils
in management of fusarium wilts. In Management of Soil-Borne Diseases
(R. Utkede, V.K. Gupta eds.). Kalyani publisher, Ludhiana, India, Pp. 301–
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