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Structure and differentiation of the cell wall of Phytophthora palmivora: Cysts, hyphae and sporangia
Abstract and Figures
Walls from cysts, hyphae and sporangia of Phytophthora palmivora consist chiefly (ca. 90% dry wt) of ß-glucans with 1,3-, 1,4- and 1,6-links. The glucans are predominatly ß-1,3-linked but there are significant differences in the relative proportion of 1,3-, 1,6- and 1,4-linked glucosyl residues among the three wall types. There are also differences in protein content, susceptibility to degradation by various ß-glucanases, and surface texture. The isolated cyst wall consists solely of a thin fabric of long, tightly interwoven, randomly oriented microfibrils. Both inner and outer surfaces of the cyst wall are distinctly microfibrillar. The hyphal wall has two different textures; the internal surface is distinctly microfibrillar while the external surface is non-fibrillar. In a germinated cyst, there is a zone of demarcation where the microfibrils of the cyst wall disappear into the smooth outer texture of the germ tube wall. An exo-ß-1,3-glucanase preferentially removed the amorphous material of the outer surface of the germ tube leaving exposed a continuous microfibrillar fabric from cyst to hyphal tube. Conceivably, the textural and structural differentiation of the cell wall may play a decisive role in cellular morphogenesis.
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... These results are consistent with the fact that P. palmivora cell walls are composed of ß-glucan mainly 1,3-, 1,6-and 1,4-links (ca. 90% substrates for various ß-glucanase and chitnase degradation, respectively (Tokunaga and Bartnicki-Garcia, 1971;Hamid et al., 2013;Mélida et al., 2013). In addition, the lower chitinase P. palmivora cell walls. ...
... According to Mélinda et al. (2013), the cell wall analyses of two plant pathogenic species, Phytophthora infestans and Phytophthora parasitica were identified as Type I. Type I cell walls consisted 85.6% β-glucans where 32 to 35% was made up of cellulose (1,4-β linked glucose; β-1,4-Glc) and about 19.7% of β-1,3-glucans (Mélinda et al. 2013). In the case of P. palmivora cell walls, Tokunaga and Bartnicki-Garcia (1971) demonstrated that walls of cysts, hyphae and sporangia of P. palmivora consisted of β-glucans with 1,3-, 1,4-and 1,6-linkages. Later work by Lippmann et al. (1974) demonstrated that chemical composition of oospore-oogonium walls (oow) of Phytophthora megasperma var. ...
Calcofluor white is a fluorochrome used for detecting β-glucans in cell walls of plant pathogenic fungi. The aim of this study was to detect β-glucans in oospores of the heterothallic Phytophthora palmivora by crossing two compatible A1 and A2 mating types on carrot agar plates with or without a supplement of aqueous French bean extract. Lack of calcofluor white induced fluorescence, in yellow to deep brown oospores, suggests a change in the type of β-glucans in the outer oospore-oogonium cell wall. This staining method is an easy, quick and visual way to monitor changes in β-glucans during oospore development.
... Accordingly, it is possible to conclude that when cellulase enzyme molecules are available in the growth medium, the ability of Phytophthora to become pathogenic would be diminished and subsequently their growth would be inhibited because of the degradation of biosynthesized cellulose in their cell walls. Especially, there are also β-glucans in Phytophthora cell wall (Tokunaga and Bartnicki-Garcia, 1971). Cellulase is an enzyme complex which is usually composed of exoglucanases, endoglucanases, and β-glucosidase (Jayasekara and Ratnayake, 2019). ...
Damping-off disease is caused by Pythium sp., Phytophthora sp., Rhizoctonia sp., and Fusarium sp., and affects seedlings at the nursery stage. Phytophthora and Pythium cell walls contain cellulose. The current study aimed to determine potential use of cellulase extracts of selected fungal species i.e. Talaromyces sp., Trichoderma sp., Aspergillus sp., A. niger, and Penicillium oxallicum to suppress these pathogens and to investigate the potential use of fungal co-culture extracts with the highest cellulase activity for seed treatment to control damping-off. The impact of cellulase extracts on pathogen growth was determined by agar well diffusion method. For seed treatment, tomato seeds were soaked in cellulase extracts from the selected co-culture for 24 h and were sown in pathogens inoculated soil samples. Cellulase extracts of Trichoderma and Aspergillus suppressed Phytophthora growth with 63% and 57% of inhibition percentages respectively. When seeds were treated with Talaromyces sp.+Aspergillus sp.+P. oxallicum co-culture extracts, the highest seedling emergence percentage was observed in Phytophthora sp. Fusarium sp., and four-pathogen mixture-inoculated soils respectively. Lowest disease incidence was observed in Phytophthora sp. inoculated soil. Results show the potential of cellulolytic fungal species; Trichoderma sp., Aspergillus sp., and co-culture of Talaromyces sp.+Aspergillus sp.+P. oxallicum in suppression of Phytophthora sp. a common causative agent of damping-off.
... β-1,3-glucanases, which are a group of PR proteins belonging to the PR-2 family, are released in the early stage of plantpathogen interaction and participate in the hydrolysis of β-1,3glucans, the main structural component of oomycete cell walls, to restrict pathogen growth and development (Doxey et al., 2007;Balasubramanian et al., 2012). Tokunaga and Bartnicki-Garcia (1971) reported that the cell walls of both sexual and asexual fruiting units (sporangia) of oomycetes are mainly composed of β-1,3-glucans. In vitro studies with pure β-1,3-glucanases and in vivo studies with transgenic plants overexpressing β-1,3-glucanases have shown that these enzymes inhibit fungi (Mondal et al., 2007) and fungi-like pathogens (Tonón et al., 2002;Nookaraju et al., 2012) both directly by catalyzing the cleavage of β-1,3-glucans and indirectly by the formation of other elicitors, which then trigger defense reactions in the host cell (Andreu et al., 1998). ...
N-acyl-homoserine lactones (AHLs), a well-described group of quorum sensing (QS) molecules, may modulate plant defense responses and plant growth. In the present study, long-chained oxo-C14-HSL (N-3-oxo-tetradecanoyl-L-homoserine lactone) was shown to a distinct potential to prime cucumber for enhanced defense responses against biotrophic oomycete pathogen Pseudoperonospora cubensis (P. cubensis) and hemibiotroph bacterium P. syringae pv. lachrymans (Psl). We provide evidence that AHL-mediated enhanced defense against downy mildew disease is based on cell-wall reinforcement by lignin and callose depositions, activation of defense-related enzymes, accumulation of ROS and phenolic compunds.
... Based on SEM, it appeared that the action of the partial purified proteins occurred during hypha development or cell wall synthesis. In case of Phytophthora, cell wall compositions were composed of 80-90% glucan, 5-10% protein, and 1-2% lipids [40]. In addition, cell-wall-associated proteins were also identified in this oomycete mold, and these proteins were proposed to be important for pathogenicity [41]. ...
Black rot disease in orchids is caused by the water mold Phytophthora palmivora. To gain better biocontrol performance, several factors affecting growth and antifungal substance production by Pseudomonas aeruginosa RS1 were verified. These factors include type and pH of media, temperature, and time for antifungal production. The results showed that the best conditions for P. aeruginosa RS1 to produce the active compounds was cultivating the bacteria in Luria-Bertani medium at pH 7.0 for 21 h at 37 °C. The culture filtrate was subjected to stepwise ammonium sulfate precipitation. The precipitated proteins from the 40% to 80% fraction showed antifungal activity and were further purified by column chromatography. The eluted proteins from fractions 9–10 and 33–34 had the highest antifungal activity at about 75% and 82% inhibition, respectively. SDS-PAGE revealed that the 9–10 fraction contained mixed proteins with molecular weights of 54 kDa, 32 kDa, and 20 kDa, while the 33–34 fraction contained mixed proteins with molecular weights of 40 kDa, 32 kDa, and 29 kDa. Each band of the proteins was analyzed by LC/MS to identify the protein. The result from Spectrum Modeler indicated that these proteins were closed similarly to three groups of the following proteins; catalase, chitin binding protein, and protease. Morphological study under scanning electron microscopy demonstrated that the partially purified proteins from P. aeruginosa RS1 caused abnormal growth and hypha elongation in P. palmivora. The bacteria and/or these proteins may be useful for controlling black rot disease caused by P. palmivora in orchid orchards.
... (X36.000) (Reprinted, by permission, fromTokunaga and Bartnicki-Garcia, 1971b) ...
... EA6 is of a proteinaceous nature. Since glucan (β-1,3, 1,4-and 1,6-links) constitute > 90% of Phytophthora cell walls (Tokunaga and Bartnicki-Garcia, 1971), we suspected that the anti-Phytophthora activiy of Pseudomonas sp. EA6 might be due to glucanases. ...
Biological control is an eco-friendly strategy for mitigating and controlling plant diseases with negligible effects
on human health and environment. Biocontrol agents are mostly isolated from field crops, and microbiomes
associated with wild native plants is underexplored. The main objective of this study was to characterize the
bacterial isolates associated with Smilax bona-nox L, a successful wild plant with invasive growth habits. Forty
morphologically distinct bacterial isolates were recovered from S. bona-nox. Based on 16S rRNA gene sequencing, these isolates belonged to 12 different genera namely Burkholderia, Pseudomonas, Xenophilus,
Stenotrophomonas, Pantoea, Enterobactriaceae, Kosakonia, Microbacterium, Curtobacterium, Caulobacter,
Lysinibacillus and Bacillus. Among them, Pseudomonas sp. EA6 and Pseudomonas sp. EA14 displayed the highest
potential for inhibition of Phytophthora. Based on sequence analysis of rpoD gene, these isolates revealed a 97%
identity with a Pseudomonas fluorescence strain. Bioactivity-driven assays for finding bioactive compounds revealed that crude proteins of Pseudomonas sp. EA6 inhibited mycelial growth of P. parasitica, whereas crude
proteins of Pseudomonas sp. EA14 displayed negligible activity. Fractionation and enzymatic analyses revealed
that the bioactivity of Pseudomonas sp. EA6 was mostly due to glucanolytic enzymes. Comparison of chromatographic profile and bioactivity assays indicated that the secreted glucanolytic enzymes consisted of β-1,3 and
β-1,4 glucanases, which acted together in hydrolyzing Phytophthora cell walls. Since the biological activity of the
crude glucanolytic extract was > 60-fold higher than the purified β-1,3 glucanase, the glucanolytic enzyme
system of Pseudomonas sp. EA6 likely acts synergistically in cell wall hydrolysis of P. parasitica.
Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1, which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amides fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control.
Because microbodies in many filamentous fungi fail to stain with the 3,3′-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalasc. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.
The changes in cytoplasmic organization up to the early stages of primary spore initial delimitation are described in sporangia of four species of Saprolegniaceae. Organelle associations characteristic of asexual and vegetative stages are present in the cytoplasm of presporangium hyphae of Achlya debaryana. Organelles are aligned parallel to the axis of hyphal growth and the apex assumes the characteristic profile of a sporangium. Dense-body vesicles possibly originate from expanded cisternae of endoplasmic reticulum at this stage. In Dictyuchus anomalus, D. pseudodictyon, and Brevilegnia minutandra, nuclei of the primary spore initial stage orient radially in the cytoplasm with nucleus-associated organelles close to the sporangium wall. Nuclei in the central region of sporangia degenerate.
Isolated cell walls of the oomycetous fungus, Sapromyces elongatus were studied by biochemical analysis, electron microscopy and X-ray diffraction. The walls were composed of more than 90% glucan with 3.7% protein, 0.1% ash and only traces of glucosamine and phosphorus. The protein component consisted of 17 amino acids including 2.5% (of total amino acids) hydroxyproline. The glucans showed the typical Oomycete pattern, which consisted of a large amount of β-1 → above; 3 and β-1 →; above; 6 linked polymers with a smaller amount of cellulose (β-1 →; above; 4 linkage). The cellulose occurred as poorly crystalline cellulose I and microfibrils were observed in wall preparations that gave cellulose X-ray reflections. The amorphous matrix of the waits was partially removed by an enzyme complex that contained β-1 →; above; 3 and β-1 →l above; 6 glucanase activities.
Chitin has long been defined as a substance that contains nitrogen, is insoluble in strong alkali solutions at 140° to 160°, and which on hydrolysis yields glucosamine and acetic acid. It has been established that some preparations of both animal and fungal origin are almost certainly straight chain, β 1–4 linked, polymers of acetylglucosamine (N-acetyl-2-amino-2-deoxy-d-glucose). On hydrolysis such a substance should yield 29,5% of its weight as acetic acid and 88% as glucosamine (106% as glucosamine hydrochloride). Its nitrogen content should be 6,9%. There is, as yet, no evidence that substances giving all the reactions of chitin described in Section B do not contain such a polymer, and X-ray evidence lends support to the conclusion that all so examined do contain it. A preparation that contains the theoretical amount of nitrogen and acetyl groups is a rarity owing to the difficulties of purification without degradation. It will be assumed in this article that materials satisfying the tests in Section B do in fact contain a pure acetylglucosamine polymer and that this is chitin. The reasons for this cautious approach will become apparent. It must be emphasised at the outset that to show that a material is “insoluble” and that on hydrolysis glucosamine is formed is no justification for assuming that it contains chitin. There are many polymers in both animals and lower plants that are insoluble under some conditions and that contain acetylglucosamine units. They are, however, mixed polymers and chitin is the only unmixed polymer of acetylglucosamine for which evidence has yet been secured.
Flagella and cleavage vesicles form during the initial stages of direct germination in the same manner as they do in indirect germination. Later, however, the flagella degenerate and cleavage of the cytoplasm is not complete. Instead, a new wall layer is deposited onto the existing sporangial wall, and this germination wall extends with the germ tube and forms the hyphal wall. Material for the germination wall first appears around vesicular aggregates concentrated at the periphery of the sporangial cytoplasm. As the wall forms the vesicular aggregates become encased in it, and the wall eventually consists of irregular deposits of wall material interspersed with the vesicular aggregates. These are clearly cytoplasmic in nature and different from lomasomes, since they are associated with ribosomes and other small elements of the cytoplasm. Likely to contribute to the wall formation are the endoplasmic reticulum, microbodies, dictyosome-derived vesicles, and possibly polyvesicular bodies and larger vacuoles with fibrillar contents. The basal plug of the sporangium forms the same way as the germination wall and also contains numerous vesicular inclusions. Fewer vesicular inclusions are found in the sporangial wall, and none have been observed in the walls of the growing hyphae. This difference in wall structure can be explained on the basis of their different growth patterns. Penetration of the sporangial plug is concomitant with prominent lomasomal activity and the cytoplasm at the hyphal tip is characterized by the presence of numerous vesicles, which are probably derived from dictyosomes.
Isolated cell walls of the oomycetous fungus, Sapromyces elongatus were studied by biochemical analysis, electron microscopy and X-ray diffraction. The walls were composed of more than 90% glucan with 3.7% protein, 0.1% ash and only traces of glucosamine and phosphorus. The protein component consisted of 17 amino acids including 2.5% (of total amino acids) hydroxyproline. The glucans showed the typical Oomycete pattern, which consisted of a large amount of β-1 → 3 and β-1 → 6 linked polymers with a smaller amount of cellulose (β-1 → 4 linkage). The cellulose occurred as poorly crystalline cellulose I and microfibrils were observed in wall preparations that gave cellulose X-ray reflections. The amorphous matrix of the walls was partially removed by an enzyme complex that contained β-1 → 3 and β-1 → 6 glucanase activities.
The appearance of the walls of apical and sub-apical regions of hyphae of predominantly 5 day cultures of Neurospora crassa, Schizophyllum commune and Phytophthora parasitica as seen with the electron microscope, employing shadowed or sectioned material, is illustrated and described in detail. The appearance of untreated or control material in buffer is compared with that exposed to various single and sequential treatments with enzymes, including laminarinase, Pronase, cellulase and chitinase, as well as various chemical treatments.
From these observations is inferred the co-axial distribution of polymers such as β1, 3-, β1, 6-glucans, glycoproteins, proteins, chitin and cellulose in the walls of each species. The distributions have both similarities and differences between the species. The significance of all these features for the growth, mechanical rigidity and integrity of a hypha is briefly discussed.