ABSTRACT
Contamination of food by aflatoxin of Aspergillus flavus is a major global problem affecting trade, quality, utility of food and human health. While all the members of A. flavus does not produce aflatoxin, sensitive, cost effective and reproducible methods for large scale screening and differentiation of toxigenic A. flavus from atoxigenic ones are scarce. Here, we made one such attempt using coconut milk agar (CMA), yeast extract sucrose agar (YESA), ammonium hydroxide vapour tests, enzyme linked immuno sorbent assay (ELISA) and polymerase chain reaction (PCR) for large scale screening of toxigenic strains of A. flavus. Fifty nine isolates of A. flavus obtained from major chilli growing regions of India were categorized into toxigenic and atoxigenic strains by using cultural, analytical and molecular methods. Forty two (71.18 %) isolates showed positive response in coconut milk agar (CMA), 17 (28.81%) isolates did not match while 23 (38.98 %) isolates showed red colour and 36 (61.01%) isolates did not produce red colour upon exposure to ammonia vapour in YESA. Out of 59 isolates, isolates CAF43 came out as highly toxigenic, as it produced 3128.20 μg kg-1 aflatoxin B1 followed by CAF 42 which produced 3035.10 μg kg-1. Among 59 isolates, eight A. flavus isolates were amplified with two regulatory (aflR and aflJ) and two structural (norA and ver1) genes at a region of 900, 450, 400 and 450 bp, respectively.
ABSTRACT
Rice sheath blight arising due to lack of systemic resistance against causative agent Rhizoctonia solani has been a major economic threat to rice growers across the globe. This warrants induced systemic resistance (ISR) in plant to make them disease resistant. Application of antibiotic producing Pseudomonads that also secrete defense inducing enzymes is known to impart resistance to plants. Here, we report induction of the induced systemic resistance (ISR) by six strains of rhizosphere fluorescent Pseudomonas possessing 2,4-diacetyl phloroglucinol (2,4-DAPG) antibiotic genes against rice sheath blight pathogen Rhizoctonia solani. In the preliminary screening, isolate EP5 (endophytic Pseudomonas fluorescens) showed 76.5% inhibition against R. solani. EP5 treated rice grains showed the highest germination of 96.6%, mean root length of 15.3 cm, shoot length of 12.6 cm and vigour index of 2104.9. Paddy grains treated with EP5 (through seed treatment followed by root dipping) upon challenge inoculation with R. solani (Rs1) gave higher activity of peroxidase (PO) 2.50 change in absorbance at 470 nm/min/mg protein), polyphenol oxidases (PPO), 2.25 change absorbance at 420 nm/ min/mg protein, phenyl alanine ammonia lyase (PAL) and 28.5 nmol transcinnamic acid/hr/mg protein. 2,4-DAPG positive fluorescent Pseudomonas strains produced PO, PPO, PAL and 28.5 nmol transcinnamic that resulted in ISR against rice sheath blight pathogen R. Solani.
ABSTRACT
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.
ABSTRACT
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-1day-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 (4-55ºC), 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 35-45ºC 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.