Prevalence variability and characterisation of Phytophthora palmivora Butl. inciting bud rot of coconut and its management using endophytes in seedlings

Abstract

The research work entitled “Prevalence, variability and characterisation of Phytophthora palmivora Butl. inciting bud rot of coconut and its management using endophytes in seedlings” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2024 with the following objectives; assessment of the prevalence of bud rot in different agroecological units; characterisation of Phytophthora palmivora causing coconut bud rot and its variability; and to evaluate fungal root endophyte Piriformospora indica and the bacterial foliar endophyte Bacillus amyloliquefaciens VLY24 in its management. The survey was conducted in three selected agroecological units of Kerala viz., AEU 8 (Southern laterites), AEU 9 (South central laterites) and AEU 11 (Northern laterites) to study the prevalence of bud rot and to collect the diseased samples. The highest (38.09%) and lowest (4.44%) disease incidence was recorded from the locations of AEU 11. The bud rot incidence in AEU 8 varied from 5.79 per cent to 28 per cent. The disease incidence of AEU 9 varied from 5.00 per cent to 34.28 per cent. The varieties, WCT, CGD, COD, Komadan and other dwarf palms belonging to the age group 2-50 years old were observed with disease incidence during the survey. Characteristic symptoms of bud rot noticed at survey locations were rotting, withering and drooping of spear leaves followed by its complete rotting with a foul smell. Ten isolates of Phytophthora spp. were obtained from infected palms of surveyed locations. Based on the molecular characterisation seven isolates were identified as P. palmivora, two as P. nicotianae and one P. meadii.A screening was carried out to identify the most virulent isolate of Phytophthora spp. based on the lesion size produced on the detached spear leaves and nuts of West Coast Tall variety upon the inoculation with mycelium disc and zoospore suspension. The screening was done for 13 Phytophthora isolates, including ten isolates from surveyed locations and three isolates obtained from ICAR-Central Plantation Crops Research Institute, Kasaragod. P. palmivora isolate 8APp from Kottukal (AEU 8) was identified as the most virulent isolate which produced largest lesions on detached leaves and nuts with a fastest lesion growth. This isolate was selected for further studies, dual culture, pot culture and field studies against P. palmivora.Variability of 13 Phytophthora isolates was studied based on the cultural, morphological characters and molecular analysis through RAPD. Significant variability was found in all the observed characters among different isolates. Phytophthora isolates exhibited a variety of growth patterns like stellate, semi-starry, chrysanthemum, light rosette and also without any specific pattern. Phytophthora palmivora isolate 8APp (Kottukal, AEU 8) showed the highest growth rate (1.33 cm/day). The lowest growth rate was observed for the isolate 9APn (Attingal, AEU 9; 0.89 cm/day).Phytophthora isolates produced hyaline coenocytic hyphae. Significant variation in width of hypha was noticed among isolates and it varied from 4.08 - 6.34 µm. The chlamydospore dimensions varied from 19.39- 31.52 µm x 18.90 - 30.65 µm which significantly varied among the isolates. Phytophthora produced ovoid, pyriform, ellipsoid and limoniform shaped sporangia and significant variation in sporangial size was noticed among the isolates. Length of sporangia varied from 24.56 - 55.56 µm; breadth varied from 19.55 - 28.58 µm and L/B ratio varied from 1.26 – 2.20.The genetic variability among Phytophthora isolates was studied by RAPD. Analysis of the RAPD pattern produced by four decamer primers revealed intra specific variability in isolates and also the variability existing in morphologically similar isolates. Evaluation of P. indica and B. amyloliquefaciens VLY24 against P. palmivora was done by dual culture assay. B. amyloliquefaciens VLY24 inhibited the mycelial growth of P. palmivora by 50.22 per cent at 10 DAI in dual culture plate. P. indica restricted the mycelial growth of P. palmivora by 36 per cent. In the interaction between B. amyloliquefaciens and P. palmivora, an inhibition zone of 1.64 cm was produced by B. amyloliquefaciens against P. palmivora; while, the type of interactions like lysis, inhibition zone and over growth was absent in the interaction between P. indica and P. palmivora. Antagonistic potential of B. amyloliquefaciens was comparatively higher than P. indica against P. palmivora. Evaluation of P. indica and B. amyloliquefaciens VLY24 against P. palmivora was carried out by pot culture studies. Colonization of B. amyloliquefaciens alone in coconut seedlings reduced the lesion size by 69.15 and 63.68 per cent in pot culture study1 and 2 respectively. Piriformospora indica + B. amyloliquefaciens colonized seedlings reduced the lesion size by 57.71 and 59.69 per cent in pot culture study1 and 2 respectively. The per cent reduction in lesion size over control in pot culture study1 and 2 by P. indica alone colonized seedlings was 20.89 and 25.85 respectively. Colonization of P. indica and B. amyloliquefaciens enhanced the growth of coconut seedlings by improving seedling height, girth, number of leaves and also early splitting of leaves. In the pot culture study 1 and 2, P. indica + B. amyloliquefaciens colonization improved the seedling height by 42 and 49 per cent, girth by 46 per cent, number of leaves by 18 per cent respectively. Colonization of P. indica alone increased the seedling height by 37 and 30 per cent, girth by 58 and 41 per cent, number of leaves by 31 and 24 per cent in pot culture 1 and 2 respectively. B. amyloliquefaciens alone colonized seedlings improved the seedling height by 23 and 24 per cent, girth by 22 and 30 per cent, number of leaves by 18 and 15 per cent respectively in pot culture 1 and 2. The highest splitting per cent of leaf (80 per cent) at 12th month stage was noticed in P. indica alone colonized seedlings and P. indica + B. amyloliquefaciens colonized seedlings in pot culture study 1. The early splitting of leaf was observed in B. amyloliquefaciens alone colonized seedlings and P. indica + B. amyloliquefaciens colonized seedlings at 9th month stage itself in pot culture study 2 and the highest splitting per cent (60%) was noticed in B. amyloliquefaciens alone colonized seedlings. The field experiments were conducted to evaluate P. indica and B. amyloliquefaciens VLY24 against natural incidence of bud rot disease in colonized seedlings. Lowest disease incidence was recorded in B. amyloliquefaciens alone colonized seedlings (11.11% and 0% in field 1 and 2 respectively), followed by P. indica+ B. amyloliquefaciens colonized seedlings (22.22% and 0 % in field 1 and 2 respectively). P. indica alone colonized seedlings recorded 22.22 per cent and 11.11 per cent DI in field 1 and 2 respectively. In field study 1 and 2, P. indica + B. amyloliquefaciens colonization improved the height by 20 and 15 per cent, girth by 27 and 25 per cent, number of leaves by 9 per cent respectively. P. indica alone colonized seedlings increased the height by 21 and 16 per cent, girth by 24 and 22 per cent, number of leaves by 13 per cent respectively in field experiment 1 and 2. B. amyloliquefaciens alone colonized seedlings did not significantly improved the growth parameters viz., height, girth and number of leaves compared to control. However, the leaf splitting per cent was significantly improved at the 18th month stage under field conditions. Biochemical mechanism of disease tolerance in B. amyloliquefaciens and P. indica colonized seedlings was studied at 24 and 72 hours after inoculation (HAI). Increased activity of defense related enzymes viz., phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, catalase and superoxide dismutase was noticed in B. amyloliquefaciens and P. indica colonized coconut seedlings. Moreover, B. amyloliquefaciens and P. indica reduced the bud rot symptoms in coconut seedlings by inhibiting the accumulation of ROS and hydrogen peroxide. The relative expression of genes involved in salicylic acid and jasmonic acid pathway was studied in B. amyloliquefaciens and P. indica colonized seedling upon P. palmivora inoculation. B. amyloliquefaciens and P. indica reduced the bud rot symptoms in colonized seedlings by the upregulation of genes involved in salicylic acid signalling (NPR1 and PR5) pathway. Upregulation of LOX2 gene involved in jasmonic acid pathway was observed in B. amyloliquefaciens and P. indica colonized coconut seedlings at 72 HAI of P. palmivora. Relative expression of OPR3 gene involved in jasmonic acid pathway was less in B. amyloliquefaciens and P. indica colonized seedlings at 24 and 72 HAI of P. palmivora compared to control diseased seedlings. The bud rot disease of coconut caused by Phytophthora spp. is widely distributed in Kerala. Variability in cultural, morphological and genetic characters exist among different Phytophthora spp. isolates obtained from three selected agroecological units of Kerala viz., AEU 8, AEU 9 and AEU 11. Sowing of coconut seed nuts in P. indica enriched medium (106 cfu g-1) followed by a foliar spray (108 cfu ml-1) of B. amyloliquefaciens VLY24 in 6 months old seedlings is effective in reducing the bud rot incidence and enhancing the growth by improving seedling height, girth, number of leaves and early splitting of the leaves. P. indica and B. amyloliquefaciens VLY24 enhance the tolerance of coconut seedlings against P. palmivora by increasing the antioxidant enzyme activity and inducing the expression of genes in salicylic acid and jasmonic acid pathways.