5.2.79
SECONDARY METABOLITES OF PSEUDOMONAS FLUORESCENS IN THE SUPPRESSION OF BACTERIAL BLIGHT OF COTTON INDUCED BY XANTHOMONAS AXONOPODIS PV. MALVACEARUM

KK MONDAL1, P DUREJA2, RP SINGH1 and JP VERMA1

1Division of Plant Pathology, and 2Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012, India

Background and objectives
Bacterial blight caused by Xanthomonas axonopodis pv. malvacearum (Xam) is the most important disease of cotton worldwide, causing up to 20-30% yield losses. Biocontrol of Xam using cotton rhizosphere and phylloplane microflora has been demonstrated [1]. Metabolites with biocontrol properties have been reported from diverse members of the beneficial rhizosphere and phylloplane microflora. The present investigation was aimed at evaluation of the role of secondary metabolites of Pseudomonas fluorescens strain PfK-26, a successful biocontrol agent isolated from cotton rhizosphere, in controlling bacterial blight disease intensity; and to understand the relative importance of different secondary metabolites.

Materials and methods
Plants and microorganisms used in this experiment were Gossypium barbadense, G. hirsutum, P. fluorescens strain PfK-26, isolated from cotton rhizosphere, Xam race-32, which is highly virulent race prevalent in India. The strain PfK-26 of P. fluorescens and race 32 of Xam used throughout this work were grown on yeast-glucose-chalk agar (YGCA) medium for short-term maintenance whereas long-term storage was done in 10% glycerol at -80C. The strain PfK-26 of P. fluorescens was selected for siderophore production on low iron succinate medium, for catechol production and HCN production. Toxicity of cyanic acid to Xam was studied. Extraction and partial purification of metabolites was done through TLC (silica gel 60 F254, Merck) using polar solvent CHCI3:MEOH (100:1, v/v); structures of these metabolites were identified using GCMS [2]). Efficacy of the metabolites on growth/susceptible disease reaction of Xam was studied both in vitro and in vivo by paper disc and injection infiltration methods, respectively.

Results and conclusions
The strain PfK-26 of P. fluorescens produced siderophores under iron-limiting conditions with the absorbance peak at 500 nm; catechol at the mid-stationary phase of bacterial growth and HCN at mid-stationary to early death phase. Cyanic acid inhibited the growth of Xam completely at 1 gM. Extraction and partial purification of metabolites through TLC resulted in the detection of four main compounds possessing Rf values of 0.72, 0.64, 0.51 and 0.46, referred to as compounds I, II, III and IV which were produced in the ratio of 3:2:1:2. Compounds I and IV are fluorescent. The compounds II and IV suppressed the growth of Xam under in vitro conditions. Both fluorescent (I and IV) as well as non-fluorescent (II) compounds inhibited the susceptible reaction on pre-inoculation to challenge inoculation (0-8 h) of Xam. The results of the present study indicated that antagonism or suppression of susceptible reaction of Xam by cotton rhizosphere strain PfK-26 of P. fluorescens is due to production of siderophores and cyanic acid; involvement of other secondary metabolites with antimicrobial properties is not ruled out. In conclusion, suppression of bacterial diseases by the rhizosphere isolates is multifactorial with bacterial secondary metabolites possessing a key role.

References
1. Laha GS, Verma JP, Singh RP, 1996. Indian Phytopathology 49, 3-8.
2. Keel C, Schnider U, Maurhofer M et al., 1992. Molecular Plant-Microbe Interactions 5, 4-13.