Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management.

Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 µg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 µg/ml) and N2-1 (16.41 µg/ml); and succeeded to sporulate even at 2000 µg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of ß-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes.

Distribution and pathogenic diversity in Fusarium udum Butler isolates: the causal agent of pigeonpea Fusarium wilt.

BACKGROUND: Fusarium wilt (Fusarium udum Butler), an important soil-borne disease of pigeonpea [Cajanus cajan (L.)], causes significant yield losses across the major pigeonpea production regions. Widespread and high diversity in F. udum hampers the breeding for pigeonpea wilt resistance. The study aimed to elucidate the pathogenic diversity and distribution of F. udum variants in major pigeonpea growing regions of India. RESULTS: The roving survey was conducted in major pigeonpea-growing states of India to collect the F. udum isolates. Pathogenic variability of 60 F. udum isolates which are selected from diverse geographical locations and pathogenicity test were performed against 11 pigeonpea host differentials cultivars [ICP 8858, ICP 8859, ICP 8862, ICP 8863, ICP 9174, C 11, BDN 1, BDN 2, LRG 30, ICP 2376 and Bahar (ICP 7197)]. The current study indicated distribution of F. udum isolates into nine variants (0, 1, 2, 3, 4, 5, 6, 7 and 8). Variant-2 and 3 were found to be widespread and predominant in most pigeonpea producing regions. Variant-7 (Karnataka) and Variant-8 (Madhya Pradesh and Maharashtra) were found highly virulent, as most of the host differentials were susceptible to these variants. Three host differential cultivars namely ICP 9174, BDN-2 and Bahar (ICP 7197) were found resistant to most of the F. udum isolates. CONCLUSION: The present study generated significant information in terms of variants of F. udum which could be used further for the deployment of location-specific wilt resistant cultivars for optimized disease-management strategies. Study is also useful for development of broad-based wilt resistant cultivars to curtail the possible epidemics.

A new host record for Candidatus Phytoplasma cynodontis (16Sr XIV-A) associated with phyllody and fasciation of linseed (Linum usitatissimum) from India.

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.

Characterization of phytotoxin producing Alternaria species isolatedfrom sesame leavesand their toxicity.

Sesame (Sesamum indicum L.), is an important oilseed crop in the tropics and subtropics, referred as "Queen of Oilseeds" owing to its high cooking quality and medicinal value. Sesame production, particularly in India, has been declining since last decade and 'Leaf blight' caused by Alternaria spp. is reported to cause yield loss up to 30-40%. Here, we investigated the fungal toxin produced by Alternaria and its pathogenicity. A total of 164 Alternaria strainswere isolated on potato dextrose agar media from the infected sesame leaves showing circular concentric rings with dark brown spots symptoms. All the isolates were screened for cultural and morphological characters. Colour of the fungus was grey to dark brown, formed smooth, raised, fluffy, and regular to irregular margins. Among 164 isolates, 43 isolates were moderately growing and 121 were fast in growth. The DNA of the isolate was amplified with ITS primers and sequence of BLAST results confirmed seven different species of Alternaria of NCBI database. Further, toxigenic potentiality of the isolates was tested with dilutions of culture filtrate (1:1 to 1:5) on sesame leaves. Among 164 isolates, 23 showed toxigenicity, varied from highly toxigenic to least toxigenic. Pathogenicity of the isolates showed that they were highly virulent to less virulent when tested by the detached leaf method. Based on the toxigenicity, the toxin was partially purified and brown coloured paste was recovered. Chemistry of the toxin was confirmed based on the IR, UV, NMR and mass spectra analyses, and it resembled the structure of alternariol mono methyl ether and altenuene which are mycotoxins in nature. Further, bioassay of toxin was carried out at different concentrations (50 to 2000 ppm) on seeds and seedlings of sesame. Maximum inhibition of seed germination of 81.1% was observed at 2000 ppm and the least was 6.67% at 50 ppm. With the increase in the concentration of toxin, the manifestation of the symptom was conspicuous and quick such as marginal, veinal necrosis, drooping and yellowing with lesion formation. From the present study, it is found that the species of Alternaria are responsible for the cause of blight disease symptoms and the toxicity of toxin produced by the pathogen was very high. The Alternaria toxin could inhibit the growth of the plant as well as seed germination rate.

PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils.

Azotobacter strains were isolated by serial dilution method and colonies were viscous, smooth, glistening, and brown to black colour on Jenson's N-free agar. Morphological and biochemical tests showed characteristic features of Azotobacter. Further, molecular analyses revealed the presence of different Azotobacter species viz., A. armeniacus, A. chroococcum, A. salinestris, A. tropicalis and A. vinelandii. The isolates were tested for their ability of nitrogen fixation, indole acetic acid (IAA), gibberllic acid production and phosphate solubilization. Four isolates (GVT-1, GVT-2 KOP-11 and SND-4) were efficient in fixation of highest amount of N2 (29.21 µg NmL(-1) day(-1)), produced IAA (25.50 µg mL(-1)), gibberllic acid (17.25 µg 25 mL(-1)) and formed larger P solubilizing zone (13.4 mm). Some of the Azotobacter strains were produced siderophores, hydrogen cyanide and were positive for ammonia production with respect to antifungal activity of Azotobacter was tested with dual culture method and A. tropicalis inhibited the growth of Fusarium, Aspergillus and Alternaria species. Azotobacter isolates were tested against salt (0-10%), temperature (4-55 degrees C), pH (5.0-10) and insecticide chloropyrifos (0-3%) tolerance study. Among them, A. chroococcum was found tolerant to a maximum of 6% NaCl with a temperature of 35-45 degrees C and to a pH up to 8. All the 4 strains showed effective growth against 3% chloropyrifos concentration. The studies revealed that the Azotobacter strains not only produced plant growth promoting substances but are also tolerant to abiotic stresses such as temperature, pH and insecticides.

Antifungal Attributes of Lactobacillus plantarum MYS6 against Fumonisin Producing Fusarium proliferatum Associated with Poultry Feeds.

Fumonisins, being common in occurrence in maize-based feeds, pose a great threat to animal and human health. The present study is aimed at determining the antifungal activity of Lactobacillus plantarum MYS6 against a fumonisin producing fungus, Fusarium proliferatum MYS9. The isolate was subjected to standard tests for determining its probiotic attributes and antifungal properties. L. plantarum MYS6 thrived well at pH 3.0 and 6.0, and exhibited strong resistance up to 3% bile. The isolate showed a high degree of cell surface hydrophobicity corresponding to its strong adhesion to chicken crop epithelial cells. Co-inoculation with the fungus on modified de Man Rogosa Sharpe medium revealed the inhibitory effect of L. plantarum MYS6 on fungal growth and biomass. Observation using scanning electron microscopy showed distortion of hyphal structures, swollen tips and disrupted conidia. Conidia germination inhibition assay restrained germination and showed deformed hyphae. The bioprotective feature of the isolate was evident by the inhibition of fungal development in maize-kernel treated with the cell free supernatant of L. plantarum MYS6. Both the isolate and its extracellular metabolites lowered fumonisin content in feed model up to 0.505 mg/Kg of feed and 0.3125 mg/Kg of feed respectively when compared to the level of 0.870 mg/Kg of feed in control. The major antifungal compounds produced by the isolate were 10-Octadecenoic acid, methyl ester; palmitic acid, methyl ester; heptadecanoic acid, 16-methyl ester; stearic acid and lauric acid. L. plantarum MYS6 reduced 61.7% of fumonisin possibly by a binding mechanism. These findings suggest the application of L. plantarum MYS6 as an efficient probiotic additive and biocontrol agent in feed used in poultry industry. Additionally, the antifungal metabolites pose a conspicuous inhibition of Fusarium growth and fumonisin production.

Detection of antimicrobial traits in fluorescent pseudomonads and molecular characterization of an antibiotic pyoluteorin.

Thirty isolates of fluorescent pseudomonads were obtained from rhizosphere of different crops in Raichur, India. The fluorescent pseudomonad strains were characterized in vitro for biochemical traits, antimicrobial traits, and pyoluteorin antibiotic production. All the isolates that showed fluorescent pigment production under UV light were rod shaped, Gram negative, positive for oxidase, catalase and citrate utilization tests, and negative for indole test. Out of 30 isolates, 07 isolates were positive for HCN production, 15 isolates were positive for H2S production, and all the isolates were positive for siderophore production. Among all the isolates, RFP-22 showed the maximum percent inhibition of mycelium (46.66 %) of Rhizoctonia solani, the pathogen, and the remaining isolates showed the moderate to least inhibition of mycelium growth of R. solani. The 16S rRNA analysis confirmed that the antibiotic positive isolates belonged to genus Pseudomonas. The amplification of 779 bp region in isolates RFP- 4 and RFP-19 corresponded to pyoluteorin antibiotic-coding pltB gene. Further characterization of pyoluteorin antibiotic through TLC and TOF-MS analysis confirmed the presence of pyoluteorin at 274.26 (g/mol) peak and 2.10 min retention time. Biochemical and molecular analyses confirmed the antagonism of Pseudomonas and isolate through pyoluteorin production.

An integrated approach for the reduction of aflatoxin contamination in chilli (Capsicum annuum L.).

An integrated approach for management of aflatoxin contamination in chilli was undertaken by evaluating the fungicides, bioagents and plant extracts against Aspergillus flavus under both in vitro and field condition. Maximum inhibition of radial growth (91.1%) was observed with 0.3% mancozeb followed by captan (85.2%). Carbendazim (73%) was effective and superior over other systemic fungicides. A complete inhibition (100%) of A. flavus was observed in neem seed kernel extract (NSKE), nimbicidin and pongamia oil at 5%. An indigenous Pseudomonas fluorescens bioagent isolate inhibited (74.9%) the growth of A. flavus over Trichoderma harzianum (70.4%). The superior performing fungicides, plant extracts and bioagents identified under in vitro were used for challenge inoculation on chilli fruits and so also for field evaluation. The captan treated fruits recorded the least infection of A. flavus (1.6%) followed by P. fluorescens (2.0%), NSKE (2.2%) and nimbicidin treated fruits (7.8%) as against control (38.3%). As regards to field evaluation, the least incidence was recorded in NSKE sprayed chilli plot (1.6%) and was on par with captan (2.2%), P. fluorescens (2.4%) and T. harzianum (2.6%) compared to control (7.4%). Hence, a pre-harvest spray of NSKE (5%) or mancozeb (0.3%) or P. fluorescens (1 × 10(8) cfu/ml) 10 days before harvest of chilli is recommended for field level management of aflatoxin.