Everything you need to know about the entry of pathogens into host plants . Learn about:- 1. The Roles of Physical and Chemical Signals in the Germination of Propagules of Plant Pathogens 2. Topographical Features 3. Adhesion 4. Breaching the Cell Wall by Mechanical Force.
Entry of Pathogens into Host Plants
The Roles of Physical and Chemical Signals in the Germination of Propagules of Plant Pathogens:
Many fungi, on encountering their host or some other solid substrate germinate, producing germ tubes which may differentiate into infection structures. These vary from being simple appressoria to complex structures such as ‘cushions’. Similarly, parasitic angiosperms such as Striga elaborate haustoria.
The stimuli provided by the host for germination, growth and the differentiation of infection structures are as follows:
Lee and Dean (1994) found a correlation between hydrophobicity of the contact surface and the formation of appressoria by the fungus, Magnaporthe grisea but with Phytophthora palmivora the situation was more complex.
Germlings of this organism that were free floating or were in contact with smooth substrates under high nutrient conditions (20 per cent pea broth) did not form appressoria regardless of the hydrophobicity of the contact surface.
In contrast, under low nutrient conditions (5 per cent pea broth), appressoria were formed on smooth substrates that were hydrophobic but not hydrophilic substrates. However, if these same surfaces were scratched, appressoria formed on the scratches, irrespective of the levels of nutrients or substrate hydrophobicity.
Xiao and co-workers (1994) reported that although conidia of M. grisea started to germinate whether they contacted a liquid or solid surface, appressoria only formed on solid surfaces and not on liquid or agar surfaces.
On freshly prepared agar surfaces, germ tubes of the fungus penetrated directly without the differentiation of appressoria but if the agar were allowed to dry partially appressoria were formed.
Several chemical components of host plants have been implicated in the germination of propagules of plant pathogens and the differentiation of infection structures. In particular, the wax on the surface of aerial parts of the plant is a rich source of diverse compounds, which may play these roles.
For example – wax from rice leaves relieved the self-inhibition of conidia of the rice blast pathogen, Magnaporthe grisea, and stimulated appressorium production. In isolates of Colletotrichum gloeosporioides that infect avocado, specific components of waxes were involved since the surface wax of this host but not of other plants triggered both conidial germination and the development of infection structures. The fatty alcohol fraction of the wax, which comprised 5 per cent of the total, was the most effective and synthetic aliphatic n-fatty alcohols with 24 or more carbons were also active.
Reciprocally, wax of avocado was ineffective in inducing differentiation of other species of Colletotrichum which are pathogenic to other plants. Species of Colletotrichum which infect ripe climacteric fruit, such as tomato, banana and avocado are responsive to the ripening hormone ethylene forming multiple appressoria on glass surfaces in the presence of micromolar concentrations of the gas. Confirmation of the role of ethylene in these processes was obtained in experiments with transgenic tomatoes that did not produce ethylene.
On these the fungus was unable to germinate or form appressoria unless exogenous ethylene was supplied. Thus, these fungal pathogens of ripe climacteric fruit recognize the hosts’ ripening signal and this allows them to form the structures necessary for the launch of a successful attack at the appropriate time.
Flavonoids exuded from the roots of legumes stimulate the spore germination of several soil-borne fungi. For example Bagga and Straney (2000) found that naringenin was a powerful stimulant of the germination of macroconidia of Nectria haematococca, stimulation occurring in the low mM range.
Other flavonoids, such as luteolin and hesperitin, were also effective but at higher concentrations. Effectiveness of the flavonoids as stimulants of spore germination was related to their ability to inhibit cAMP phosphodiesterase activity and thus increase cAMP levels.
Signalling compounds, passed between parasitic plants and their hosts, have been reviewed by Estabrook and Yoder (1998). Seeds of Striga normally germinate only when in close proximity to roots of host plants. However, some non-host plants also stimulate germination and, in fact, the first germination stimulant to be isolated, strigol, was from a non-host plant, cotton.
SXSg from Sorghum bicolour was the first stimulant to be isolated from a host plant but it is quickly auto-oxidized to the inactive quinone sorgoleone. Subsequently, Siame et al. (1993) reported that strigol was the major germination stimulant of S. asiatica from roots of maize (Zea mays) and proso millet (Panicum milaceum) and a minor component of the stimulant activity of sorghum. The major stimulant from sorghum was sorgolactone, a compound closely related to strigol.
When in proximity to a host, germinating seed of Striga asiatica differentiate an attachment organ, the haustorium. A number of compounds from host plants induce haustorium differentiation and these include the flavonoids, xenogosins A and B, ferulic acid and 2, 6-dimethoxy-p-benzoquinone, (DMBQ) this last being oxidatively released from the host root by an enzyme of the pathogen.
White rot caused by Sclerotium cepivorum is one of the most important diseases of onion. Work summarized by Coley-Smith (1990) and carried out in his laboratory over a number of years has demonstrated that the germination of microsclerotia of the fungus is stimulated by alk (en)yl-l-cysteine sulphoxides from the host.
In most agricultural soils, nutrients are limiting and, as a result, many soil inhabiting organisms, including pathogenic fungi, are essentially dormant. Dormancy may be broken by the addition of nutrients such as those supplied by germinating seeds and plant roots. Despite the widespread occurrence of this phenomenon, relatively few studies have been made of the compounds responsible.
Stimulants may also play an important role in the establishment of infection by aerial organisms. In particular, pollen and intact anthers are a rich source of nutrient and may enhance the virulence of facultative pathogens.
For example – Fusarium graminearum causes head blight of wheat but plants are only susceptible when warm, moist weather coincides with anthesis. When test plants, from which anthers had been removed, were inoculated with an aerosol of macroconidia of the fungus they remained essentially free of infection, whereas the fungus grew prolifically on the extruded anthers of control plants and caused heavy infection.
In a cup plate assay of anther extracts, hyphae from germinating macroconidia of F. graminearum were 70 per cent longer than controls. The stimulants responsible were isolated and identified as choline and glycinebetaine with EC50 concentrations of 10 and 30 parts per billion, respectively.
Two other Fusarium species were stimulated at similar concentrations but not F. nivale which has now been reclassified as Microdochium nivale. When the compounds were applied to slivers of filter paper as substitutes for anthers they promoted infection.
The effect of choline and glycinebetaine on F. graminearum has been reinvestigated. They found that the compounds are transported into hyphae of the fungus by different permeases and are converted into a common active component which increases the hyphal growth unit but not fungal biomass. (The hyphal growth unit is defined as the total length of the hyphae of a colony divided by the number of hyphal tips).
In other words, choline and glycinebetaine reduce branching relative to that in control colonies, allowing growth to be concentrated in extension. This could be advantageous to the pathogen since it would increase the probability of rapid entry into the host, thus avoiding desiccation should the weather become dry.
The topologies of plant surfaces provide signals to many fungal pathogens. For example, rust fungi usually enter their hosts through stomata, their topology triggering the development of infection structures.
Elegant work by Staples’ group has shown that a simple ridge, 0.5 mm high on a flat surface, was optimal for the differentiation of infection structures of Uromyces appendiculatus, the bean rust fungus. A ridge of almost exactly this height (0.487 + 0.07mm) was found on guard cells of the host, Phaseolus vulgaris. However, when the germ tube of the rust grew over the ridge it appeared to be flattened, suggesting the application of force.
Moreover, experiments in which hyphae were perturbed with micropipettes demonstrated that the area that was most responsive for appressorium formation was 0-10 mm behind the apex of the germ tube, perturbation more than 40mm from the apex being entirely ineffective.
Similar work has been done with 26 other rust species in which four types of behaviour were recognized. In group 1, which contained the important cereal rusts Puccinia graminis f. sp. tritici and P. recondita, no appressoria were formed on any membrane whether they were smooth or possessed a single ridge, apart from one isolate of P. recondita which formed a few appressoria erratically.
However, more recent work by Read and co-workers (1997) has shown that P. graminis f. sp. tritici and P. hordei do differentiate infection structures at high frequency (83-86 per cent) over multiple ridges that were closely spaced (optimally 1.5mm and 2.0 mm high), such topology reflecting the close spacing of cell junctions associated with guard cells of cereals.
Moreover, the frequency was increased by the presence in aqueous solution of the leaf alcohol trans-2-hexen-1-ol to 88 per cent. In Allen and co-worker’s group 2, which contained U. appendiculatus, there was a relatively sharp optimum height for the ridge of about 0.5mm at which more than 80 per cent of germ tubes differentiated infection structures.
A third group consisted of fungi which responded with increased appressorium differentiation to increased height of ridges but there was no decline once the maximum had been reached.
Allen and co-worker’s final group of rust fungi, exemplified by Phakopsora pachyrhizi, a pathogen of soybean, formed appressoria on ridges of all heights as well as smooth polystyrene membranes and silane-treated glass.
For rust fungi which enter via stomata, locating a stoma may be facilitated by responding to other topological signals. For example – germ tubes of P. graminis f. sp. tritici orient themselves at right angles to leaf veins which, owing to the manner of their distribution, maximizes the chance of the tube encountering a stoma.
Fungi that penetrate plants directly also respond to the topography of their hosts. For example, Rhizoctonia solani produces infection cushions both on hypocotyls of cotton seedlings and on artificial replicas of hypocotyls.
Fungi which enter their host plants directly without the aid of a wound or a vector must adhere firmly to them and this is particularly true of those that use mechanical force. Jones and Epstein (1989) found that macroconidia of the squash pathogen, Nectria haematococca, adhered to polystyrene Petri dishes if they were harvested at 248C but not if they were harvested at 18C although the attachment process itself appeared to be temperature insensitive. Adhesion was also inhibited by sodium azide and cycloheximide, suggesting a requirement for respiration and protein synthesis.
In experiments with another fungus, Uromyces appendiculatus, adhesion of germlings was directly related to the hydrophobicity of the substrate.
Kuo and Hoch (1996) have investigated adhesion of the pycnidiospores of Phyllosticta ampelicida, the causal agent of black rot of grape. In this species, adhesion was a prerequisite for germination but, in contrast to the work with macroconidia of Nectria haematococca, the attachment of spores to poorly wettable surfaces such as polystyrene was not inhibited by sodium azide.
Moreover, adhesion occurred in water within a few minutes and in less than 0.03 s if the water was acidified. Both of these observations suggest that, unlike macroconidia of Nectria haematococca, attachment is not dependent on metabolic activities of the spores. On wettable surfaces such as nutrient- and water-agars or heat-treated glass the pycnidiospores did not attach firmly and did not germinate.
Adhesion is also a necessity for fungi that employ appressoria for the direct penetration of plants. The chemical nature of the glue that sticks the appressoria of Magnaporthe grisea to surfaces has been reported to consist of a mixture of lipids, proteins, sugars and water but unidentified substances made up nearly a quarter of the material investigated.
Adhesion is also crucial to the successful parasitism of plants by bacteria. Many bacteria produce fimbriae and they play a role, for example, in the attachment of Pseudomonas syringae pv. phaseolicola to bean leaves as well as Pseudomonas solancearum to walls of tobacco leaf cells.
Mutation of the chvB locus of Agrobacterium tumefaciens gave rise to phenotypes that were non-motile, avirulent and could not attach to host roots. However, motility, virulence and attachment were restored if the mutants were grown in a medium of high osmolarity containing Cations.
Under these conditions, the mutants were able to produce an active 14-kDA outer membrane protein, termed rhicadhesin, which is also important in the attachment of Rhizobium leguminosarum bv. viciae to pea roots.
The haustoria of parasitic angiosperms such as Striga must also adhere firmly to the roots of their hosts in order to penetrate them and experiments with monoclonal antibodies suggested that pectin, which accumulated between the pathogen and the host root, fulfilled this function.
Although for many plant pathogens a capacity to breach the cell walls of their hosts is not required for entry since they rely on wounds, natural openings or vectors, many fungal pathogens achieve entry by mechanical force or enzyme activity or a combination of both.
One method fungi have developed for applying considerable pressure on a restricted area is to produce melanized appressoria which adhere tightly to surfaces and within which massive turgor pressures are developed.
In the rice blast pathogen, Magnaporthe grisea, germinating conidia differentiate melanized appressoria in response to the physical cues of hydrophobicity and hardness as well as chemical signals. Synthesis of melanin is crucial since buff mutants, which are defective in melanin production, are non-infective. Melanin is permeable to water but is impermeable to solutes, including glycerol.
De Jong and coworkers (1997) found that this sugar alcohol accumulated to molar concentrations in appressoria allowing the generation of osmotic pressures as high as 8.0MPa (80 bar – roughly 40 times the pressure of a car tyre).
Restriction enzyme mediated integration (REMI) is a mutagenesis technique that has been used to identify a number of genes involved in pathogenesis, including penetration. Clergeot and co-workers (2001) have described a non-pathogenic mutant of M. grisea, named punchless, and obtained by this technique.
The gene disrupted, PLS1, encodes a putative integral membrane protein of225 amino acids with homology to the tetraspanin family. The authors suggest that PLS1 is essential for the differentiation of the penetration peg.
A considerable literature has accumulated that implicates degradative enzymes in pathogenesis or virulence. Early work was particularly concerned with pectic enzymes, which are likely to be important not only directly in ingress and destruction of structural materials, but also indirectly as a source of nutrient for the pathogen, since the depolymerization of pectic substances to monomers or oligomers of a low degree of polymerization would be readily assimilated.
However, partial depolymerization may give rise to oligomers that function as elicitors of defence reactions. More recently, other enzymes such as lipases, cutinases and proteases have been investigated, in some instances with particular reference to the ability of an organism to penetrate its host. A further point for consideration is that some enzymes are able to kill cells.
Before the routine application of molecular biology techniques to plant pathology, the case for the involvement of degradative enzymes in pathogenesis or virulence was usually made on the basis of six criteria set out by Cooper (1983), to which two others have been added more recently –
(1) The ability to produce enzymes in vitro,
(2) Detection of the enzymes in infected tissue,
(3) Depletion of plant material such as the middle lamella,
(4) Correlation of enzyme production with virulence or pathogenicity,
(5) Reproduction of symptoms of the disease with purified enzymes,
(6) Reduction of symptoms in vivo when enzyme activity is inhibited. To these must now be added the following –
(7) Fusing the promoter of the gene specifying the enzyme of interest to a reporter gene such as GUS (encoding glucuronidase),
(8) Genetically engineering alterations in enzyme production and demonstrating corresponding alterations in pathogenicity or virulence.
Such alterations may take the form of gene complementation of a deficient mutant, heterologous gene expression, antisense gene expression and, most directly, gene disruption.
The first of these points is the weakest. Most fungal and bacterial pathogens of plants produce enzymes that degrade plant materials in vitro, but so do a great many saprophytic species since they are requirements for metabolizing the dead or dying vegetation that constitutes most of the carbonaceous material which is deposited on the soil surface. Furthermore, enzymes produced by an organism in culture may differ considerably from those produced in the plant.
Thus, the simple demonstration of the production enzymes in vitro must be regarded as only an indication that they may have a role to play in vivo or pathogenesis. In the case of necrotrophs, for example – some enzymes may be required purely for the saprophytic phase of growth. Indeed, the ability of many plant pathogens to secrete a multiplicity of degradative enzymes has often thwarted their unequivocal demonstration as pathogenicity or virulence factors.