Efficacy of pink pigmented facultative methylotrophs for the management of leaf blight disease in Amaranthus (Amaranthus tricolor L.)

Abstract

Amaranthus (Amaranthus tricolor L.), ("poor man's spinach") is one of the widely cultivated tropical leafy vegetables of Southern India. It is attacked by the most destructive disease, leaf blight by Rhizoctonia solani Kuhn, causing more than 90 per cent of yield loss. Chemical management of this pathogen causes health hazards due to pesticide residue. PPFM naturally occurring on the phyllosphere of amaranthus have great value in giving protection against leaf diseases, on which no detailed systematic studies have been conducted in Kerala. In this context, the present study was undertaken with the objectives of identification of Pink Pigmented Facultative Methylotrophs (PPFM) from amaranthus phyllosphere and to evaluate their efficiency for the management of leaf blight disease in amaranthus. The experiment was carried out in Pepper Research Station, Panniyur, during March 2022 – November 2022. Purposive sampling surveys were conducted in amaranthus growing fields of five different AEUs in Kannur district of Kerala viz.Thalassery, Kuttiyattoor, Panniyur, Punnad and Kanichar from April to May, 2022. The disease incidence and severity were maximum in the Kuttiyattoor area with 89.40 and 74.60 per cent, respectively. The fungus was isolated from leaf samples and the pathogenicity was proved. Symptomatology was studied both under natural and artificial conditions. The fungus was characterized and identified as Rhizoctonia sp. by studying their cultural and morphological characteristics. Highly virulent isolate, LB-3 was selected for further studies. PCR amplification of large subunit (LSU) region of the fungal genome followed by sequencing and in silico analysis confirmed that the suspected fungus, LB3 as Rhizoctonia solani. A total of 10 isolates of Pink Pigmented Facultative Methylotrophs (PPFM) from amaranthus phyllosphere were isolated and cultural, morphological and biochemical characterization was done. All the isolates were screened for their antagonistic potential against selected R. solani using dual culture technique. PPFM-2 and PPFM-5 showed 53.18 and 53.69 (in solid medium) and 77.77 and 74.04 (in liquid broth) per cent inhibition respectively. Hence, these two most promising isolates were selected for further field studies. The bacterial isolates PPFM-2 and PPFM-5 were confirmed as Microbacterium testaceum and Methylobacterium hispanicum through the amplification of 16S rRNA region of the genome followed by sequencing and in silico analysis. Under in vitro conditions, antagonistic activity of PPFM-2 and PPFM-5 were compared with biocontrol agents, botanical and fungicide against R. solani. Dual culture assay showed that PPFM-2 (52.77 %) and PPFM-5 (53.88 %) were more effective than Pseudomonas fluorescens (43.42 %) and less effective than Trichoderma asperellum (72.50 %) for inhibition of R. solani. Poisoned food technique showed that PPFM-2 and PPFM-5 were in line with mancozeb 75 WP, as both showed 100 per cent inhibition to R. solani. And they were more effective than Raksha at 0.6 (36.00 %), 1.0 (74.40 %) and 2.0 (82.20 %) per cent concentration. Efficacy of selected strains of PPFMs (M. testaceum and M. hispanicum), biocontrol agents (T. asperellum and P. fluorescens), botanical (Ready to Use Neem Oil Garlic Soap, Raksha) and fungicide (Mancozeb 75 WP) against R. solani under field conditions were tested. The natural disease incidence in the field was delayed to 28 days by M. hispanicum and to 23 days by M. testaceum. Maximum per cent reduction over control for disease incidence and disease severity was showed by M. testaceum and M. hispanicum. Biometric observations at 30, 45 and 65 days after transplanting, showed maximum increase over control for plant height, number of leaves per plant, fresh and dry weight of shoots and shoot root ratio in PPFM treated plots. In contrast, the root length as well as fresh and dry weight of roots were maximum in P. fluorescens treatment. Recording the PPFM population in the phyllosphere at harvest, it is revealed that plants with the highest population of PPFM displayed significantly enhanced biometric characters as compared to plants with a substantially lower population.