S. Mathiyazhagan

Induction of phenolics and defense-related enzymes in coconut (Cocos nucifera L.) roots treated with biocontrol agents

The effect of soil application of biocontrol agents (Pseudomonas fluorescens, Trichoderma viride and T. harzianum) in combination with chitin on induction of phenolics and defense enzymes in coconut roots infected with Ganoderma lucidum, the causal agent of Ganoderma disease, was investigated. Soil application of these biocontrol formulations in combination with chitin induced a significant increase in the activities of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), chitinase and b-1,3-glucanase in the G. lucidum infected palms. Activities of both PAL and PO reached maximum levels within 3 d while the activity of PPO reached the maximum level 6 d after application of a mixture of P. fluorescens, T. viride and chitin. Isozyme analysis revealed that unique PO3 and PPO2 isozymes were induced in coconut palms treated with P. fluorescens + T. viride + chitin. Accumulation of phenolics was recorded 3 d after treatment and reached maximum levels 9 d after treatment application. Activity of chitinase was significantly increased from the third day after treatment imposition and continued to increase up to 9 to 12 d in all treatments. Chitinase isozyme analysis revealed that a unique Chit3 isoform was induced in coconut roots treated with P. fluorescens + T. viride + chitin. The b-1,3-glucanase activity was maximum 9 d after treatment application. The mechanisms by which P. fluorescens + T. viride + chitin reduced the incidence of Ganoderma disease in coconut may be related to its ability to induce defense mechanisms in coconut palms.

PGPR MEDIATED MANAGEMENT OF STEM BLIGHT OF PHYLLANTHUS AMARUS (SCHUM AND THONN) CAUSED BY CORYNESPORA CASSIICOLA (BERK AND CURT) WEI

Bacillus subtilis (BSCBE4), Pseudomonas chlororaphis (PA23), endophytic P. fluorescens (ENPF1) inhibited the mycelial growth of stem blight pathogen Corynespora casiicola (Berk and Curt)Wei under in vitro. All these bacterial isolates produced both hydroxamate and carboxylate type of siderophores. But the siderophore production was maximum with the isolate ENPF1. Delivering of talc based formulation of BSCBE4 through seedling dip and foliar application effectively reduced stem blight disease incidence and increased the dry matter production under pot culture and field conditions. Application of BSCBE4, PA23 and ENPF1 increased the defense related enzymes such as peroxidase, polyphenol oxidase, chitinase and β-1,3 glucanase in P. amarus up to ten days after challenge inoculation with C. cassicola. Native gel electrophoretic analysis revealed that challenge inoculation of pathogen with BSCBE4 and PA23 induced both peroxidase and polyphnol oxidase isoforms.

Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode

Antifungal antibiotic from Pseudomonas chlororaphis isolate PA23 was identified as Phenazine using TLC and HPLC. Phenazine recorded the highest inhibition zone of 21 mm with 35.55% percent inhibition of mycelial growth of Pythium aphanidermatum over control. It had a significant effect on the hyphal morphology of P. aphanidermatum and on spore germination of Botryodiplodia theobromae and Alternaria solani. Disorganization of hyphal morphology of P. aphanidermatum includes vacuolization, cell content degeneration and hyphal lysis. Similarly interaction of phenazine with Rhizoctonia solani resulted in abnormal swelling of hyphal tips was noticed in the hyphal tips. Similarly the germination of sclerotia of Macrophomina phaseolina, R. solani and Sclerotium rolfsii were completely inhibited by phenazine at a concentration 50 μl. Incubation of the eggs of the root knot nematode Meloidogyne incognita in 30 μl concentration of phenazine, completely suppressed the hatching of juveniles.

Development of bioformulations of antagonistic bacteria for the management of damping off of Chilli ( Capsicum annuum L )

Several isolates of PGPR belonging to the species of Bacillus subtilis and Pseudomonas sp. were screened for its effectiveness in controlling damping off of chilli (Capsicum annuum L). Among the different isolates, Bacillus subtilis (CBE4) and Pseudomonas chlororaphis (BCA + ) proved to be effective against Pythium aphanidermatum under in vitro. Talc based formulation of these PGPR isolates were prepared and various treatments namely seed treatment and soil application were given for assessing their ability to control damping off disease under glass house condition. The treatment with the isolate CBE4 recorded a least per cent disease incidence of 15.66 followed by 16.07% in treatment with BCA + . Significant increases in plant growth was induced by treatment with PGPR isolates CBE4 and BCA + . The efficacies of these PGPR isolates are equivalent to the standard fungicide Ridomil. In spite of its direct action these PGPR isolates triggered the defense related enzymes involved in phenyl propanoid pathway and phenolics. Higher activities of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and, β-1,3 glucanase were observed in CBE4 and BCA  +  pre treated chilli plants challenged with P. aphanidermatum. The talc based formulations had a shelf life of more than 4 months.

Genetic variability and aflatoxigenic potential of Aspergillus flavus isolates from maize

Abstract Various isolates of Aspergillus flavus collected from infected maize seeds were tested for their ability to produce aflatoxin B1 (AFB1) in vitro. The amount of AFB1 produced by the toxigenic isolates of A. flavus was ranged from 0.57 to 82.91 ng/ml. Among the 51 isolates of A. flavus tested, the isolate AFM1 produced the highest amount of (82.91 ng/ml) AFB1. Nineteen isolates did not produce aflatoxin B1. The genetic variability among the A. flavus isolates was investigated by PCR amplification of internal transcribed spacer (ITS) including ITS1, ITS2 and the intervening 5.8S rRNA region of ribosomal DNA followed by sequencing. PCR amplification of the ITS regions of the A. flavus isolates (AFM1, AFM3 and AFM5) by using ITS1 and ITS4 primers gave products of approximately 580 base pairs in length. The nucleotide sequences were compared with ITS sequences of A. flavus from Genbank database. Analysis of the genetic coefficient matrix derived from the nucleotide sequences of the ITS regions of A. flavus isolates showed that minimum and maximum percent similarities among the tested A. flavus strains were in the range of 12 and 99%, respectively. Phylogenetic analysis of ITS sequences by the unweighted pair group method arithmetic average (UPGMA) identified three main clusters (A, B and C). Cluster A consisted of only one strain and cluster C consisted of three strains including AFM5. The sequence of AFM5 was clustered with the sequence of a clinical isolate of A. flavus (AY677676) and about 76% similarity index was observed between them at molecular level. All the remaining strains belonged to cluster B, which includes AFM1 and AFM3. The genetic variability among A. flavus isolates should be considered when A. flavus isolates are required for testing in resistance breeding programmes.

Biological control of onion leaf blight disease by bulb and foliar application of powder formulation of antagonist mixture

Abstract Three antagonists: Pseudomonas fluorescens (Pf1), Bacillus subtilis and Trichoderma viride, were tested alone and in combination for suppression of onion leaf blight (Alternaria palandui) disease under glasshouse and field conditions. The average mean of disease reduction was 24.81% for single strains and 42.44% for mixtures. In addition to disease suppression, treatment with a mixture of antagonists promoted plant growth in terms of increased plant height and ultimately bulb yield. Though seed treatment of either single strain or strain mixtures alone could reduce the disease, subsequent application to root, leaves or soil further reduced the disease and enhanced the plant growth. The mixture consisting of Pseudomonas fluorescens Pf1 plus Bacillus subtilis plus Trichoderma viride was the most effective in reducing the disease and in promoting plant growth and bulb yield in greenhouse and field tests.

Endophytic Pseudomonas fluorescens Endo2 and Endo35 induce resistance in black gram (Vigna mungo L. Hepper) to the pathogen Macrophomina phaseolina

Abstract The effect of endophytic Pseudomonas fluorescens isolates Endo2 and Endo35 on induced systemic disease protection against dry root rot of black gram (Vigna mungo L. Hepper) caused by Macrophomina phaseolina was investigated under glasshouse conditions. When the bacterized black gram plants were inoculated with dry root rot pathogen, the activities of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) were stimulated in addition to accumulation of phenolics and lignin. Activity of phenylalanine ammonia-lyase (PAL) reached the maximum 24 h after pathogen challenge inoculation, whereas the activities of PO and PPO reached the maximum at 72 h and 48 h, respectively. Isoform analysis revealed that a unique PPO3 isozyme was induced in bacterized black gram tissues inoculated with the pathogen. Phenolics were found to accumulate in bacterized black gram tissues challenged with M. phaseolina one day after pathogen challenge. The accumulation of phenolics reached maximum at the third day after pathogen inoculation. Similar observation was found in the lignin content of black gram plants. In untreated control plants, the accumulation of defence enzymes and chemicals started at the first day and drastically decreased 3 days after pathogen inoculation. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restricting invasion of Macrophomina phaseolina in black gram roots.

Antiviral action of Harpulia Cupanioides and Mirabilis Jalapa against tomato spotted wilt virus (TSWV) infecting tomato

Mirabilis jalapa and Harpulia cupanioides remain as an unexploited potential to be tapped, as sources of antiviral principles. The antiviral protein from M. jalapa (MAP) and H. cupanioides (HAP) were highly effective in inhibiting TSWV at 60 and 80% saturation. A minimum concentration of 400 μg/ml of MAP was sufficient to inhibit TSWV. HAP at 800 μg/ml recorded 98.41% inhibition of lesion formation. The plants treated with M. jalapa and H. cupanioides challenged with TSWV did not show significant variation in absorbance values when compared to uninoculated healthy plants, indicating the absence of detectable virus through indirect DAS ELISA thus indicating the effectiveness of AVPs. Pre-application of MAP and HAP induced the activity of phenols, PO, PPO and PAL leading to the suppression of TSWV on local lesion and systemic host.

Influence of phylloplane colonizing biocontrol agents on the black spot of rose caused by Diplocarpon rosae

Abstract An attempt was made to study the biocontrol efficacy of antagonistic microorganisms in phylloplane of rose cv. Edward to manage the black spot (Diplocarpon rosae) disease. Eight antagonistic microorganisms were tested in vivo against the black spot pathogen. Among these, Trichoderma viride and Pseudomonas fluorescens pf1 reduces the mycelial growth significantly. These two biocontrol agents were evaluated for their ability to induce defense-related enzymes and chemicals in plants. Increased activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO) and total phenolics were recorded in all the biocontrol agents treated leaves. P. fluorescens Pf1 recorded early and increased synthesis of the entire defense-related enzymes and total phenol within 6 days. The application of biocontrol agents induced the defense-related enzymes involved in phenyl propanoid pathway in addition to direct antagonism, which collectively contribute for enhanced resistance against invasion of Diplocarpon rosae in rose.

Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I17 produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I17 which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.