Electrotaxis of zoospores of Phytophthora palmivora at physiologically relevant field strengths

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. We show here that zoospores of the plant pathogen Phytophthora palmivora exhibit anodal electrotaxis in electrical fields ≥0.5 V m⁻¹ comparable in size to the physiological fields around roots. An experimental protocol for applying weak electrical fields and quantifying electrotaxis is described. In this system, zoospore suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of encysted zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. The electrotactic response saturated at fields above 10 V m⁻¹ was inhibited in media of osmotic strength below 400 Osmol m⁻³, was maximal at pH 7.5 and increased at high zoospore densities. These data suggest that electrotaxis may be a useful adjunct to chemotaxis in root targeting by zoospores.     

Moments of weaknesses – exploiting vulnerabilities between germination and encystment in the Phytomyxea

Attempts at management of diseases caused by protozoan plant parasitic Phytomyxea have often been ineffective. The dormant life stage is characterised by long-lived highly robust resting spores that are largely impervious to chemical treatment and environmental stress. This review explores some life stage weaknesses and highlights possible control measures associated with resting spore germination and zoospore taxis. With phytomyxid pathogens of agricultural importance, zoospore release from resting spores is stimulated by plant root exudates. On germination, the zoospores are attracted to host roots by chemoattractant components of root exudates. Both the relatively metabolically inactive resting spore and motile zoospore need to sense the chemical environment to determine the suitability of these germination stimulants or attractants respectively, before they can initiate an appropriate response. Blocking such sensing could inhibit resting spore germination or zoospore taxis. Conversely, the short life span and the vulnerability of zoospores to the environment require them to infect their host within a few hours after release. Identifying a mechanism or conditions that could synchronise resting spore germination in the absence of host plants could lead to diminished pathogen populations in the field.     

Encystment of Pythium aphanidermatum Zoospores is Induced by Root Mucilage Polysaccharides, Pectin and a Monoclonal Antibody to a Surface Antigen

The infection of roots by the pathogenic Oomycete Pythium aphanidermatuminvolves interactions between the fungal zoospores and rootsurface mucilage polysaccharides. After initial recognitionat the root surface the zoospores are triggered to encyst duringwhich adhesive glycoproteins are secreted followed by a fibrillarcyst wall. In this paper a simple in vitro assay has been usedto assess the ability of a variety of macromolecules to inducezoospore encystment. Mucilage polysaccharides of the cress rootsurface trigger encystment. Whole mucilage was fractionatedby gel filtration and a fraction low in uronic acid, containing5% fucose, was shown to be more effective in triggering encystmentthan a uronic acid-rich fraction. Encystment can also be inducedby commercial pectin. The lectin Con A, and PA1, one of a rangeof monoclonal antibodies specific for zoospore surface antigens,also triggered encystment. In Western blotting experiments PA1recognizes protein epitopes of a 75 kDa surface antigen. Theresults suggest that at least one mechanism of zoospore triggeringmay involve a specific zoospore surface receptor. Key words: Pythium aphanidermatum, recognition, encystment, zoospore, mucilage, root, monoclonal antibodies, polysaccharides     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Pattern swimming of Phytophthora citricola zoospores: an example of microbial bioconvection

The genus Phytophthora, belonging to the class Oomycota, comprises a group of over fifty fungus-like plant pathogens in both managed and unmanaged ecosystems. A unique feature of the oomycete lifecycle is a zoosporic stage in which motile, unicellular propagules, serving as the primary agents of dispersal, are produced and released in the presence of water. In Petri dish suspensions, zoospores frequently exhibit 'pattern swimming', whereby they spontaneously form concentrated swimming masses, visible to the naked eye, even in the absence of a chemical or electrical gradient. The nature of this behaviour is unclear, but is of interest because of the potential for auto-attraction and implications for cohort recruitment during infection. Similar behaviour observed in a variety of motile bacteria, algae, and protists is attributed to 'bioconvection' that results from instability in fluid density due to the organisms' upward-swimming tendency and greater-than-water density. In this investigation, we determined that Phytophthora citricola zoospore 'pattern swimming' is unrelated to phototaxis, surface tension-driven (Marangoni) convection, or auto-attraction and that the observed convective pattern, directional swimming, and depth- and concentration dependence are consistent with bioconvection.     

Phytophthora parasitica biofilm formation: installation and organization of microcolonies on the surface of a host plant

Zoospores of the oomycete Phytophthora parasitica establish microbial spheroid microcolonies and biofilms on the surface of wounded leaves of their host, Nicotiana tabacum . The formation of microcolonies involves the movement of some zoospores towards attractants from wound sites, followed by their irreversible adsorption and the formation of a cluster of cells. These cells drive the migration of a second wave of zoospores (several hundreds cells) by setting up an external chemotactic gradient leading to massive zoospore encystment and cyst-orientated germination. Zoospores that are still swimming at this stage circulate within the nascent biofilm by opening channels. Concomitantly, the cell population secretes various substances to elaborate an extracellular mucilage. Embedded within the extracellular matrix, biofilm cells are organized into a structured community as coacervates. The granular surface is composed of individual cysts, located on the outside of the microcolony. Hyphae from these cysts plunge downwards towards the dense core formed by the founder cells. This report is the first to show the installation and organization of a biofilm formed by eukaryotic cells on plant surfaces. The P. parasitica microcolonies constitute heterogeneous microenvironments for the embedded and circulating cells. They may affect plant–pathogen interactions by serving as reservoirs for pathogenic microorganisms, as protecting niche against host defences or as structures for infecting populations.     

Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus

Atlantic menhaden Brevoortia tyrannus develop characteristic skin ulcers in response to infection by the oomycete Aphanomyces invadans. To investigate pathogenicity, we conducted a dose response study. Juvenile menhaden were inoculated subcutaneously with 0, 1, 5, 10, 100, and 500 secondary zoospores per fish and monitored for 37 d post-injection (p.i.). Survival rates declined with increasing zoospore dose, with significantly different survivorship curves for the different doses. Moribund and dead fish exhibited characteristic ulcerous lesions at the injection site starting at 13 d p.i. None of the sham-injected control fish (0 zoospore treatment) died. The LD50 (lethal dose killing 50% of exposed menhaden) for inoculated fish was estimated at 9.7 zoospores; however, some fish receiving an estimated single zoospore developed infections that resulted in death. Menhaden were also challenged by aqueous exposure and confirmed that A. invadans was highly pathogenic by this more environmentally realistic route. Fish that were acclimated to culture conditions for 30 d, and presumably free of skin damage, then aqueously exposed to 100 zoospores ml(-1), exhibited 14% lesion prevalence with 11% mortality. Net-handled fish that were similarly infected had a significantly higher lesion prevalence (64%) and mortality (64%). Control fish developed no lesions and did not die. Scanning electron microscopy of fish skin indicated that zoospores adhered to intact epidermis, germinated and penetrated the epithelium with a germ tube. Our results indicate that A. invadans is a primary pathogen of menhaden and is able to cause disease at very low zoospore concentrations.     

Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.     

禾谷多粘菌游动孢子超微结构观察

本文描述了寄生在大麦根部的禾谷多粘菌Polymyxa graminis Led.的次生游动孢子的超微结构,包括核、内质网、高尔基体、线粒体、脂质粒、排泄泡、小囊、具膜小囊、核糖体以及鞭毛基体(Kinetosome)和鞭毛杆等细胞器。游动孢子中未见微体。同时也在电镜下观察了游动孢子接触和穿透根细胞时所形成的管腔(Rohr)和棘杆(Stachel)以及游动孢子穿透细胞壁的详细过程。     

The effect of the weight of the seedling-derived tuber on subsequent clonal generations in a potato breeding programme

Cultures of Polymyxa graminis were maintained in roots of barley plants grown in sand at different temperatures using Wisconsin soil temperature tanks. At 17 – 20°C, the minimum time from inoculation with cystosori to the production of zoospores from the inoculated roots was 2 – 3 wk. At 11 – 20°C many zoospores were produced but the incubation period was longer at the lower temperatures. Above 20°C little fungal development occurred. The duration of motility of zoospores ranged from c. 1 h to > 24 h. Bovine serum albumen (BSA) prolonged motility but glycine and glucose had no effect or, at higher concentrations, were toxic. Zoospores were rapidly immobilised by zinc ions in solution at or above 10μg/ml. In some experiments BSA added to the zoospore suspension greatly increased transmission of barley yellow mosaic virus (BaYMV) while glucose, glycine and ovalbumen decreased it. When seedlings were incubated with zoospore suspensions for 24 h at different temperatures, BaYMV transmission was high (> 60%) at 10, 15 and 20°C but there was little at 5 or 25°C. In experiments to determine the time taken for zoospore penetration, seedlings were incubated in suspension for different periods of time and then rinsed in zinc sulphate solution to kill free zoospores. Between 3 and 3·5 h was needed for zoospores to establish infection. Transmission occurred equally to plants of various ages between 3 days and 7·5 wk.     

Behaviour of Phytophthora cinnamomi Zoospores on Roots of Four Avocado Cultivars

Scanning electron microscopy was used to study the behaviour of Phytophthora cinnamomi zoospores on the roots of three tolerant avocado cultivars. Duke 7, G6 and Martin Grande, and a susceptible Edranol cultivar. Zoospores were attracted to the region of cell elongation and encysted on the roots of all cultivars studied. Adhesion of the zoospores appeared to be aided by root slime. Cysts usually produced one germ tube which penetrated the root directly, or formed an appressorium-like swelling before penetration occurred. Extensive growth of germ tubes occurred where zoospores germinated some distance behind the region of elongation. Cysts germinating behind this region often formed branched germ tubes and more than one appressorium-like swelling. There were no clear differences in the type of pre-penetration structures, formed by zoospore cysts, on the roots of the different avocado cultivars.     

Ultrastructural observations of mature and encysting zoospores ofPythium proliferum de Bary

Summary The process of zoospore maturation and encystment inP. proliferum was studied by electron microscopy. General ultrastructural features of the mature, swimming zoospore were found to be similar to those previously described for other oomycetes in both the attachment and ultrastructure of the flagella as well as the type and distribution of cellular organelles. Associated with extensive areas of RER in the mature zoospores were unusual, electrondense, bar-like structures. These structures were found in the groove region of young zoospores and at the periphery of encysting zoospores. Their possible function is discussed. The five main types of vesicles observed during encystment, as seen grouped in this study, along with the vesicles described in previous studies of oomycete encystment, were in table form and individually discussed. Interesting correlations appear to exist in the types of vesicles that are present within the oomycetes studied thusfar.     

Phage-Displayed Peptides as Developmental Agonists for Phytophthora capsici Zoospores

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.     

Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.)

Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of B. cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots. In vitro, B. cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes. B. cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels. However, when present at high levels on seeds, B. cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied B. cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain. This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low. B. cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence. These results suggest that B. cepacia AMMDR1 controls P. aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.     

Evidence that vector transmission of a plant virus requires conformational change in virus particles

Transmission of Cucumber necrosis virus (CNV) by zoospores of its fungal vector, Olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. Previous work has shown that specific components of both CNV and zoospores are required for successful CNV/zoospore recognition. Here, we show that limited trypsin digestion of CNV following in vitro CNV/zoospore binding assays, results in the production of specific proteolytic digestion products under conditions where native CNV is resistant. The proteolytic digestion pattern of zoospore-bound CNV was found to be similar to that of swollen CNV particles produced in vitro, suggesting that zoospore-bound CNV is in an altered conformational state, perhaps similar to that of swollen CNV. We show that an engineered CNV mutant (Pro73Gly) in which a conserved proline residue (Pro73) in the beta-annulus of the CP arm is changed to glycine is resistant to proteolysis following in vitro zoospore binding assays. Moreover, Pro73Gly particles are transmitted only poorly by O.bornovanus. Together, the results of these studies suggest that CNV undergoes conformational change upon zoospore binding and that the conformational change is important for CNV transmissibility.     

Evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides

Despite the importance of vectors in natural dissemination of plant viruses, relatively little is known about the molecular features of viruses and vectors that permit their interaction in nature. Cucumber necrosis virus (CNV) is a small spherical virus whose transmission in nature is facilitated by zoospores of the fungus Olpidium bornovanus. Previous studies have shown that specific regions of the CNV capsid are involved in transmission and that transmission defects in several CNV transmission mutants are due to inefficient attachment of virions to the zoospore surface. In this study, we have undertaken to determine if zoospores contain specific receptors for CNV. We show that in vitro binding of CNV to zoospores is saturable and that vector zoospores bind CNV more efficiently than nonvector zoospores. Further studies show that treatment of zoospores with periodate and trypsin reduces CNV binding, suggesting the involvement of glycoproteins in zoospore attachment. In virus overlay assays, CNV binds to several proteins, whereas CNV transmission mutants either fail to bind or bind at significantly reduced levels. The possible involvement of specific sugars in attachment was investigated by incubating CNV with zoospores in the presence of various sugars. Two mannose derivatives (methyl alpha-D-mannopyranoside and D-mannosamine), as well as three mannose-containing oligosaccharides (mannotriose, alpha3,alpha6-mannopentaose, and yeast mannan) and L-(-)-fucose, all inhibited CNV binding at relatively low concentrations. Taken together, our studies suggest that binding of CNV to zoospores is mediated by specific mannose and/or fucose-containing oligosaccharides. This is the first time sugars have been implicated in transmission of a plant virus.     

Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone     

Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138-146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.     

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.