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Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam
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ThesisItem Embargo Evaluation of potential rhizospheric fungal antagonists against banded leaf and sheath blight of maize caused by Rhizoctonia solani f. sp. sasaki (Kuhn.) Exner(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-06) Debbarma, Manashi; Rajesh, T.Rhizosphere represents the most dynamic ecosystem providing a close association between plant root and rhizosphere associated microbial communities. Among the microbial communities colonizing the rhizosphere, Rhizospheric Fungi (RF) hold prominent position but are less explored than that of rhizospheric bacteria. RF plays a vital role in agricultural and environmental process as they can impact the ecological balance, plant growth, suppress or stimulate colonization as well as antagonism of plant pathogens. Therefore, the present investigation was aimed to isolate and evaluate the potential antagonists from maize rhizosphere against Banded leaf and Sheath blight of maize caused by Rhizoctonia solani f. sp. sasaki. Through extensive surveys conducted in different maize growing areas of Meghalaya, soil samples were collected from maize rhizosphere region from eight (8) districts viz., Ribhoi, West Garo Hills, North Garo hills, West Khasi Hills, West Jaintia hills, South West Garo Hills, East Khasi Hills and East Jaintia Hills. A total of eighty (80) fungi were isolated and recovered from rhizospheric soil by serial dilution method, of which majority of isolates showed higher inhibition of 50-80% against R. solani, belongs to the genus Trichoderma sp. (20 isolates) followed by Penicillium sp. (18), Aspergillus sp. (10), Phoma sp. (10), Acremonium sp. (6), Fusarium sp. (5), Chaetomium sp. (4), Metarrhium sp. (2) and Pythium sp. (5). Further, eighty (80) isolates were identified based on morphological and microscopic characteristics. Out of twenty (20) Trichoderma isolates, twelve (12) isolates were found most effective based on their antagonistic potential and were selected for further studies based on antimicrobial and plant growth promotion (PGP) traits. Potential Trichoderma (12) isolates were further identified based on molecular method using ITS primers and accession number was obtained from NCBI. In addition to that, systemic fungicides were evaluated against R. solani f. sp. Sasaki using poisoned food technique, of which carbendazim @ 0.1% showed higher inhibition of 94.44% against R. solani f. sp. sasaki. Among these twelve (12) isolates, only one best RF antagonist viz., RF 44 (T. harzianum) as liquid bioformulation was further evaluated for its efficacy against BLSB of maize (R. solani f. sp. sasaki) under field condition. Various mode of bioformulation delivery either as individually or in combination of seed and soil and foliar were evaluated. In total eleven (11) treatments were evaluated, of which the treatment combination of soil + seed + foliar application + liquid was found most effective in managing the disease showing only 29% disease incidence as compared to control which showed 71% disease incidence. In addition to that, yield per plant i.e., 0.65 kg/plant was also found significantly higher as compared to control i.e., 0.32 kg/plant. Therefore, the potential RF antagonist (RF 44) can be used as effective biological control agent against BLSB of maize which further requires field trial under different locations in this region.ThesisItem Embargo Management of rhizome rot of ginger (Zingiber officinale Rosc.) in Meghalaya(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2021-09) Satyanarayana, Maaragaani S.V.; Devi, R. K. TombisanaIndia, a land of spices marked by the globe for our rich spice heritage. Ginger being one of the members in that, exerted a huge demand in open market. This crop plays a prominent role amid the rural livelihood and prosperity. In contempt of these highlights, this is crop out layered with certain biotic ailments in form of a menace ‘rhizome rot’. A study was conducted in order to identify the status and cause of the disease, evaluate the pathogen’s virulence potential, explore the diverse microhabitat of ginger rhizosphere, evaluate the potent native antagonist and fungicides along with cultural and integrative tactics to manage the disease in Meghalaya. In survey, Ri-Bhoi (54.24) recorded highest disease incidence. The causal agents were identified as Fusarium oxysporum f. sp. zingiberi (Foz) and Fusarium solani. All the three major pathogens P. aphanidermatum, Foz and R. solanacearum proved their virulence capability in pathogenicity test depicted a disease incidence and yield loss by 98.61 and 100 per cent respectively. Diverse rhizospheric microflora belongs to genus Acidovorax spp, Azohydromonas spp, Bacillus spp, Pantoea spp, Stenotrophomonas spp, Acremonium spp, Penicillum spp, Trichoderma spp. were found habituated with the crop in this region. Amid tested native isolates and fungicides, P. citrinum, Trichoderma neotropicale, Hypocrea lixii, Bacillus velezensis, B. subtilis and B. amyloliquefaciens proved their antagonistic potential and carbendazim+mancozeb was found efficient against Foz in vitro. The bacterial biocontrol agents exhibited a positive compatible response under co-culture studies under laboratory conditions. The results emanated from the bi-seasonal field experiment by the integrative tactics clearly indicated that, soil amendment with organic manures such as neem cake @250kg/ha or mustard cake @100 kg/ha either with pre sowing treatment of rhizomes with hot water @50oC for 30 minutes or with T. neotropicale @5kg/ha were found effective when challenged against the disease and could reduce the disease incidence by 23-52 per cent, and also significantly improved the agronomical and yield attributes. The technology developed in the present study would serve as an economically feasible and effective integrated strategy for the management of rhizome rot of ginger.ThesisItem Open Access Characterization of phylloplane microflora of tomato (Solanum lycopersicum L.) and their role against major foliar fungal diseases(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2020-07) Hajong, Monika; Rajest, T.Phylloplane is a leaf surface, which harbours a diverse kind of microbial populations. Foliar disease management was not as successful as compared in rhizosphere as the microbial growth on leaves are restricted by severe environmental conditions. Phylloplane microflora play a vital role in agricultural and environmental process as they can impact the ecological balance, plant growth, suppress or stimulate colonization and infection of the plant pathogens. Therefore, the aim of this investigation was to isolate and evaluate the potential strains from tomato phylloplane against the major foliar fungal diseases namely early blight, late blight and Septoria leaf spot. The surveys were conducted from different tomato growing areas of 7 districts namely Ri-bhoi, East Khasi Hills, West Jaintia Hills, East Jaintia Hills, East Garo Hills, West Garo Hills and South West Garo Hills of Meghalaya for the collection of tomato leaf samples. Total, 173 bacteria and 96 fungi were isolated from collected samples by following 3 different methods. Out of 3 different isolation methods, leaf impression method recovered the maximum microbial populations followed by leaf washing and without dilution plating method and leaf washing and serial dilution plating method. Bacteria were found predominant in tomato phylloplane compared to fungi. All the bacterial and fungal isolates were tentatively identified and grouped based on morphological and biochemical tests. The predominant bacteria tentatively identified were whitish colony (102 isolates) followed by yellow-pigmented bacteria (28 isolates), bacteria with cream colonies (20 isolates), fluorescens bacteria (10 isolates), pink-bacteria (8 isolates) and branching-like bacteria (5 isolates). The predominant fungi found were Phoma sp. followed by Trichoderma sp., Pythium sp., Penicillium sp., Fusarium sp., Acremonium sp., Aspergillus sp., Chaetomium sp., Cladosporium sp. Rhizopus sp., Botryodiplodia sp., Alternaria sp. and Camarosporium sp. Out of 173 bacterial isolates only 10 isolates were found effective by showing at least 50% growth inhibition of foliar pathogens under in vitro condition. The results of present investigation suggested that majority of the phylloplane fungi isolated from tomato are pathogenic in nature and only Penicillium sp. and Trichoderma sp., were found most effective by showing at least 50% growth inhibition of pathogens under in vitro condition. Only 10 bacterial and 10 fungal isolates were selected as antagonists against the pathogens based on their in vitro tests such as dual culture assay, antimicrobial and PGP traits. These 10 bacterial strains were further identified based on molecular method using 16S rDNA gene sequenced. The fungal strains were identified by National Centre for Fungal Taxonomy (NCFT), New Delhi. Among these 20 strains, only 2 best strains namely B. altitudinis ASH2 and P. chrysogenum ALH1L were further selected for talc-based bioformulation preparation. These talc-based bioformulations were further evaluated under pot and field condition against the late blight disease of tomato (P. infestans). Various mode of bioformulation delivery was used either in individual as seed treatment or soil treatment or root-dip treatment or foliar spray and in combination. Total 15 treatments were used under field condition including the control, and it was found that the combination treatment of B. altitudinis ASH2 and P. chrysogenum ALH2L (seed + soil + root-dip + foliar spray) was most effective in managing the late blight disease by recording only 8.33% and 41.67% disease incidence as compared to negative control (58.33%) even on 90th DAT. Also, maximum yield was recorded with these 2 treatments (48.36 and 46.28 t/ha/plot) as compared to negative control plot (23.94 t/ha/plot). Similarly, combination treatment with both the bioformulations respectively under pot experiment in both the seasons were also found the lower disease incidence with higher yield as compared to the control plant.ThesisItem Open Access Characterization of arbuscular mycorrhizal fungi and endophytic bacillus in tomato roots and their antagonism against Fusarium Wilt.(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2021-07) Devi, Nongthombam Olivia; Devi, R. K. TombisanaTomato (Solanum lycopersicum M.) is one of the most popular vegetable ranking next to potato and widely grown in Meghalaya. Among the soil borne diseases, Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (FOL) is a devastating disease causing wilting and death of tomato thereby inflicting major yield losses. Arbuscular mycorrhizal fungi (AMF) are found to be associated with 80-90% of plants including tomato. Bacillus endophytes and AMF are known to promote plant growth and suppress soil borne pathogens. In the present investigation, AMF spores were isolated from 20 sample sites coming under six districts and four agroclimatic zones of Meghalaya with highest number of spores/50 g soil recovered from Umsning (680.67 spores). A total of 41 AMF species were recovered and identified from 20 sample sites. Daistong with 15 AMF species had the highest species richness. Funneliformis mosseae, Glomus aggregatum, Glomus intraradices and G. fasciculatum were the dominating AMF species having isolation frequencies (IF) of more than 60%, whereas G. australe, F. badium, A. melleae and A. scrobiculata had IF of more than 45%. All the tomato roots collected from the 20 sample sites were colonized by AMF with Umiet (41%) recorded the highest colonization per cent. From trap culture, Umiet had the maximum spore density (1103 spore/50 g soil). All 41 AMF species isolated from field soil were also recovered from trap culture inoculum along with an additional 6 species viz. Archaeospora trappie, Acaulospora longula, Appendisphaera centroreticulata, F. geosporum, G. boreale, and G. viscosum from trap culture soil. Umiet with 19 AMF species had the maximum species richness. G. intraradices had the highest IF of 70% followed by F. mosseae, G. aggregatum, and G. fasciculatum, while AMF species with more than 50% IF were F. badium, A. mellae, A. scrobiculata and G. australe. AMF root colonization in trap plant maize was maximum in Umiet derived inoculum (92%). All the 47 AMF species showed variation in shape, size, colour and hyphal form attached to the spore walls. Out of the 12 dominating AMF species screened for growth promotion of tomato plant in pot experiment, F. mosseae and G. fasciculatum performed best in terms of plant and mycorrhizal parameters. A total of 130 endophytic Bacillus were isolated from tomato roots collected from 20 sample sites of Meghalaya. Based on biochemical tests, morphological character and bacterial identification software ABIS online all the 130 isolates were tentatively identified up to species level. The isolates belonged to the genera Bacillus, Paenibacillus and Viridibacillus with maximum of 17 isolates each tentatively identified as Bacillus amyloliquefaciens and B. thuringiensis. Out of 130 Bacillus isolates, 12 isolates showed more than 27.44% inhibition of growth of FOL in dual culture method of which isolates ERBS51 (58.43%) and ERBS10 (55.68%) recorded the maximum inhibition of FOL. Further, antagonistic test of the 12 potential isolates revealed ERBS51 (55.83% and 91.52%) followed by ERBS10 (43.37% and 87.28%) as the best one with highest FOL inhibition in sealed plate method and antagonistic activity in liquid media respectively. The 12 potential isolates were identified as Bacillus sp. (ERBS4, ERBS10, ERBS29, ERBS47, ERBS75, ERBS118), while other as B. velezensis (ERBS51), B. cereus (ERBS69 and ERBS119) and B. subtilis (ERBS80), B. swezeyi (ERBS115) and B.subtilis sub sp. spizizenii (ERBS130) using 16s rRNA gene with bacterial universal primer(27F/1492R) and Bacillus specific primers (BCF1/BCR2). All the 12 isolates were grouped together in Neighbour-joining phylogenetic tree. Majority of the isolates were positive for iturin, surfactin, and bacillomycin genes. The 12 isolates were further screened for hydrolytic enzyme production, antimicrobial and plant growth promotion (PGP) activities and found to have varying results. Isolate ERBS51 was found to be positive for majority of the attributes such as siderophore, ammonia production, phosphate, ZnCO3, ZnCl solubilisation, alpha amylase, cellulase, protease, pectinase and lipase production. ERBS51 (95% and 1472.50) followed by ERBS10 (88% and 1287.54) treated tomato seeds had the maximum germination and vigour index respectively. Pot and field experiment were conducted with F. mosseae, G. fasciculatum, ERBS51 (Bacillus velezensis) and ERBS10 (Bacillus sp.) both individual and combinations as treatments. The combined application gave better result than individual microbes as treatment. Among all the treatments given as single inoculum and all the possible combinations of the four microbes,F. mosseae+G. fasciculatum+B. velezensis+Bacillus sp. treatment as consortium with recorded highest disease reduction in wilt severity in both pot (77.44%) and field (66.74%) experiments respectively. F. mosseae+G. fasciculatum+B. velezensis+Bacillus sp. treatment also recorded high plant and yield attributes in both the conditions. F. mosseae, G. fasciculatum, B. velezensis and Bacillus sp. as a consortium could be used as a part of integrated management of Fusarium wilt of tomato in Meghalaya.ThesisItem Embargo Evaluation of biocontrol potential of Beauveria bassiana (Balsamo) Vuillemin against major rice pathogens(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2021-12) Deb, Lipa; Devi, R. K. TombisanaBeauveria bassiana (Ascomycota: Hypocreales: Cordycipitaceae), a widely studied entomopathogenic fungus recently drawn attention worldwide as potential biocontrol agent not only against insect-pests but also against several plant pathogens. As potential alternative to injudicious chemicals uses in crop protection, B. bassiana plays multifarious roles as plant disease antagonist, endophytes, plant growth promoter as well as beneficial rhizosphere colonizers providing an excellent opportunity as biocontrol agent in plant disease management. The present study was conducted with an aim to evaluate biocontrol potential of native B. bassiana isolates against major rice pathogens by studying its antimicrobial, growth promoting and colonizing ability in rice. A total of fifty-three (53) native isolates of B. bassiana were obtained from six major districts of Meghalaya, identified based on morphological and microscopic characteristics further confirmed by PCR-based detection by using universal ITS (ITS1, ITS4) primers at 550 bp amplicon size. Under in vitro assay, out of 53 tested isolates, only 15 isolates of B. bassiana were screened based on their efficient antagonistic potential against three (3) fungal viz., Rhizoctonia solani, Pyricularia oryzae, Bipolaris oryzae and one (1) bacterial pathogen viz., Xanthomonas oryzae pv. oryzae of rice with per cent inhibition recorded maximum up to 71.15%, 63.33%, 62.59% and 84.00% respectively. Under antimicrobial studies, screened B. bassiana isolates (15) showed ability to release volatile inhibitory metabolites delimiting the growth of R. solani (86.48%), P. oryzae (67.78%), B. oryzae (90.18%) and X. oryzae pv. oryzae, respectively. Majority of the isolates showed ability to produce cell wall degrading enzymes (CWDEs) viz., caesinases (68.41%), cellulase (77.26%), lipase (61.66%), xylanase (81.54%), α-amylase (77.78%), protease (72.22%) and pectinase (76.46%) in addition to iron-chelating siderophore compounds (74.74%). Presence of virulent genes viz., Bbchit1, Cdep1, Bbhog1 and Bbjen1 were detected in majority of B. bassiana isolates by performing PCR amplification using gene-specific primers at 198 bp, 249 bp, 193 bp and 198 bp, respectively. However, under plant growth promotion (PGP) studies, majority of B. bassiana isolates showed ability to produce ammonia (NH3) and inole-3-acetic acid (IAA) of concentration maximum up to 3.49 μg/ml and 6.16 μg/ml respectively. In addition to that, B. bassiana isolates also qualified as potential phosphorus (P), potassium (K) and zinc (Zn) solubilizers with per cent solubilization efficiency recorded maximum up to 68.68%, 81.74% and 64.36%, respectively. Based on potential antimicrobial and PGP traits, only five (5) isolates viz., Bb4, Bb16, bb25, Bb44 and Bb53 were selected for further studies. Potential B. bassiana isolates showed ability to tolerate aluminium (Al) and iron (Fe) stress conditions with maximum tolerance limit up to 100 ppm and 275 ppm, respectively under different acid (pH) condition to the lowest up to pH 3.0, respectively. Further, potential B. bassiana isolates viz., Bb4, Bb16, Bb25, Bb44 and Bb53 were found compatible among each other and developed into liquid-based microbial consortium (Bb-MC). Biopriming of rice seeds with Bb-MC showed considerable increase in seedling growth parameters as compared to seed treatment with single isolates and control at 10th DAS and 25th DAS. Per cent colonization (%) of B. bassiana was recorded maximum in stems (96.00%) followed by leaves (92.00%) and roots (28.00%) on 14th dpi through combined application of seed, root and foliar spray techniques. However, association of B. bassiana isolates viz., Bb4, Bb16 and Bb44 as potential colonizer in rice was further confirmed by light microscopy studies and PCR-based detection assay. In addition to that, application of Bb-MC also performed better under pot and field conditions against sheath blight disease R. solani of rice by combined treatment of seed, seedling and four (4) foliar sprays of 10 ml Bb-MC in 1 litre water at 15 days interval after transplanting. Reduced disease incidence and severity of 22.87% and 37.07% was recorded against maximum yield of 2.83 tonne/ha as compared to inoculated control with 74.23% and 94.27%, respectively with yield of 1.36 tonne/ ha. With these findings, the multifaceted role played by B. bassiana as biocontrol agent as investigated in the present study paved substantial evidence that B. bassiana in addition to its entomopathogenic behaviour also act as potential plant disease antagonist, thus, providing a new dimension in crop protection by introducing newer concept in Integrated plant disease management programmes along with overall health of plant.ThesisItem Embargo Investigation on grey leaf blight disease of mango (Mangifera indica L.) in North-East India(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2021-12) Das, Tanmoy; Devi, R. K. TombisanaMango (Mangifera indica L.) belongs to family Anacardiaceae and is the most important commercially grown fruit crop of India. India is a prominent exporter of fresh mangoes to the world. The country has exported 49,658.68 MT of fresh mangoes to the world during the year 2019-20. Among the important varieties and hybrids Amarapali, Himsagar and Mallika are grown in NE India. Among various fungal diseases, grey leaf blight is an important disease and commonly observed in NE India. Increasing intensity year by year may become major hurdle. Looking to the seriousness of the disease and economic importance of the crop, a study was conducted to understand the incidence of grey leaf blight of Mango, assess the epidemiology of Pestalotiopsis mangiferae and develop an effective disease management practice against grey leaf blight of mango in NE India. A survey was carried out in five mango growing states viz., Assam, Arunachal Pradesh, Manipur, Meghalaya and Tripura. During survey Tripura recorded the highest (64.21%) grey leaf blight incidence followed by Assam (63.15%) and the lowest incidence was recorded from Meghalaya (51.64%). During survey it was recorded that among hybrid varieties Amrapali is the major growing variety in Tripura. Effect of five different temperatures on growth and sporulation of P. mangiferae was studied and significantly higher mycelial growth (85.15 mm) and excellent sporulation (3.4x1041.94%) was found effective against P.mangiferae followed by Garlic (36.18%). Pseudomonas fluorescens (87.16%) showed best antagonistic potential against P. mangiferae followed by Trichoderma harzianum (86.48%). Among the fungicides tested in vitro carbendazim 12%+mancozeb 63% (100%) and cymoxanil 8%+mancozeb 64% (100%) proved to be the best. The results of two years field experiment in Tripura (2018-2019) showed that both carbendazim 12%+mancozeb 63% (82.16%) cymoxanil 8%+mancozeb 64% (74.43%) were found highly effective against grey leaf blight of mango. 6 ml-1) was observed at 27 ±1°C followed by 30°C (84.26 mm and 3.1x106 ml-1) were observed. The field study in Tripura (2018 and 2019) showed first grey leaf blight infection in March and infection rise from June. The correlation matrix shows the incidence of grey blight is negatively correlated with max. and min. temperatures but positively correlated with max. and min. relative humidity and rainfall. Four phytoextracts were screened in vitro by poisoned food technique. 41.94%) was found effective against P.mangiferae followed by Garlic (36.18%). Pseudomonas fluorescens (87.16%) showed best antagonistic potential against P. mangiferae followed by Trichoderma harzianum (86.48%). Among the fungicides tested in vitro carbendazim 12%+mancozeb 63% (100%) and cymoxanil 8%+mancozeb 64% (100%) proved to be the best. The results of two years field experiment in Tripura (2018-2019) showed that both carbendazim 12%+mancozeb 63% (82.16%) cymoxanil 8%+mancozeb 64% (74.43%) were found highly effective against grey leaf blight of mango.ThesisItem Open Access Bio-efficacy of Trichoderma formulation against damping-off caused by Pythium spp. and Rhizoctonia solani Kuhn. on tomato (Solanum lycopersicum)(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019-11) Biam, Markidahun; Majumder, DipaliDamping-off is one of the most prevalent and damaging disease of tomato nurseries in Meghalaya, caused by Pythium spp. and Rhizoctonia solani Kuhn. These pathogens can kill both germinating seeds and young seedlings. Management of damping off by integration of biocontrol agents, specially the Trichoderma spp., not only can reduce doses of fungicides but will be a better management strategy against the damping off of pathogens. Considering the fact, present investigation was formulated and aimed on to evaluate bio-efficacy of Trichoderma formulation against Pythium spp. and Rhizoctonia solani kuhn. cause damping off in tomato. Out of 180 soil samples collected from different habitats (crop rhizosphere, compost manure, forest, jhum areas, pig manure, coal mine and lime stone) of 11 districts of Meghalaya, 97 Trichoderma isolates were identified based on their morphology. Dual culture assays of the twenty screened isolates against damping-off pathogens revealed that the 4 Trichoderma isolates viz. TR55, TR66, TR122 and TR136 were found highly effective in inhibiting Pythium spp. with percent inhibition of 89.26, 88.15, 88.89 and 87.78 respectively, whereas only 2 isolates viz. TR55 and TR122 were effective against R. solani with percent inhibition of 87.41 and 86.48, respectively. Isolate TR55 recorded as the best antagonist against both pathogens causing damping off. All 20 screened isolates were positive for ACC deaminase production whereas 17 were positive for chitinase production. Isolate TR 106 and TR 136 were the best in chitinase production with purple colour zone of 9 cm in diameter. Out of 20 screened isolates tested for other functional attributes (determining antagonistic potentials), 16 isolates were found positive for siderophore and ammonia production, whereas 13 isolates were positive for HCN production. Screening for plant growth promotion traits of 20 isolates revealed that all the isolates produced IAA and Phosphorous with values ranging from 0.33 to 4.96 μg/ml and 0.03 to 0.98 μg/ml, respectively. Presence of antibiotic biosynthetic genes in the 20 isolates by using gene specific primers detected that 12 isolates were positive for β-1,6-Glucanase (Tvbgn3), 10 isolates for Trichodiene synthase (tri5), 14 isolates for Serine protease (prb1) and 17 isolates for Endochitinase (ech42). Only 4 Trichoderma isolates showed presence of all the 4 antibiotic biosynthetic genes tested. Observations of the anamorphic characteristics of the Trichoderma isolates showed variation in anamorphic characters viz., size of phialides, phialospore and conidiophores among the isolates. The sequence analysis of the internal transcribed spacer regions 1 and 2 (using universal primer ITS1 and 4) of the rDNA of the 10 potential isolates showed that they belonged to 2 Trichoderma species viz., T. hamatum (TR 55, TR 66, TR 87 and TR 122) and T. harzianum (TR 64, TR 88, TR 106, TR 109 and TR 136) with amplicon at 600bp in all isolates. Dendogram was generated using alignments containing the ITS region revealed formation of two main clusters with closeness ranging from 13 to 96 per cent. The biopriming of tomato seeds with 4 Trichoderma isolates (TR55, TR66, TR122 and TR136) showed considerable increase in germination percentage and vigour index over control, with the highest germination percentage and vigour index recorded in TR55 (75.13 per cent and 47.99 per cent, respectively). Among the treatments (seed, soil and seed plus soil) highest reduction in incidences of pre-emergence and post emergence damping-off was obtained with the isolate, TR55 (seed plus soil treatment). It also exhibited increase in the growth parameters such as root length, plant height, number of leaves, number of flowers, fresh as well as dry fruit weight and yields of tomato; variety Hybri-017 in the treated crops as compared to the untreated control. The rhizosphere colonization of 4 potent isolates of Trichoderma spp. viz., TR55, TR66, TR122 and TR136 showed that their rate of colonization increases up to 45 days after sowing and reduced at 60 days after sowing in all the treatments. Colonization was highest in TR 55 (3.73 × 106) followed by TR 122 (3.63× 106), TR 66 (3.50× 106) and TR 136 (3.37× 106). Native Trichoderma isolates TR55, TR66 and TR122 performed better in terms of functional attributes viz., disease suppression, growth promotion, rhizosphere colonization, self-life in talc formulation, could be further evaluated under different climatic condition of the state for development of effective Trichoderma formulations found effective can be recommended as a component of integrated disease management practice to manage damping off of tomato in the nursery beds in Meghalaya.ThesisItem Open Access Management of Penicillium rot of Khasi Mandarin (Citrus reticulata Blanco) by using native Bacillus subtilis isolates(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019-11) Tariang, Janshame; Majumder, DipaliMandarin orange is an important horticulture crop in India next to banana. Amongst a variety of mandarin, Khasi mandarin is widely cultivated in the North-Eastern (NE) states of India, particularly in the border areas of Meghalaya which is very popular due to its unique quality, fruit colour, sugar-acid blend and shelf life. Although Meghalaya is one of the largest producers of orange in the country, due to the problem of post-harvest diseases, there have been considerable losses to the harvested fruits and the average yield of Khasi mandarin is alarmingly low. Several losses of the fruit have been reported due to post-harvest disease Penicillium rot caused by Penicillium. spp. The present investigation was formulated with an aim to minimise the loss of the Khasi mandarin fruit due to the post-harvest disease Penicillium rot by using native B. subtilis isolates. Samples were obtained from 15 different natural habitats (crop rhizosphere, hot springs, pig manure, river bank deposits, citrus fruit surfaces, mixed manure, coal mines, limestone mines, forest, leave mould, vermi-compost, fishery ponds, oyster mushroom compost, jhum area and from bio-extract) of the 8 districts of Meghalaya (West Jaintia Hills, East Khasi Hills, Ri- Bhoi, West Garo Hills, North Garo Hills, West Khasi Hills, East Jaintia Hills and South West Garo Hills). A total of 260 isolates was collected, maximum of which was obtained from West Jaintia Hills district (94 isolates) and a majority was isolated from the rhizosphere of crops (181 isolates). Based on the morphological characters of the colonies and biochemical test viz. Gram staining test, catalase and oxidase test, 95 isolates were initially designated as B. subtilis. Further molecular identification of these isolates was done using 16S rRNA intervening sequence Bacillus genus specific primer BCF1/BCR2 which identified 69 isolates as Bacillus spp. To further confirm the isolates as B. subtilis, species specific primer EN1F/RN1R was used which further confirmed 12 isolates as B. subtilis (Bs 80, Bs 167, Bs 174, Bs 190, Bs 193, Bs 197, Bs 216, Bs 217, Bs 219, Bs 256, Bs 257 and COB5Y1). These 12 B. subtilis isolates were studied for their antimicrobial traits and all the 12 isolates were found to be negative for HCN production, negative to the presence of antibiotic biosynthetic genes coding for iturin D and fengycin B, whereas 6 isolates were positive for catalase production test and 10 isolates were positive for protease production test. Six B. subtilis isolates viz. Bs 167, Bs 197, Bs 217, Bs 219, Bs 256 and COB5Y1 were considered potential based on the antimicrobial test. Bio-assay of these 12 B. subtilis isolates against Penicillium rot pathogen of Khasi mandarin fruit revealed that maximum disease inhibition per cent against Penicillium spp. was recorded for the isolate COB5Y1 (88.11%), followed by isolate Bs 167 (80.82%), Bs 197 (54.82%) and Bs 217 (40.19%). Based on in-vitro evaluation, isolates COB5Y1, Bs 167, Bs 197 and Bs 217 were considered potential B. subtilis isolates amongst 12 B. subtilis isolates tested. Four isolates (COB5Y1, Bs 167, Bs 197 and Bs 217) were selected for field evaluation based on their performance against antimicrobial test and dual culture test. Amongst these four B. subtilis isolates, two isolates Bs 167 and COB5Y1 showed maximum disease inhibition per cent with 78.75% (21.25% disease incidence) and 65% (35% disease incidence) respectively even after storage of the fruit for upto 30days when the bio-agent was applied as liquid formulation at post-harvest stage of the fruit. There was no significant difference with the point of inoculation of the pathogen for disease development. B. subtilis isolates (COB5Y1, Bs 167, Bs197 and Bs 217) were also found to have no adverse effect on the post-harvest fruit quality such as mass loss, TSS and acidity of the fruit. The viability test revealed that B. subtilisisolates were found most viable when stored at 4°C upto 180 days. Two isolates Bs 167 and COB5Y1 were regarded as effective antagonists that could serve as potential bio-agent against Penicillium rot of Khasi mandarin in Meghalaya which need further evaluation under field condition Meghalaya.
