Characterization of field isolates of Fusarium spp. from eggplant in India for species complexity and virulence.

Eggplant wilt, despite emerging as a severe disease in India, the etiology must be better studied for its species' complexity and variability. The identity of fungal isolates associated with eggplants of India was established morphologically followed by sequencing and phylogenetic analysis. Three species, Fusarium falciforme, Fusarium incarnatum and Fusarium proliferatum, were observed for the first time in India. The isolates were tested for pathogenicity. Though all of them were pathogenic, the isolates displayed varying degrees of virulence. In further studies, the genetic relatedness of the isolates for virulence was assessed with candidate avirulent (SIX effectors), virulent (Fow1 and Fow2) and SSR markers. The SIX effector genes could not delineate the virulent isolates and were expressed in some non-F. oxysporum isolates for the first time. Likewise, the virulent genes, Fow1 for expression across the isolates and Fow2 for random expression across the isolates, were unsuitable markers for identifying the virulent groups. Hence, the F. oxysporum and F. solani isolates were genotyped with SSR markers. Though the clustering did not correlate with their virulence levels, the dendrogram grouping revealed variability among the F. oxysporum and F. solani isolates. This study concludes that although multiple species of Fusarium are associated with eggplant wilt in India, only F. oxysporum and F. solani are widespread in the surveyed areas. Though the three markers could not delineate the race specificity of the isolates, only the SSR makers could identify the genetic variability and hence, would help screen eggplant germplasm for fusarium wilt resistance.

Biocontrol of sheath blight of rice (Oryza sativa L.) through alteration in expression dynamics of candidate effector genes of Rhizoctonia solani AG1-IA during pathogenesis.

In genome analyses of Rhizoctonia solani AG1-IA causing sheath blight (ShB) of rice, many genes were identified to have a hypothetical role in pathogenesis. To understand their roles in pathogenesis, their expressions during fungal infection were studied. An aggressive R. solani strain, RIRS-K, was first identified among six isolates, RIRS-K, RIRS-17, RIRS-S, RIRS-T, RIRS-MU and RIRS-FD, for inducing a maximum relative lesion height (RLH) of 32.7% on a ShB susceptible cultivar, Pusa Basmati-1. Hypothetical pathogenicity genes (52 nos) identified by in silico analyses of the publicly available genomic database of the pathogen strain were evaluated in Pathogen-Host Interaction (PHI) blast and RIRS-K. Though PHI blast identified 26 genes as potential ones, only 8 were constitutively expressive in RIRS-K cultured in a minimal broth. Among them, only expressions of AG1IA_06195, AG02692, AG04508, and AG05730 were induced in the rice plant inoculated with RIRS-K and, hence, were identified as the candidate ones. The candidate genes were highly expressed in the aggressive strain (RIRS-K) in comparison to the less aggressive one (RIRS-17). In further testing of their expressions in the highly aggressive fungal strain, RIRS-K infecting PB-1 pre-colonized by a potent biocontrol consortium comprising of Bacillus subtilis (S17TH), Pseudomonas putida (TEPF-Sungal-1), and Trichoderma harzianum (S17TH), the disease scoring and gene expression studies indicated that the candidate genes were downregulated. The studies, therefore, speculated that these genes might play a role in pathogen aggressiveness and ShB development.

Physiological race characterisation of Fusarium oxysporum strains infecting tomato employing candidate pathogenicity genes and host resistance.

Physiological races of 14 strains of Fusarium oxysporum f.sp. lycopersici were established by PCR profiling SIX gene expressions. No amplification of the SIX4 (Avr1) gene was observed in any of the 14 strains. Based on amplification of the SIX3 (Avr2) gene, 6 strains were distinguished as race 2. Race 2 strains are known to contain identical SIX3 sequences and differ from race 3 strains by single point mutations. Hence, based on polymorphic amplicons of the SIX3 gene detected by stringent PCR conditions, 8 strains were identified as race 3. The identity of the physiological races of the strains was validated by inoculating on three germplasm lines, EC-814916, FEB-2 and Pusa Rohini carrying I-2, I-3 and no I gene, respectively. The race 2 and race 3 strains were avirulent on EC-814916 and FEB-2 lines, respectively. All the 14 fungal strains were pathogenic on Pusa Rohini, the Fusarium wilt susceptible cultivar lacking R genes and exhibited different levels of virulence. In evaluating two other potential pathogenicity genes, Fow1 and Fow2 as markers for virulence, their expressions were observed among both the races of the Fol strains, and hence are not potential candidates for physiological race discrimination. However, strong expressions of the genes in the root tissues inoculated with the highly virulent strain, TOFU-IHBT in comparison to the uninoculated control indicated their roles in fungal pathogenicity. To understand the role of these pathogenicity genes in countering the host defence mechanisms, their expressions in response to ROS and phenolics, the earliest known defence mechanisms of host plants were assessed. In H2O2, the Fow2 gene expressed 1.4-fold greater than that of the control. On the contrary, in relation to the control, the expressions of Fow1 were strongly repressed exhibiting 0.7-to 0.8-fold lesser at 0.1 mM through 3 mM concentrations than that of the control indicating that the gene is modulated by the phenolic acid indicating the roles of Fow2 and Fow1 in alleviating oxidative stress and targeted by the phenolic acid, respectively.

Identification of a new phytotoxic compound from culture filtrates of an aggressive Rhizoctonia solani AG 1A isolate inducing sheath blight of rice.

Phytotoxins produced by Rhizoctonia solani AG1-1A (Anastomosis Group 1 Subgroup 1A) play a significant role in developing sheath blight disease in rice. A phytotoxin in the partially purified ethyl acetate fraction from the culture filtrate of a highly aggressive R. solani (RIRS-K) isolate, with Indian Type Culture Collection (ITCC) number 7479, infecting rice that could incite necrotic symptoms characteristic of the fungus was identified. The role of the crude toxin in the pathogenicity and virulence of the fungal pathogen on rice was first established by artificial inoculation assay under controlled conditions. The crude ethyl acetate extract obtained from the culture filtrate of RIRS-K was first fractionated by column chromatography. Further purification of the bioactive fraction was carried out by using bioassay-guided fractionation, and a toxic fraction was obtained. The most bioactive fraction was analyzed by GC-MS analysis, and 3-butylpyridine (3-BP) was identified as a major compound in the active fraction by comparing its mass spectrum with NIST library and its standard. The purified bioactive fraction and standard (3-BP) toxicity was further validated and compared at 1000 ppm. The result showed that both the bioactive fraction and the 3-BP have caused necrosis, similar to the one incited by R. solani. This study showed that 3-BP is one of the major compounds responsible for the necrosis development in the rice plant during ShB disease and is a hitherto unexplored toxin of R. solani in rice.

Metabolomic analysis of sheath blight disease of rice (Oryza sativa L.) induced by Rhizoctonia solani phytotoxin.

AIM: To understand the mechanism of necrosis incited by a host-selective phytotoxin designated as Rhizoctonia solani toxin (RST) identified to be a potential pathogenic factor of R. solani AG1 IA, causing sheath blight (ShB) of rice. METHODS AND RESULTS: The metabolomic changes induced by the phytotoxic metabolite in a ShB susceptible rice cultivar were elucidated by gas chromatography-mass spectrometry analysis and compared with that of the pathogen to identify rice metabolites targeted by the phytotoxin. The profiles of about 29 metabolites with various physiological roles in rice plants have been identified worldwide. Unsupervised and supervised multivariate chemometrics (principal component analysis and partial least squares-discriminant analysis) and cluster (Heat maps) analyses were used to compare the metabolites obtained from chemical profiles of the treatments with sterile distilled water (SDW) control. The results indicated that the rice plant expressed more metabolites in response to the pathogen than the phytotoxin and was lowest in SDW control. The key metabolites expressed in rice in response to the treatments were investigated by the variable importance in projection (VIP) analysis using p < 0.05 VIP >15. The analysis identified 7 and 11 upregulating metabolites in the phytotoxin and the pathogen treatments, respectively, compared to the untreated control. Among the phytotoxin-treated and the pathogen inoculated samples, the phytotoxin-treated sample recorded upregulation of six metabolites, whereas nine metabolites were upregulated in the pathogen-inoculated samples. These upregulating metabolites are speculated for the necrotic symptoms characteristic to both the phytotoxin and pathogen. In this analysis, hexadecanoic acid and dotriacontane were highly expressed metabolites specific to the phytotoxin and pathogen-treated samples, respectively. Besides upregulation, the metabolites also have a VIP score of >1.5 and hence fulfilled the criteria of classifying them as reliable potential biomarkers. In the pathway analysis, hexadecanoic acid and dotriacontane were identified to be involved in several important biosynthetic pathways of rice, such as the biosynthesis of saturated fatty acid and unsaturated fatty acids cutin, suberin and wax. CONCLUSIONS: The study concludes that though certain metabolites induced by the phytotoxin in the susceptible variety during necrosis shares with that of the pathogen, the identification of metabolites specific to the phytotoxin in comparison to the pathogenic and SDW controls indicated that the phytotoxin modulates the host metabolism differently and hence can be a potential pathogenicity factor of the ShB fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to lack of knowledge on the pathway genes of RST and in the absence of an ShB-resistant variety, understanding differentially expressed metabolic changes induced in the susceptible variety by the phytotoxin in comparison to that of the pathogenic and uninoculated controls enables us to identify the key metabolite changes during the ShB infection. Such metabolomic changes can further be used to infer gene functions for exploitation in ShB control.

Genome-Wide Identification and Characterisation of Cytokinin-O-Glucosyltransferase (CGT) Genes of Rice Specific to Potential Pathogens.

Cytokinin glucosyltransferases (CGTs) are key enzymes of plants for regulating the level and function of cytokinins. In a genomic identification of rice CGTs, 41 genes with the plant secondary product glycosyltransferases (PSPG) motif of 44-amino-acid consensus sequence characteristic of plant uridine diphosphate (UDP)-glycosyltransferases (UGTs) were identified. In-silico physicochemical characterisation revealed that, though the CGTs belong to the same subfamily, they display varying molecular weights, ranging from 19.6 kDa to 59.7 kDa. The proteins were primarily acidic (87.8%) and hydrophilic (58.6%) and were observed to be distributed in the plastids (16), plasma membrane (13), mitochondria (5), and cytosol (4). Phylogenetic analysis of the CGTs revealed that their evolutionary relatedness ranged from 70-100%, and they aligned themselves into two major clusters. In a comprehensive analysis of the available transcriptomics data of rice samples representing different growth stages only the CGT, Os04g25440.1 was significantly expressed at the vegetative stage, whereas 16 other genes were highly expressed only at the reproductive growth stage. On the contrary, six genes, LOC_Os07g30610.1, LOC_Os04g25440.1, LOC_Os07g30620.1, LOC_Os04g25490.1, LOC_Os04g37820.1, and LOC_Os04g25800.1, were significantly upregulated in rice plants inoculated with Rhizoctonia solani (RS), Xoo (Xanthomonas oryzae pv. oryzae) and Mor (Magnaporthe oryzae). In a qRT-PCR analysis of rice sheath tissue susceptible to Rhizoctonia solani, Mor, and Xoo pathogens, compared to the sterile distilled water control, at 24 h post-infection only two genes displayed significant upregulation in response to all the three pathogens: LOC_Os07g30620.1 and LOC_Os04g25820.1. On the other hand, the expression of genes LOC_Os07g30610.1, LOC_Os04g25440, LOC_Os04g25490, and LOC_Os04g25800 were observed to be pathogen-specific. These genes were identified as the candidate-responsive CGT genes and could serve as potential susceptibility genes for facilitating pathogen infection.

Corrigendum: Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

[This corrects the article DOI: 10.3389/fpls.2021.748013.].

Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium.

Though the vascular wilt of tomato caused by the species of Fusarium is globally reported to be a complex disease in certain countries, for example, India, our studies indicated that the disease is caused by either Fusarium oxysporum f. spp. lycopersici (Fol) or Fusarium solani (FS) with the Fol being widely prevalent. In assessing the genetic diversity of 14 Fol strains representing the four Indian states by the unweighted pair group method with arithmetic averaging using Inter Simple Sequence Repeat (ISSR) amplicons, the strains distinguished themselves into two major clusters showing no correlation with their geographic origin. In pot experiments under polyhouse conditions, the seed dressing and soil application of a talc-based formulation of a biocontrol treatment, TEPF-Sungal-1 (Pseudomonas putida) + S17TH (Trichoderma harzianum) + CG-A (Chaetomium globosum), which inhibited Fol, was equally effective like the cell suspensions and was even better than the fungicidal mixture (copper oxychloride-0.25% + carbendazim-0.1%) in promoting the crop growth (52.3%) and reducing vascular wilt incidence (75%) over the control treatment, despite the challenge of inoculation with a highly pathogenic TOFU-IHBT strain. This was associated with significant expressions of the defense genes, indicating the induction of host resistance by a biocontrol consortium. In field experiments on two locations, the bioconsortium was highly effective in recording maximum mean fruit yields (54.5 and 60%) and a minimum mean vascular wilt incidence (37.5%) in comparison to the untreated control. Thus, Chaetomium-based bioconsortium demonstrated consistency in its performance across the two experiments in 2 years under the two field conditions.

Brassica nigra essential oil: In-vitro and in-silico antibacterial efficacy against plant pathogenic and nitrifying bacteria.

The present study was aimed to examine the antibacterial potential of Brassica nigra essential oil (BNEO) against Ralstonia solanacearum, causal agent of bacterial wilt and Nitrosomonas sp., the nitrifying bacteria. In poisoned food assay, BNEO showed 100% growth inhibition of R. solancearum at ≥ 125 µg mL-1. Revalidation of findings by volatile assay employing inverted Petri plate technique exhibited 100% bacterial growth inhibition caused by vapors of BNEO, even at 50 µg mL-1 concentration. In the broth microdilution assay, the BNEO exhibited significant antibacterial activity only at higher concentrations (>500 µg mL-1). At 500 µg mL-1, BNEO showed 80% bacterial growth inhibition over control, which was at par with that of streptomycin (5 µg mL-1). In resazurin microtitre-plate assay, the maximum concentration of BNEO, at which color change occurred was 512 µg mL-1 (T9), and thus 512 µg mL-1 was concluded as the minimum inhibitory concentration (MIC). BNEO effectively inhibited the activity of Nitrosomonas spp. with 30-65% nitrification inhibition at the dose of 400 mkg-1 of Urea-N. Homology modeled protein targets assisted computational tool-based novel analysis helped to understand that the antibacterial potency of BNEO is due to preferable binding efficiency of allyl isothiocyanate (AITC), the major active ingredient of BNEO.

Deciphering the Behavioral Response of Meloidogyne incognita and Fusarium oxysporum Toward Mustard Essential Oil.

Environmental concerns related to synthetic pesticides and the emphasis on the adoption of an integrated pest management concept as a cardinal principle have strengthened the focus of global research and development on botanical pesticides. A scientific understanding of the mode of action of biomolecules over a range of pests is key to the successful development of biopesticides. The present investigation focuses on the in silico protein-ligand interactions of allyl isothiocyanate (AITC), a major constituent of black mustard (Brassica nigra) essential oil (MEO) against two pests, namely, Meloidogyne incognita (Mi) and Fusarium oxysporum f. sp. lycopersici (Fol), that cause severe yield losses in agricultural crops, especially in vegetables. The in vitro bioassay results of MEO against Mi exhibited an exposure time dependent on the lethal concentration causing 50% mortality (LC50) values of 47.7, 30.3, and 20.4 µg ml-1 at 24, 48, and 72 h of exposure, respectively. The study revealed short-term nematostatic activity at lower concentrations, with nematicidal activity at higher concentrations upon prolonged exposure. Black mustard essential oil displayed excellent in vitro Fol mycelial growth inhibition, with an effective concentration to cause 50% inhibition (EC50) value of 6.42 µg ml-1. In order to decipher the mechanism of action of MEO, its major component, AITC (87.6%), which was identified by gas chromatography-mass spectrometry (GC-MS), was subjected to in silico docking and simulation studies against seven and eight putative target proteins of Mi and Fol, respectively. Allyl isothiocyanate exhibited the highest binding affinity with the binding sites of acetyl cholinesterase (AChE), followed by odorant response gene-1 (ODR1) and neuropeptide G-protein coupled receptor (nGPCR) in Mi, suggesting the possible suppression of neurotransmission and chemosensing functions. Among the target proteins of Fol, AITC was the most effective protein in blocking chitin synthase (CS), followed by 2,3-dihydroxy benzoic acid decarboxylase (6m53) and trypsinase (1try), thus inferring these as the principal molecular targets of fungal growth. Taken together, the study establishes the potential of MEO as a novel biopesticide lead, which will be utilized further to manage the Mi-Fol disease complex.

Chitinase Expression Due to Reduction in Fusaric Acid Level in an Antagonistic Trichoderma harzianum S17TH.

To study the effect of reduction in phytotoxin level on fungal chitinases, antagonistic Trichoderma spp. were screened for their ability to reduce the level of fusaric acid (FA), the phytotoxin produced by Fusarium spp. A T. harzianum isolate S17TH was able to tolerate high levels of FA (up to 500 ppm) but was unable to reduce the toxin to a significant level (non-toxic) added to minimal synthetic broth (MSB). However, the isolate was able to reduce 400 ppm FA in the liquid medium after 7 days to a non-toxic level and displayed similar level of antagonism over the control (without FA). In studies of the effect of the reduction in FA (400 ppm) level on chitinase gene expression in PCR assays, nag1 was significantly repressed but ech42 expression was only slightly repressed. Chitinase activity was either reduced or absent in the extracellular proteins of MSB supplemented with 400 ppm FA, which could be attributed to the effect of residual FA or its breakdown products through unknown mechanisms. Selection of S17TH as a toxin insensitive isolate that could commensurate the negative effect on chitinase activity makes it a potential antagonist against Fusarium spp.

Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum.

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-ß-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 µM ml(-1) and 0.119 µM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.

Cloning and characterization of a bifunctional glycosyl hydrolase from an antagonistic Pseudomonas putida strain P3(4).

A fluorescent pseudomonad strain P3(4) showing chitinolysis on chitinase detection agar and antagonism against Fusarium oxysporum f.sp dianthi causing vascular wilt of carnation was isolated from pea rhizosphere soil. PCR primers specific for glycosyl hydrolase family 5 (GH5) of Pseudomonas putida isolate KT2440 amplified a 947 bp fragment of the GH5 gene from P3(4). Cloning of this gene into Escherichia coli M15 using an expression vector pQE-30UA and screening on chitin and chitosan detection agar identified one positive clone (Pchi(+) ). Sequence analysis of the cloned insert revealed an open reading frame of 947 nucleotides corresponding to a protein of 315 amino acids with a predicted molecular mass of 38.0 kDa. The deduced amino acid sequence of the open reading frame (gene product/GH) showed 83-84% homology to the GH5 of P. putida strains F1 and KT2440, respectively. The purified enzyme was homogenous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was visualized as single fluorescent band in native gel assay with 4-methylumbelliferyl-N -acetyl-ß;-D-glucosaminide and glycol chitosan, respectively. For hydrolysis of 4-nitrophenyl-N -acetyl-ß;-D-glucosaminide (pNP-(GlcNAc) and colloidal chitosan, the enzyme had an optimal temperature of 40 °C, and was stable within the temperature range of 10 °C to 40 °C. The enzyme showed an optimal pH of 3.5, with maximum stabilities at 5.0 and 5.5 for hydrolysis of pNP-(GlcNAc) and colloidal chitosan, respectively. Fe(3+) and Cu(2+) stimulated chitinase and chitosanase activities by 74.2 and 51.4%, respectively. The purified GH displayed 70 and 45% inhibition of spore germination of the pathogenic fungi, Fusarium oxysporum f.sp. dianthi and Alternaria solani, respectively.

Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Alternaria solani infecting Tomato.

Antagonistic Bacillus spp. displaying in vitro production of siderophore, chitinase, and ß-1,3-glucanase were identified from dual culture assays. In independent greenhouse studies, seed bacterization and soil application of Bacillus atrophaeus S2BC-2 challenge inoculated with Fusarium oxysporum f.sp. lycopersici (FOL) and Alternaria solani (AS) recorded low percent disease index of 25.3 and 28.7, respectively, over nonbacterised pathogen control (44.3 and 56.4). The low disease incidence corroborated with tomato growth promotion with high vigor index (8,041.2) and fresh plant weight (82.5 g) on challenge inoculation with FOL. Analysis of root and leaf samples in rhizobacterial treatment challenged with FOL and AS revealed maximum induction of chitinase (1.9 and 1.7 U/mg of protein, respectively) and ß-1,3-glucanase (23.5 and 19.2 U/mg of protein, respectively). In native gel activity assays, the rhizobacterial treatment on challenge inoculation strongly expressed three high intensity PO isoforms along with one low intensity isoform. In studies on genetic diversity of the Bacillus strains by repetitive extragenomic palindromic-polymerase chain reaction (REP-PCR) and amplified rDNA restriction analysis (ARDRA) patterns, ARDRA was more highly discriminant than REP-PCR and allowed grouping of the strains and differentiation of the antagonistic strains from other isolates.

The fungal pathogen Cochliobolus heterostrophus responds to maize phenolics: novel small molecule signals in a plant-fungal interaction.

The transcription factor ChAP1 of the fungal pathogen of maize, Cochliobolus heterostrophus, responds to oxidative stress by migration to the nucleus and activation of antioxidant genes. Phenolic and related compounds found naturally in the host also trigger nuclear localization of ChAP1, but only slight upregulation of some antioxidant genes. ChAP1 thus senses phenolic compounds without triggering a strong antioxidant response. We therefore searched for genes whose expression is regulated by phenolic compounds and/or ChAP1. The C. heterostrophus genome contains a cluster of genes for metabolism of phenolics. One such gene, catechol dioxygenase CCHD1, was induced at least 10-fold by caffeic and coumaric acids. At high phenolic concentrations (≥ 1.6 mM), ChAP1 is needed for maximum CCHD1 expression. At micromolar levels of phenolics CCHD1 is as strongly induced in chap1 mutants as in the wild type. The pathogen thus detects phenolics by at least two signalling pathways: one causing nuclear retention of ChAP1, and another triggering induction of CCHD1 expression. The low concentrations required for induction of CCHD1 indicate fungal receptors for plant phenolics. Symbiotic and pathogenic bacteria are known to detect phenolics, and our findings generalize this to a eukaryotic pathogen. Phenolics and related compounds thus provide a ubiquitous plant-derived signal.