Prevalence of bitter gourd mosaic complex and evalution of beneficial fungal root emdophyte, Piriformospora indica for its management

Abstract

The doctoral research work, entitled “Prevalence of bitter gourd mosaic complex and evaluation of beneficial fungal root endophyte, P. indica for its management” was done with the objectives to assess the prevalence of bitter gourd mosaic complex (BGMC) in the major six bitter gourd cultivating agroecological units (AEUs) of Kerala; detect the virus(es) associated with the disease; manage the disease using beneficial fungal root endophyte P. indica; and elucidate the role of anti oxidants and stress phytohormones in P. indica-mediated tolerance to the disease. Roving survey was conducted across 18 locations in Kerala, India during the summer months of 2021 to assess the prevalence of BGMC disease in various AEUs. The study covered six AEUs and involved fields with a minimum area of 15 cents, cultivating different varieties of bitter gourd, including KAU-released Preethi, hybrids and local varieties. The disease incidence (DI) and severity as vulnerability index (VI) were recorded based on viral symptoms observed in the fields. BGMC was prevalent in all surveyed locations, with 14 locations reporting DI above 50 per cent, and seven locations recording 100 per cent. The highest incidence and severity of the disease were observed in AEU 8 (Thiruvananthapuram) and AEU 1 (Alappuzha). Preethi, a widely cultivated variety in Kerala, showed the highest susceptibility (DI 99.04 per cent and VI-65.01). The infected plants exhibited a range of symptoms including mosaic, stunting, yellowing of leaves, downward and upward leaf curling, blistering, puckering, chlorosis and leaf hairiness. The prevalence of symptoms varied across AEUs, with mosaic and stunting being the commonly observed. Less common symptoms included rosetting, vein banding and vein clearing. The virus(es) identification revealed the association of virus(es) with specific symptoms. Tomato leaf curl virus (ToLCV) infection was characterized by upward leaf curling and hairiness; whereas papaya ring spot virus (PRSV) caused leaf yellowing, blistering and puckering; while cucumber mosaic virus (CMV) was linked to mottling and leaf distortion. 174 Immunological (DAS-ELISA and DIBA) and molecular (PCR and RT-PCR) diagnostic methods were used to detect ToLCV, PRSV and CMV. The study revealed that ToLCV and PRSV were prevalent across all AEUs, with PRSV being the most dominant virus (77.78 %) followed by ToLCV (50 %); while CMV was detected in fewer locations (16.67 %). The virus infections occurred either as single, double or triple; with single infections being the most common (61.11 %). Double infections were found in 33.33 per cent of locations, while triple infections were rare (5.56 %). The pattern of infection varied between AEUs, with single infections dominating in AEU 6 and 11, and multiple infections being predominant in AEU 8. In both pre-inoculation and post-inoculation studies, the influence of P. indica on viral disease progression in bitter gourd was evaluated. Pre-inoculation studies involved colonizing bitter gourd plants with P. indica prior to the viruses inoculation (ToLCV, PRSV and CMV) by grafting at different intervals; while post-inoculation studies assessed the colonization of plants after the viruses inoculation. Results indicated that P. indica significantly delayed the symptom appearance and reduced the incidence and severity of BGMC compared to non-colonized plants. Viral titre levels were lower in P. indica-colonized plants, especially when virus inoculation was done at 10 or 15 days after colonization (DAC). Moreover, yield parameters like flowering, fruit setting and yield were significantly improved in P. indica-colonized plants compared to the viruses infected as well as absolute controls. Field studies assessed the effect of P. indica on BGMC incidence in the colonized and control plants in two seasons. P. indica significantly delayed the viral symptoms. The control plants had symptoms at 12.80 days in summer and 17.6 days in rabi; while the colonized plants showed symptoms at 28.27 days and 30.53 days respectively. The disease incidence was lower in the colonized plants in both seasons (46.67 % in summer and 33.33 % in rabi) compared to controls (100 %). Assessment of the yield and yield parameters revealed earlier flowering, higher fruit set (71.36% in summer and 74.95% in rabi), and increased yields (2.89 kg in summer and 3.07 kg in rabi) in the colonized plants compared to control plants. These results demonstrate 175 that P. indica effectively manages BGMC with enhanced growth and yield in bitter gourd plants. Biochemical assays were undertaken to assess the accumulation of reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂), the activities of catalase, superoxide dismutase (SOD) and peroxidase at 2-, 15-, and 30- days of post-treatment. The results demonstrated that the virus(es)-inoculated plants had significantly elevated levels of ROS and H₂O₂; while the highest catalase, peroxidase and SOD activities were observed in P. indica-colonized treatments. These findings underscore the potential of P. indica in enhancing oxidative stress tolerance and its utility as a biocontrol agent in managing viral diseases in bitter gourd. The transcript levels of salicylic acid (NPR1, PR1), abscisic acid (AAO3, ABA2) and ethylene (ETR1, EIN2) signalling genes were quantified through semi quantitative RT-PCR. The results indicated that P. indica significantly upregulated NPR1 and PR1 expressions in the viruses-inoculated plants compared to control, thus, enhancing induced systemic resistance in plants. Additionally, P. indica upregulated ABA-signalling and modulated ethylene signalling genes in the viruses-infected plants. These findings suggest that P. indica contributes to enhanced phytohormonal responses, facilitating tolerance to the viruses in bitter gourd plants. Seeds from both the infected and healthy plants were raised in insect-proof condition. Subsequent viruses detection at different interval revealed absence of all the three viruses both in seedlings / plants raised from the viruses infected plants. Therefore, this study concludes that ToLCV, PRSV and CMV are not seed transmissible in Preethi variety of bitter gourd. Moreover, none of these viruses was detected in embryos or cotyledons of seeds from symptomatic plants. BGMC is more prevalent with high disease incidence and severity in the selected AEUs of Kerala; and caused by ToLCV, PRSV and CMV either singly or in combinations. The disease can be effectively managed in the field by the beneficial fungal root endophyte P. indica, without considerable yield loss and with ecological sustainability.