Solani Cell Walls Research Articles

Biocontrol and Action Mechanism of Bacillus subtilis Lipopeptides' Fengycins Against Alternaria solani in Potato as Assessed by a Transcriptome Analysis.

Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01’s fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01’s secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins’ biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.

Heat-Stable Antifungal Factor and Expression Patterns of Protease by Chitinase-Producing Bacterium, Pedobacter sp. PR-M6

This study was conducted to investigate the protease expression patterns of the novel chitinase-producing bacterium Pedobacter sp. PR-M6. Protease were detected on SDS-PAGE gel as condition of 4℃, 25℃ and 37℃ to activate of enzymes obtained from incubation of LB, NB and 1% skim milk medium at 4℃, 20℃ and 30℃ after 21 days of incubation, respectively. Pedobacter sp. PR-M6 displayed proteolytic activity on SDS-PAGE gels containing 0.1% (w/v) gelatin, and protease isozymes of PR-M6 were expressed as two major bands (P7 and P8) and seven minor bands (P1, P2, P3, P4, P5, P6, and P9). Protease isozymes of PR-M6 were inhibited at 4℃ and 25℃. The two protease isozymes P5 and P6 were newly expressed in the LB culture medium at 20℃. To investigate the properties of a heat-stable antifungal factor (HSAF), PR-M6 stain was cultured to obtain the products of biosynthesis for incubation in LB medium. HSAF exhibited active biosynthesis at 20℃ for incubation in LB medium. Crude HASF obtained from LB20 medium showed hyphal growth inhibition of Rhizoctonia solani. Inhibition rate of R. solani growth was as high as 67% at 24 h and 47.8% at 48 h after incubation with 1,000 ppm of crude HSAF. Trypan blue stained the inside of the edge for destructed R. solani cell walls after crude HSAF treatment. These results indicated that HSAF and protease from Pedobacter sp. PR-M6 could be used as biocontrol agents against phytopathogens in agriculture.

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.

FsFKS1, the 1,3-β-Glucan Synthase from the Caspofungin-Resistant FungusFusarium solani

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.

A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases

Genome-wide analysis of chitinase genes in the Hypocrea jecorina (anamorph: Trichoderma reesei) genome database revealed the presence of 18 ORFs encoding putative chitinases, all of them belonging to glycoside hydrolase family 18. Eleven of these encode yet undescribed chitinases. A systematic nomenclature for the H. jecorina chitinases is proposed, which designates the chitinases corresponding to their glycoside hydrolase family and numbers the isoenzymes according to their pI from Chi18-1 to Chi18-18. Phylogenetic analysis of H. jecorina chitinases, and those from other filamentous fungi, including hypothetical proteins of annotated fungal genome databases, showed that the fungal chitinases can be divided into three groups: groups A and B (corresponding to class V and III chitinases, respectively) also contained the so Trichoderma chitinases identified to date, whereas a novel group C comprises high molecular weight chitinases that have a domain structure similar to Kluyveromyces lactis killer toxins. Five chitinase genes, representing members of groups A-C, were cloned from the mycoparasitic species H. atroviridis (anamorph: T. atroviride). Transcription of chi18-10 (belonging to group C) and chi18-13 (belonging to a novel clade in group B) was triggered upon growth on Rhizoctonia solani cell walls, and during plate confrontation tests with the plant pathogen R. solani. Therefore, group C and the novel clade in group B may contain chitinases of potential relevance for the biocontrol properties of Trichoderma.

Proteomic response of the biological control fungus Trichoderma atroviride to growth on the cell walls of Rhizoctonia solani

Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea, therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of the R. solani cell walls. Twenty-four protein spots were identified using matrix-assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and N-terminal sequencing. Identified up-regulated proteins include known fungal cell wall-degrading enzymes such as N-acetyl-beta-D: -glucosaminidase and 42-kDa endochitinase. Three novel proteases of T. atroviride were identified, containing sequence similarity to vacuolar serine protease, vacuolar protease A and a trypsin-like protease from known fungal proteins. Eukaryotic initiation factor 4a, superoxide dismutase and a hypothetical protein from Neurospora crassa were also up-regulated as a response to R. solani cell walls. Several cell wall-degrading enzymes were identified from the T. atroviride culture supernatant, providing further evidence that a cellular response indicative of biological control had occurred.

Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum.

We investigated the regulation of chit33 expression in Trichoderma harzianum CECT 2413. This gene encodes the Chit33 endochitinase, which is a major component of the fungus' chitinolytic enzyme system and is important for biocontrol. To this end, both Northern analysis and reporter gene fusions of a 1.4-kb fragment of the 5'-upstream sequences of chit33 to the Aspergillus niger goxA gene (encoding glucose oxidase) and the Aquorea victoria green fluorescent protein were used. Northern analysis and data obtained with the reporter systems were compatible, thus showing that the 1.4-kb fragment bears all necessary information for the regulation of chit33 gene expression. chit33 is weakly expressed during growth on chitin and Rhizoctonia solani cell walls. The addition of N-acetylglucosamine transiently induced chit33 expression in resting cells of the fungus. The addition of either glucose or glycerol prevented induction of chit33 gene expression by chitin or cell walls. Incubation of T. harzianum in the presence of low concentrations (0.1%, w/v) of glucose and high concentrations (38 mM) of ammonium sulfate, or in the presence of high concentrations (1%, w/v) of glucose and low concentrations (0.38 mM) of ammonium sulfate also stimulated chit33-mRNA accumulation, although to a lower degree than induction by N-acetylglucosamine. Transfer of T. harzianum cultures to either 40 degrees C or 4 degrees C initiated a very rapid expression of chit33 in the absence of an inducer, yet only at very low levels (5%) of the induced control. Confrontation experiments, using the gfp gene as a reporter and R. solani as a host, showed that chit33 is expressed only during but not before the stage of overgrowth on R. solani. These data show that Chit33 is an enzyme involved in mycoparasitism; and its formation is controlled by induction, by either carbon or nitrogen starvation and, to a low degree, also under conditions of temperature stress.

Constitutive hydrolytic enzymes are associated with polygenic resistance of tomato to Alternaria solani and may function as an elicitor release mechanism

Foliar resistance to early blight disease of tomato, caused by the necrotrophic fungus Alternaria solani, is inherited in a complex quantitative manner. Our previous studies revealed that three moderately-resistant tomato breeding lines with different sources of early blight resistance, all possessed higher constitutive and more rapid accumulation of PR proteins, including specific antifungal isozymes of chitinase and β-1,3-glucanase than susceptible genotypes. In the present study, additional early blight resistant tomato breeding lines and susceptible genotypes were investigated for their constitutive levels of PR proteins. All resistant lines possessed higher constitutive levels of chitinase and β-1,3-glucanase than susceptible genotypes supporting earlier reported findings. Constitutive and pathogen-induced expression of PR genes during early stages of the interaction was found to be much higher in the highly resistant breeding line NC 24-E than a susceptible line.In vitro elicitor release experiments were performed with purified A. solani cell walls treated with constitutive total enzyme preparations isolated from resistant and susceptible plants. Enzyme preparations from resistant and susceptible genotypes differed in their ability to produce hypersensitive response (HR) elicitors from fungal cell walls: the enzyme preparations from resistant breeding lines released HR elicitors, while enzyme preparations from susceptible lines did not. Additionally, experiments with a partially purified preparation of basic tomato chitinases demonstrated that these enzymes were able to release HR elicitors from germinating spores of A. solani, but not mature, intact cell walls. The possibility that constitutively produced hydrolytic enzymes may act as an elicitor-releasing mechanism in resistance to early blight of tomato is discussed.

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls.

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.

Enzyme Activity and Electrophoretic Profile of Extracellular Protein Induced in Trichoderma spp. by Cell Walls of Rhizoctonia solani

Summary: 1,3-β-D-Glucanase and chitinase activities were induced in Trichoderma harzianum when glucose or cell walls of Rhizoctonia solani (anastomosis group AG2) were used as sole carbon source. Electrophoresis showed that more proteins were induced by cell walls than by glucose. The composition, as revealed by electrophoresis, of the T. harzianum extracellular proteins was similar when grown on R. solani AG2 and AG4 cell walls but different for R. solani AG1 cell walls. Several major proteins were induced in all strains of T. harzianum but there were strain differences in the number and intensity of other proteins. The number of induced proteins was less for four strains of T. viride and their composition was different from those of five strains of T. harzianum. The results indicate that chitinase, 1,3-β-D-glucanase and a large number of other extracellular proteins may be involved in the degradation of R. solani cell walls.

Chitosan as a Component of Pea-Fusarium solani Interactions

Chitosan, a polymer of beta-1,4-linked glucosamine residues with a strong affinity for DNA, was implicated in the pea pod-Fusarium solani interaction as an elicitor of phytoalexin production, an inhibitor of fungal growth and a chemical which can protect pea tissue from infection by F. solani f. sp. pisi. Purified Fusarium fungal cell walls can elicit phytoalexin production in pea pod tissue. Enzymes from acetone powders of pea tissue release eliciting components from the F. solani f. sp. phaseoli cell walls. Hydrochloric acid-hydrolyzed F. solani cell walls are about 20% glucosamine. The actual chitosan content of F. solani cell walls is about 1%. However, chitosan assays and histochemical observations indicate that chitosan content of F. solani spores and adjacent pea cells increases following inoculation. Dormant F. solani spores also accumulate chitosan. Concentrations of nitrous acid-cleaved chitosan as low as 0.9 microgram per milliliter and 3 micrograms per milliliter elicit phytoalexin induction and inhibit germination of F. solani macroconidia, respectively. When chitosan is applied to pea pod tissue with or prior to F. solani f. sp. pisi, the tissue is protected from infection.