5.2.81
BIOLOGICAL CONTROL OF CITRUS FRUIT SOFT ROT USING ALGINATE FORMULATION OF BACILLUS SUBTILIS AF1

K MANJULA and AR PODILE

Department of Plant Sciences, University of Hyderabad, Hyderabad 500 046, India

Background and objectives
Bacillus subtilis AF1 isolated from pigeonpea wilt non-conducive soil has plant growth-promoting and emergence-promoting activity. B. subtilis AF1 also promotes nodulation and was antagonistic towards major fungal pathogens of plants. B. subtilis AF1 extensively colonizes the surface of the fungal pathogen Aspergillus niger and lyses the mycelium. Formation of reproductive structures was delayed in A. niger during interaction with B. subtilis AF1. One of the factors contributing to the mycolytic activity was found to be a 1,4,O-N-acetyl glucosaminidase. The objective of the present study was to compare the efficiency of culture filtrate proteins, partially purified 1,4,O-N-acetyl glucosaminidase and alginate formulation of B. subtilis AF1 for control of post-harvest citrus fruit soft rot.

Materials and methods
Crude culture filtrate proteins and partially purified 1,4,O-N-acetyl glucosaminidase of B. subtilis AF1 were tested for in vitro inhibitory effect against the citrus fruit soft rot pathogen A. niger spores in microtitre plates. Late log phase culture of B. subtilis AF1 grown in potato dextrose broth was added with 2% (w/v) sodium alginate and was dropped in to 0.1 M CACI2 solution under aseptic conditions. The alginate beads formed and entrapped B. subtilis AF1 cells were allowed to stabilize for 1 h and dried under sterile air flow after washing with sterile distilled water. The dried beads were stored at 30C for 4 months before their usage for post-harvest citrus fruit soft rot control. Potassium phosphate buffer at 0.2 M concentration was used for dissolving the beads and the resulting mixture was diluted with sterile saline for treating the citrus fruits. The disease control with alginate and fresh culture of B. subtilis AF1 treatments was compared. Similarly culture filtrate proteins and partially purified 1,4,O-N-acetyl glucosaminidase were used for control of citrus fruit soft rot and the results were correlated with their in vitro inhibitory effect on germination of A. niger spores in microtitre plate assays.

Results and conclusions
Culture filtrate proteins, partially purified 1,4-O-N-acetyl glucosaminidase, inhibited the germination of A. niger spores for 32 h. The inhibition of spore germination by partially purified 1,4,O-N-acetyl glucosaminidase and culture filtrate proteins did not differ at 0.01 mg concentration indicating the involvement of some other factors in antagonistic activity of B. subtilis AF1. Percentage disease development in alginate, culture filtrate proteins and partially purified 1,4,O-N-acetyl glucosaminidase-treated fruits were compared with control fruits which were treated with pathogen alone. 94% of the fruits were found to be diseased in control whereas only 8% were diseased in alginate and fresh culture treatments. The number of soft rot lesions and lesion size were smallest in case of alginate treatment which is similar in fresh culture treatment. Crude culture filtrate proteins were more effective than partially purified 1,4,O-N-acetyl glucosaminidase in controlling the disease. Alginate formulation of B. subtilis can efficiently be used for control of post-harvest citrus fruit soft rot.