Management of leaf rot disease in coconut palms through biocontrol agents

Abstract

The doctoral research, “Management of Leaf Rot Disease in Coconut Palms through Biocontrol Agents,” was conducted with the following objectives: to identify and characterize the major fungal pathogens associated with leaf rot disease (LRD) in coconut palms; to evaluate antagonistic and phylloplane microflora; and to develop a promising microbial consortium for managing LRD pathogens at the field level. A comprehensive survey was undertaken across 110 locations in three southern districts of Kerala Thiruvananthapuram, Kollam, and Pathanamthitta encompassing agro- ecological units (AEU) 1, 8, and 9 on the natural incidence of LRD. The disease incidence of LRD showed a decreasing trend moving northward from Thiruvananthapuram, Kollam, and Pathanamthitta. The highest leaf rot incidence (%) (DI) was recorded at the Athiyannur block of Thiruvananthapuram district (47.18%; AEU 8), and the lowest DI was recorded at the Mallapally block of Pathanamthitta district (22.6%; AEU 9). Characteristic symptoms of leaf rot include deterioration of the spindle leaves, rotten tips of the leaflets, extensive rotting of leaves, and a fan-like appearance of the crown. West Coast Tall (WCT) is more susceptible to LRD, followed by the Chowghat Orange Dwarf (COD) variety. Among the hybrids, T X D was the most susceptible. Specimens from symptomatic palms were collected, and causal pathogens were isolated and purified using hyphal tip and single spore isolation techniques. Morphological identification was supplemented by molecular characterization using ITS1/ITS4 primers, confirming a fungal complex involving Colletotrichum theobromicola, Fusarium equiseti, and Exserohilum rostratum as the major pathogens. Pathogenicity tests on detached spindle leaves substantiated Koch’s postulates, validating the role of these pathogens in both individual and combined inoculations. The combined inoculation assay revealed synergistic interactions among the pathogens, leading to more severe lesion formation, suggesting a multi-pathogen etiology. Simultaneously, phylloplane microflora from healthy leaflets of LRD-affected and unaffected palms were isolated through serial dilution and leaf impression techniques. The in vitro antagonistic potential of these isolates, CPBM-2, CPBM-4, CPBM-14,

CPBM-1, CPBM-7 including native Trichoderma spp., Pseudomonas fluorescens (PN026), Bacillus amyloliquefaciens (VLY24), and Piriformospora indica, was evaluated using dual-culture techniques. Antagonistic index was calculated based on lysis, inhibition zones, and overgrowth parameters, following the protocol described by Nakkeeran et al. (2005). The results demonstrated that several isolates exerted significant inhibitory effects on LRD pathogens, with CPBM-2, CPBM-4, and CPBM- 14 being the most effective agents. Compatibility studies among the promising isolates identified three mutually non-antagonistic strains suitable for consortia formulation. Molecular characterization of these strains further confirmed their identity Bacillus velezensis (CPBM-2), B. velezensis (CPBM-4), B. amyloliquefaciens (CPBM-14) using 16S rRNA sequencing. Their efficacy was validated via detached spindle leaf assays, where lesion size was significantly reduced in the treated samples. Field trials at the Coconut Research Station, Balaramapuram (2021–2023), using a randomized block design (RBD) and the West Coast Tall variety, evaluated the field performance of the promising isolates individually and in combination. Ten treatments, including a chemical check (Copper Oxychloride (COC) 0.25%) and an untreated control, were tested. Biocontrol agents were applied as both soil and foliar sprays (CFU @ 10⁸ mL⁻¹). Periodic biometric parameters, such as palm height, collar girth, and number of leaves, were recorded, and the colonization of microbes within plant tissues was confirmed through 3,3′-Diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) staining and scanning electron microscopy (SEM). In this study, a significant induction of plant defense enzymes, specifically phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), and catalase (CAT), was observed in palms treated with biocontrol agents. Biochemical assays conducted at 24- and 72-hours post-inoculation demonstrated a marked increase in enzyme activity, which correlated with enhanced plant defense responses. Semi- quantitative molecular expression analysis indicated the upregulation of Lipoxygenase 2 (LOX2) and 12-oxophytodienoate reductase 3 (OPR3) (jasmonic acid pathway) and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and Pathogenesis-related protein 5 (PR5) (salicylic acid pathway), signifying the activation of resistance pathways.

A talc-based microbial consortium, comprising the three most potent and compatible isolates B. velezensis (CPBM-2), B. velezensis (CPBM-4), B. amyloliquefaciens (CPBM-14), was developed and evaluated under field conditions. Various application strategies, including quarterly, half-yearly, and annual applications, both foliar and soil, were tested for efficacy. The quarterly application of microbial consortia as foliar treatment consistently resulted in the lowest disease incidence and lesion metrics, on par with chemical control (COC 0.25 % - quarterly intervals). In conclusion, this study identified and characterized the major fungal pathogens associated with LRD and underscored the significant role of phylloplane biocontrol agents in suppressing the LRD both in vitro and in vivo. The synergistic application of compatible antagonists as a microbial consortium not only reduced disease severity but also enhanced the defense mechanisms of coconut palms.