PhD

The study on ‘Endophytic microorganism mediated systemic resistance in cocoa against Phytophthora palmivora (Butler) Butler’ was carried out during 2005-2010. The pathogen causing pod rot of cocoa was isolated from infected pods, and its pathogenicity established. Based on cultural and morphological characters, it was identified as Phytophthora palmivora (Butler) Butler. Endophytes were isolated from samples of feeder roots, tender shoots, leaves and pods of cocoa collected from various locations of major cocoa growing areas of the state. The population of endophytic microflora varied among different locations and parts of the plant, and in general, the population was more in roots. Bacteria and fluorescent pseudomonads were more abundant than filamentous fungi and yeasts.
Out of the 325 endophytic isolates comprising of 116 bacteria, 153 fluorescent pseudomonads, 34 yeasts and 22 fungi, 82 were found exerting antagonism towards the pathogen. These antagonistic endophytes were further evaluated in in vitro by dual culture and by inoculation on detached cocoa pods, and leaves. It was found that, 25 isolates were more efficient antagonists. These included endophytic isolates of 12 fluorescent pseudomonads, nine bacteria and four fungi. The 25 efficient endophytic antagonists were evaluated for growth promoting ability in cocoa seedlings. It was observed that eight isolates had a profound effect on growth promotion. Hence, these were selected as potential endophytes and were subjected to various tests to study the attributes underlying their antagonistic and growth promoting effects. The potential endophytes consisted of isolates of two bacteria, five fluorescent pseudomonads and one fungus. These eight potential endophytes along with two reference cultures were evaluated in in vitro for various attributes, which underlay their beneficial effects. It was found that, isolates EB-35, EB-40, and EF-81 produced more ammonia. Phosphate soulubilizing ability was maximum for EB-35. The isolates EB-35, EB-40 and EB-65 produced high quantity of IAA. High score for antagonistic index was obtained by EB-31 and EB-35. Vigour index of coca seedlings was also high for EB-31 and EB-35. The plant growth promoting index worked out based on aforementioned attributes was high for five isolates viz., EB-31, EB-35, EB-40, EB-65 and EF-81, which were selected as promising endophytes and were subjected to further studies and in vivo evaluation. The promising endophytes were found to produce volatile and non-volatile inhibitory metabolites against the pathogen. Maximum inhibition through volatile production was with EB-35 and EB-31. While through non-volatiles, the maximum inhibition was by EB-35 and EB-40. Of the four promising bacterial endophytes, three emitted fluorescence under UV light and EB-31 and EB-65 produced more siderophores under iron limiting condition.
A pot culture experiment was conducted to study the induction of systemic resistance and suppression of Phytophthora infection in cocoa seedlings by the promising endophytes. Here also, endophytic isolates effected growth promotion in cocoa seedlings and reduction in infection. Induction of systemic resistance was studied by assay of defense related compounds and enzymes. In general, the study revealed more accumulation of phenols and proteins in treated seedlings. Higher activity of PO, PPO, and β-1,3-glucanase was also noticed. Native PAGE analysis revealed six isoforms of PO and seven of PPO. More isoforms were present in endophyte treated plants. Promising endophytes were evaluated for efficiency in reducing Phytophthora pod rot in field in comparison with two reference cultures and chemicals. During the first phase of experiment, viz., after first spraying, maximum reduction in disease was observed in EB-65 and EF-81. Whereas, after second spraying least incidence was in EB-31. After two weeks of third spraying, minimum disease was observed in Pf1 and EB-35. However, the isolate EB-31 was the most efficient one which recorded the least disease incidence during most of the period under observation especially when the disease was at its peak.
Based on cultural, morphological and biochemical characters coupled with results of molecular characterization, the promising bacterial endophytes were identified as Pseudomonas putida (EB-31), Bacillus subtilis (EB-35), P. plecoglossicida (EB-40) and P. aeruginosa (EB-65). The isolate EF-81 was identified as Penicillium minioluteum. In the radiotracer experiment, it was found that EB-35 and EB-65 entered the cocoa seedlings when applied on leaves and also inside the pods on application on the intact surface.