Fungal Pathogen Rhizoctonia Solani Research Articles

Effects of biocontrol agents on suppression of damping-off in Cucumis sativus L. caused by Rhizoctonia solani

Biocontrol agents (Pseudomonas aeruginosa HB-15 and Pantoea sp. HB-22, alone or in combination) were applied on cucumber (Cucumis sativus L.) plants to evaluate their effect on suppressing damping-off caused by the fungal pathogen Rhizoctonia solani. Plant biomass, percent root mortality, phosphorus uptake, and the levels of stress-indicators NH3-NH4+ and proline in the plants were measured following the treatments. Additionally, gene expression induced by the biocontrol agents was compared to the gene expression induced by jasmonic acid and salicylic acid treatments. The biocontrol treatments increased fresh weight and phosphorus uptake, and reduced root mortality of the cucumber plants inoculated with the pathogen. No differences in plant ecophysiological responses were detected among biocontrol agent treatments. Concentrations of NH3-NH4+ and proline in the leaves and roots were higher in the control than in the biocontrol treatments. Salicylic acid- and Pantoea sp. HB-22-treated cucumbers showed increased expression of the acidic peroxidase-encoding gene and the phenylalanine ammonia homologue, while the P. aeruginosa HB-15 treatment did not induce an increase in expression of any of the genes studied.

Genes of the de novo and Salvage Biosynthesis Pathways of Vitamin B6 are Regulated under Oxidative Stress in the Plant Pathogen Rhizoctonia solani.

Vitamin B6 is recognized as an important cofactor required for numerous metabolic enzymes, and has been shown to act as an antioxidant and play a role in stress responses. It can be synthesized through two different routes: salvage and de novo pathways. However, little is known about the possible function of the vitamin B6 pathways in the fungal plant pathogen Rhizoctonia solani. Using genome walking, the de novo biosynthetic pathway genes; RsolPDX1 and RsolPDX2 and the salvage biosynthetic pathway gene, RsolPLR were sequenced. The predicted amino acid sequences of the three genes had high degrees of similarity to other fungal PDX1, PDX2, and PLR proteins and are closely related to other R. solani anastomosis groups. We also examined their regulation when subjected to reactive oxygen species (ROS) stress inducers, the superoxide generator paraquat, or H2O2, and compared it to the well-known antioxidant genes, catalase and glutathione-S-transferase (GST). The genes were differentially regulated with transcript levels as high as 33 fold depending on the gene and type of stress reflecting differences in the type of damage induced by ROS. Exogenous addition of the vitamers PN or PLP in culture medium significantly induced the transcription of the vitamin B6 de novo encoding genes as early as 0.5 hour post treatment (HPT). On the other hand, transcription of RsolPLR was vitamer-specific; a down regulation upon supplementation of PN and upregulation with PLP. Our results suggest that accumulation of ROS in R. solani mycelia is linked to transcriptional regulation of the three genes and implicate the vitamin B6 biosynthesis machinery in R. solani, similar to catalases and GST, as an antioxidant stress protector against oxidative stress.

Biocontrol of <i>Rhizoctonia solani</i> K1 by Iturin A Producer <i>Bacillus subtilis</i> RB14 Seed Treatment in Tomato Plants

Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentration of bacterial cells for the treatment was about 108 cfu/ml. Treated tomato seeds showed 99% germination index similar to the untreated seeds. Scanning Electron Microscopic observations showed a clear evidence of the presence of B. subtilis RB14 on tomato seed surface. Clear inhibition zone was observed using treated seed in dual plate assay against R. solani K1. B. subtilis RB14 treated seed showed 80% reduction in disease incidence during in vivo plant experiments. B. subtilis RB14 produces lipopeptide antifungal antibiotic iturin A which could suppress R. solani K1. The phenomenon was supported by our observation where we found significant amount of iturin A from the root zone soil of the seed treated plants.

Rhizoctonia solani에 의한 인삼 모잘록병 방제용 종자처리 약제 선발

Damping-off is a critical disease on ginseng seedling, which caused by the fungal pathogen Rhizoctonia solani. The disease has been prevented by tolclofos-methyl for the last 20 years. However, the tolclofos-methyl usually detected on the harvested roots of 6-year-old ginseng. Herein, we tried to select an alternative pesticide which not only must be safe but also efficiently inhibits the fungal pathogen. Four fungicides (fludioxonil, flutolanil, pencycuron, and thifluzamide) were applied to their inhibition efficacy against the pathogen. In in vitro test, fludioxonil treatment showed 80% inhibition activity for 25 days. Thifluzamide and flutolanil showed the activity for 10 days. Pencycuron showed for 1 days. In addition, the fludioxonil was more effective to control the pathogen comparing to other three fungicides in field. The incidence of damping-off was reduced to 71% by fludioxonil treatment. The level of the fungicide residue in seedling was 0.44 mg/kg, which value will be a negligible level in final products after 5 years. Consequently, the fludioxonil is a conceivable alternative for tolclofos-methyl to cope with R. solani.

Diversity and Activity of Lysobacter Species from Disease Suppressive Soils.

The genus Lysobacter includes several species that produce a range of extracellular enzymes and other metabolites with activity against bacteria, fungi, oomycetes, and nematodes. Lysobacter species were found to be more abundant in soil suppressive against the fungal root pathogen Rhizoctonia solani, but their actual role in disease suppression is still unclear. Here, the antifungal and plant growth-promoting activities of 18 Lysobacter strains, including 11 strains from Rhizoctonia-suppressive soils, were studied both in vitro and in vivo. Based on 16S rRNA sequencing, the Lysobacter strains from the Rhizoctonia-suppressive soil belonged to the four species Lysobacter antibioticus, Lysobacter capsici, Lysobacter enzymogenes, and Lysobacter gummosus. Most strains showed strong in vitro activity against R. solani and several other pathogens, including Pythium ultimum, Aspergillus niger, Fusarium oxysporum, and Xanthomonas campestris. When the Lysobacter strains were introduced into soil, however, no significant and consistent suppression of R. solani damping-off disease of sugar beet and cauliflower was observed. Subsequent bioassays further revealed that none of the Lysobacter strains was able to promote growth of sugar beet, cauliflower, onion, and Arabidopsis thaliana, either directly or via volatile compounds. The lack of in vivo activity is most likely attributed to poor colonization of the rhizosphere by the introduced Lysobacter strains. In conclusion, our results demonstrated that Lysobacter species have strong antagonistic activities against a range of pathogens, making them an important source for putative new enzymes and antimicrobial compounds. However, their potential role in R. solani disease suppressive soil could not be confirmed. In-depth omics'–based analyses will be needed to shed more light on the potential contribution of Lysobacter species to the collective activities of microbial consortia in disease suppressive soils.

Diversity and functions of volatile organic compounds produced by Streptomyces from a disease-suppressive soil

In disease-suppressive soils, plants are protected from infections by specific root pathogens due to the antagonistic activities of soil and rhizosphere microorganisms. For most disease-suppressive soils, however, the microorganisms and mechanisms involved in pathogen control are largely unknown. Our recent studies identified Actinobacteria as the most dynamic phylum in a soil suppressive to the fungal root pathogen Rhizoctonia solani. Here we isolated and characterized 300 isolates of rhizospheric Actinobacteria from the Rhizoctonia-suppressive soil. Streptomyces species were the most abundant, representing approximately 70% of the isolates. Streptomyces are renowned for the production of an exceptionally large number of secondary metabolites, including volatile organic compounds (VOCs). VOC profiling of 12 representative Streptomyces isolates by SPME-GC-MS allowed a more refined phylogenetic delineation of the Streptomyces isolates than the sequencing of 16S rRNA and the house-keeping genes atpD and recA only. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and significantly enhanced plant shoot and root biomass. Coupling of Streptomyces VOC profiles with their effects on fungal growth, pointed to VOCs potentially involved in antifungal activity. Subsequent assays with five synthetic analogs of the identified VOCs showed that methyl 2-methylpentanoate, 1,3,5-trichloro-2-methoxy benzene and the VOCs mixture have antifungal activity. In conclusion, our results point to a potential role of VOC-producing Streptomyces in disease suppressive soils and show that VOC profiling of rhizospheric Streptomyces can be used as a complementary identification tool to construct strain-specific metabolic signatures.

Down-regulation of rice serpin gene OsSRP-LRS exaggerates stress-induced cell death

Serine protease inhibitors (serpins) and their cognate proteases control diverse physiological processes, including program cell death and immune response, in insects and mammals. Arabidopsis AtSerpin1 inhibits pro-cell-death proteases and protects cells from excessive cell death induced by pathogens. The rice genome contains at least 14 serpin coding genes, but a biological role is not known for any of them. We show here that expression of rice serpin1 (OsSRP-LRS; the closest homolog of AtSerpin1 in rice) is induced by necrotrophic fungal pathogen Rhizoctonia solani inoculation, UV and salt treatment. The transgenic RNAi lines having reduced expression of OsSRP-LRS are morphologically unaffected under the stress-free condition, but show exaggerated cell death upon pathogen, UV or salt treatment. Our results suggest that OsSRP-LRS negatively regulates stress-induced cell death in rice.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BackgroundImproved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq.ResultsApproximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10 % of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation.ConclusionsThis RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1758-z) contains supplementary material, which is available to authorized users.

Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds.

The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al., 2011). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six non-ribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third lipopeptide, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.

The Novel Lipopeptide Poaeamide of the Endophyte Pseudomonas poae RE*1-1-14 Is Involved in Pathogen Suppression and Root Colonization.

Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.

Antibacterial and Antifungal Activities of Cyanobacterial Strains Isolated from Hot Springs in Oman

In this study, cyanobacterial microbial mats from five hot springs in Oman, namely Al Kasfah Rustaq, Al Thwara Nakhl, Al–Ali Hammam, Gala and Bowsher, were characterized using direct microscopy. Nine monoclonal cyanobacterial cultures were obtained and their extracts in butanol, dichloromethane (DCM) and hexane were screened for antibacterial and antifungal activities. Direct microscopy revealed the presence of 12 different unicellular and filamentous morphotypes, with different distribution in the various mats. Temperature seems to be one of the most important parameters that accounts for the differences in cyanobacterial composition of the mats. Cells of the nine isolates and their aqueous supernatants were subsequently extracted with butanol, DCM and hexane. Dried extracts were tested against nine bacterial (i.e. gram +ve Staphylococcus aureus, Bacillus subtilis and gram –ve, Escherichia coli, Klebsiella pneumoniae, Salmonella choleraesuis, S. enterica, Psuedomonas aeruginosa, Providencia stuartii, and Acinetobacter calcoaceticus) and two fungal pathogens (Rhizoctonia solani and Pythium sp.). All isolates exhibited antibacterial and antifungal activities, which depended mainly on the type of cyanobacterial culture, type of solvent used and the pathogen tested. The highest antibacterial activity was observed in Phormidium species, and butanol was found to be the most appropriate solvent to extract bioactivity from these cyanobacterial species. The results of this study suggest that thermal springs in Oman harbor diverse types of cyanobacteria, which may constitute an important source of antibacterial and antifungal compounds. Further investigation is needed to purify these compounds and find their chemical compositions and modes of action.

Fungal invasion of the rhizosphere microbiome

The rhizosphere is the infection court where soil-borne pathogens establish a parasitic relationship with the plant. To infect root tissue, pathogens have to compete with members of the rhizosphere microbiome for available nutrients and microsites. In disease-suppressive soils, pathogens are strongly restricted in growth by the activities of specific rhizosphere microorganisms. Here, we sequenced metagenomic DNA and RNA of the rhizosphere microbiome of sugar beet seedlings grown in a soil suppressive to the fungal pathogen Rhizoctonia solani. rRNA-based analyses showed that Oxalobacteraceae, Burkholderiaceae, Sphingobacteriaceae and Sphingomonadaceae were significantly more abundant in the rhizosphere upon fungal invasion. Metatranscriptomics revealed that stress-related genes (ppGpp metabolism and oxidative stress) were upregulated in these bacterial families. We postulate that the invading pathogenic fungus induces, directly or via the plant, stress responses in the rhizobacterial community that lead to shifts in microbiome composition and to activation of antagonistic traits that restrict pathogen infection.

Assessment of Wilt Intensity and Identification of Causal Fungal and Bacterial Pathogens on Hot Pepper (Capsicum annuum L.) in Bako Tibbe and Nonno Districts of West Shewa Zone, Ethiopia

Pepper wilt disease intensity was assessed on-farm in Bako Tibbe and Nonno districts of West Shewa Zone, Ethiopia during the main cropping season of October 2012. The wilt causing pathogens were identified from 50 diseased samples collected from the two districts. Of the 120 hot pepper fields surveyed, 116 fields were found to be infected with wilt disease. The overall percent prevalence and incidence of wilt disease was 96.7 and 86.4%, respectively. Identification and pathogenicity tests revealed that Ralstonia solanacearum and four fungal wilt pathogens (Rhizoctonia solani, Fusarium spp., Phytophthora spp. and Verticillium spp.) were detected in the surveyed fields. The percentage of occurrence of Rhizoctonia solani, Fusarium spp., Phytophthora spp. and Verticillium spp. were 45.0, 17.48, 12.59 and 11.89%, respectively; whereas, the frequency of R. solanacearum was 100%. Wilt disease in pepper in these two districts was caused by more than one wilt causing pathogen, thus management strategies should focus on these complex pathogens.

Production of Phenyl Lactic Acid (PLA) by Lactic Acid Bacteria and its Antifungal Effect

Phenyllactic acid (PLA) which is known as antimicrobial compound can be synthesized through the reduction of phenylpyruvic acid (PPA) by lactate dehydrogenase (LDH) of lactic acid bacteria (LAB). LAB producing PLA was isolated from Korea Kimchi and identified to Lactobacillus plantarum SJ21 by 16 rRNA gene sequence analysis. Cell-free supernatant (CFS) from L. plantarum SJ21 was assessed for both the capability to produce the antimicrobial compound PLA and the antifungal activity against four fungal pathogens (Rhizoctonia solani, Aspergillus oryzae, Botrytis cinerea, and Collectotricum aculatum). PLA concentration was investigated to be 3.23mM in CFS when L. plantarum SJ21 was grown in MRS broth containing 5mM PPA for 16 h. PLA production also could be promoted by the supplement of PPA and phenylalanine in MRS broth, but inhibited by the supplement of 4-hydroxyphenylpyruvic acid and tyrosine as precursors. Antifungal activity demonstrated that all fungal pathogens were sensitive to 5% CFS (v/v) of L. plantarum SJ21 with average growth inhibitions ranging from 27.32% to 69.05% (p -1 (2.11 mM) to 0.7 mg mL -1 (4.21 mM) at pH 4.0. The inhibition ability of CFS against the pathogens was not affected by heating or protease treatment. However, pH modification in CFS to 6.5 caused an extreme reduction in their antifungal activity. These results may indicate that antifungal activities in CFS were caused by acidic compounds like PLA or organic acids rather than proteins or peptides molecules.

Identification of signatory secondary metabolites during mycoparasitism of Rhizoctonia solani by Stachybotrys elegans.

Stachybotrys elegans is able to parasitize the fungal plant pathogen Rhizoctonia solani AG-3 following a complex and intimate interaction, which, among others, includes the production of cell wall-degrading enzymes, intracellular colonization, and expression of pathogenic process encoding genes. However, information on the metabolome level is non-existent during mycoparasitism. Here, we performed a direct-infusion mass spectrometry (DIMS) metabolomics analysis using an LTQ Orbitrap analyzer in order to detect changes in the profiles of induced secondary metabolites of both partners during this mycoparasitic interaction 4 and 5 days following its establishment. The diketopiperazine(s) (DKPs) cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid were detected as the primary response of Rhizoctonia 4 days following dual-culturing with Stachybotrys, whereas only the latter metabolite was up-regulated 1 day later. On the other hand, trichothecenes and atranones were mycoparasite-derived metabolites identified during mycoparasitism 4 and 5 days following dual-culturing. All the above secondary metabolites are known to exhibit bioactivity, including fungitoxicity, and represent key elements that determine the outcome of the interaction being studied. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se or their chemical analogs, and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles.

Ieodoglucomide C and Ieodoglycolipid, New Glycolipids from a Marine-Derived Bacterium Bacillus licheniformis 09IDYM23.

Chemical examination of the ethyl acetate extract from the fermentation broth of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new glycolipids, ieodoglucomide C (1) and ieodoglycolipid (2). The structural characterization of 1 and 2 was achieved by extensive spectroscopic evidence, including 2D NMR experiments. A combination of chemical derivatization techniques followed by NMR studies, LC-MS data analysis and a literature review was deployed for the establishment of the stereo-configurations of 1 and 2. Compounds 1 and 2 exhibited good antibiotic properties against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa with MICs ranging from 0.01 to 0.05 μM. Furthermore, the antifungal activity of 1 and 2 was evaluated against plant pathogenic fungi Aspergillus niger, Rhizoctonia solani, Botrytis cinerea and Colletotrichum acutatum as well as the human pathogen Candida albicans. Compounds 1 and 2 inhibited the mycelial growth of these pathogens with MIC values of 0.03-0.05 μM, revealing that these compounds are good candidates for the development of new fungicides.

Identification of QTLs and possible candidate genes conferring sheath blight resistance in rice (Oryza sativa L.).

Sheath blight, caused by the pathogenic fungus Rhizoctonia solani Kühn, is one of the most devastating diseases in rice. Breeders have always faced challenges in acquiring reliable and absolute resistance to this disease in existing rice germplasm. In this context, 40 rice germplasm including eight wild, four landraces, twenty- six cultivated and two advanced breeding lines were screened utilizing the colonized bits of typha. Except Tetep and ARC10531 which expressed moderate level of resistance to the disease, none could be found to be authentically resistant. In order to map the quantitative trait loci (QTLs) governing the sheath blight resistance, two mapping populations (F2 and BC1F2) were developed from the cross BPT-5204/ARC10531. Utilizing composite interval mapping analysis, 9 QTLs mapped to five different chromosomes were identified with phenotypic variance ranging from 8.40 to 21.76%. Two SSR markers namely RM336 and RM205 were found to be closely associated with the major QTLs qshb7.3 and qshb9.2 respectively and were attested as well in BC1F2 population by bulk segregant analysis approach. A hypothetical β 1–3 glucanase with other 31 candidate genes were identified in silico utilizing rice database RAP-DB within the identified QTL region qshb9.2. A detailed insight into these candidate genes will facilitate at molecular level the intricate nature of sheath blight, a step forward towards functional genomics.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-0954-2) contains supplementary material, which is available to authorized users.

Synthesis, Characterisation, and Antifungal Activities of Novel Benzodiazaborines

Eight new fluoro- and methoxy-substituted benzodiazaborines have been prepared by a simple condensation reaction in high-to-excellent yields. All new compounds have been characterised by several physical methods, including X-ray diffraction studies on three examples. All new compounds were examined for antifungal activities against five species of potentially pathogenic fungi (Aspergillus niger, Aspergillus fumigatus, Rhizoctonia solani, Verticillium albo-atrum, and Verticillium dahliae). While substitution of the aromatic group derived from the 2-formylphenylboronic acid group had an effect on bioactivities, substitution on the parent thioamide C(=S)NH2 group of the starting thiosemicarbazide greatly reduced activities.

Heterologous expression of the antifungal β-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Lignin and lignans in plant defence: Insight from expression profiling of cinnamyl alcohol dehydrogenase genes during development and following fungal infection in Populus

Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignol, the main component of lignin. Lignins, deposited in the secondary cell wall, play a role in plant defence against pathogens. We re-analysed the phylogeny of CAD/CAD-like genes using sequences from recently sequenced genomes, and analysed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus trichocarpa healthy and infected plants. Three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora sp.), varying in lifestyle and pathogenicity, were used for plant infection. Phylogenetic analyses showed that CAD/CAD-like genes were distributed in classes represented by all members from angiosperm lineages including basal angiosperms and Selaginella. The analysed genes showed different expression profiles during development and demonstrated that three genes were involved in primary xylem maturation while five may function in secondary xylem formation. Expression analysis following inoculation with fungal pathogens, showed that five genes were induced in either stem or leaves. These results add further evidence that CAD/CAD-like genes have evolved specialised functions in plant development and defence against various pest and pathogens. Two genes (PoptrCAD11 and PoptrCAD15), which were induced under various stresses, could be treated as universal markers of plant defence using lignification or lignan biosynthesis.