Thesis Abstracts

Survey of postharvest fruit rot of banana was conducted in Gorkha bazar and different locations of Kathmandu. Isolation and identification of fungi associated with the fruit rot was done.  A number of fungi such as Alternaria, Fusarium, Diplodia, Stemphylium, and Colletotrichum spp. were isolated from banana samples. Of them, Colletotrichum spp. was repeatedly isolated and tested for pathogenicity, which was proved to be pathogenic on banana fruits causing post- harvest fruit rot of banana. Thus, the Colletotrichum spp. was selected for management experiments. Cinnamon oil, neem oil, mustard oil, coconut oil and castor oil were tested for their efficacy against the pathogen both under in-vivo and in-vitro conditions. The in-vivo test comprised two experiments one with and the other without inoculation with Colletotrichum spp. Incidence of disease was reduced on oil-treated banana fruits and their storage life was increased significantly. Percent disease intensity (PDI) of untreated (control) bananas reached up to 100 in both inoculated and non-inoculated bananas while that of cinnamon-treated reached 34.44, 42.22, 55.56 at 4 th , 6 th , and 8 th days of storage followed by castor oil which reached 41.11, 43.33 and 60 at 4 th , 6 th , 8 th days of storage in case of bananas inoculated with Colletotrichum spp. In the case of non-inoculated banana, PDI of neem- and coconut-treated bananas reached 44.4, 50, and 50 at 10 th , 12 th and 14 th followed by mustard- and castor-treated bananas which reached to 50, 50, and 50 at 10 th , 12 th and 14 th days. Results showed that fruits treated with oils were better maintained and had lower disease severity scores, whereas non-treated fruits suffered increased deterioration. Physiological weight losses were recorded in all treatments increased with number of days in storage. The treatment had no significant effect on weight loss of the banana fruit throughout the storage period. The mycelium growth of Colletotrichum spp. was observed significantly highest on potato dextrose agar (53.57 mm) followed by oat flakes agar (52.02 mm), V8 juice (50.04 mm), and lowest in ripe banana agar (31.75 mm). In the in-vitro evaluation of different oils, the highest percent radial mycelial growth inhibition (98.15% at 1000 ppm) was shown by cinnamon oil which was followed by mustard (40.00% at 1000 ppm) and neem oil (38.70% at 1000 ppm).

Field and laboratory experiments for screening of strawberry varieties against leaf spot (Pestalotia longisetula) and its management were conducted. The laboratory experiments were conducted at Nepal Plant Disease and Agro Associates, Balaju, Kathmandu. The field experiments were conducted at Berry World Farm, Dhapakhel and Godawari, Lalitpur during 2019-2020. For management experiment at Dhapakhel, three botanical extracts (ginger, garlic, and turmeric), cow urine, compost tea and their combination treatments along with a fungicide (Saaf, mancozeb 63% + carbendazim 12%) and untreated control (water) were used. The experiment was laid out in a randomized complete block design with three replications. A total of four sprays were applied at 15 days intervals starting from the first week of transplantation. All the extracts at three different concentrations (10, 25 and 50%) and Saaf at 10, 25, 50, 100, 200, 500 ppm were evaluated in laboratory experiments for their efficacy against P. longisetula by food poison technique. Also, the treatments were evaluated on detached leaves under laboratory conditions by inoculation with the pathogen. Field evaluation of strawberry cultivars against the Pestalotia disease was conducted at Godawari. The cultivars included Sweet Sensation, Ruby, Winter Dawn, Florida Beauty, EMCO 32 and EMCO 33. In the field experiment, the lowest disease intensity (48.89%) was recorded in the garlic-treated plots and the highest disease intensity (84.81%) was observed in untreated plots (control). The highest (42.36%) disease control was recorded with garlic-treated plots, followed by cow urine (35.36%), ginger (32.34%) and turmeric (31.88%) -treated plots. In the laboratory experiment, garlic and cow urine alone and combination of cow urine, garlic, ginger and turmeric at 25 and 50% concentration gave 100% control of P. longisetula followed by turmeric. Saaf at 50 ppm or more showed 100% inhibition of growth of this fungus and 51.85 and 77.04% inhibition were obtained with 10 and 25 ppm, respectively. In detached leaf experiment in laboratory, the lowest disease intensity was observed in leaf sprayed with garlic (40.70%) and ginger extract (40.70%) when leaf sprayed with just water (control) reached 100% infection at 12^th day.

Late blight, caused by Phytophthora infestans, is the most devastating disease of potato, which can cause 100% crop loss under favorable conditions. With an objective of evaluating efficacy of native Trichoderma isolates in managing late blight of potato this study was carried out. Laboratory experiments for isolation and screening of Trichoderma, mass multiplication of Trichoderma isolates, growth of P. infestans in different culture media and detached leaf assay were carried out at Nepal Plant Disease and Agro Associates, Balaju, Kathmandu. Field experiment for the management of late blight was conducted at Thansing, Nuwakot during 2020-2021. Eighty-three soil samples from multiple ecosystems of Likhu Rural Municipality, Nuwakot were collected and isolation of Trichoderma was done in Trichoderma selective medium (TSM). Overall 79 Trichoderma isolates were obtained and among them, 39 were selected based on mycelia growth and sporulation at 25 °C on PDA for detached leaf assay against P. infestans in vitro. These isolates were further screened on PDA at 15 °C, 25 °C and 35°C to optimize temperature for better mycelial growth and sporulation and finally10 isolates were selected. These finally selected isolates were tested for mass multiplication in three substrates (finger millet grains, maize grains and rice husk+rice bran). Along with a commercial T. viride (Sanjeevani) these isolates, were compared in a field experiment against P. infestans under natural epiphytotic condition on potato, variety MS 42-3. The experiment was laid out in RCBD with three replications and spray of distilled water was used as control treatment. A total of five sprays (10⁹ spores per ml for the test isolates, and 10 g per lit for Sanjeevani) were applied at 15 days intervals starting from the second week after plant emergence. Five different culture media – pea agar, V8 agar, rye B agar, carrot agar and PDA were evaluated for the good growth of P. infestans. Among the total isolates, 39 isolates sporulated well (full plate covered) within 7 days at 25 °C. Of the 39 isolates, 25 isolates sporulated well within 14 days at 15°C. However, at 35°C, all 39 isolates produced abnormal compact colonies with irregular margin and scanty sporulation. In mass culture of ten selected isolates of Trichoderma, isolate 36/2B only colonized and sporulated (100%) in all three substrates used while 24/2B isolate failed to colonize and sporulated (complete) in any of the substrates. For culture of P. infestans, pea agar yielded highest colony growth (7.67 cm), followed by potato dextrose agar (4.7 cm), carrot agar (3.59 cm) and V8 agar (2.52 cm) at 18±2 ˚C after 20 DAI. The fungus did not grow on rye agar. In detached leaf assay, the effect of Trichoderma isolates on suppression of lesion size caused by P. infestans was significant. Five of the 39 isolates significantly reduced the lesion size by 40-46%, followed by seven isolates by 30-39%. In the field study, the effects of Trichoderma isolates were found significant for plant height, ground coverage, disease severity and tuber yield. Isolates 2/2A increase plant height, ground coverage and tuber yield by 41.22%, 30.31% and 72.33% respectively followed by isolates TL-81 (28.26%, 19.04% and 50.81% resp.) over the control. The highest reduction in disease severity was found with the isolates 2/2A (37.33%) and TL-81 (37.21%) which was statistically par with ‘21/2B’ (30.37%). Among tested isolates 2/2A and TL-81 performed better in the field and laboratory experiments and are found potential for management of P. infestans of potato.

Pestalotia leaf spot, caused by Pestalotia longisetula, is one of the economically important common diseases of strawberry, characterized by necrosis of leaves from the edge. Both field and laboratory experiments were conducted to screen six strawberry cultivars against P. longisetula and to manage the disease through application of biocontrol agents during 2019/20. Diseased leaf samples from strawberry farms of Berry World Pvt. Ltd. at Godawari and Hattiban were collected and processed for isolation and identification of associated pathogens in the laboratory of Nepal Plant Disease and Agro Associates (NPDA) at Balaju, Kathmandu. The screening experiment was carried out in a randomized complete block design (RCBD) with three replications and six treatments (cultivars: Sweet Sensation, EMCO 32, Florida Beauty, EMCO 33, Winter Dawn and Ruby Gems) at Godawari, Lalitpur under natural epiphytotic conditions. Also, the cultivars were evaluated against the disease by detached leaf assay at the NPDA laboratory. The management experiment was conducted at Dhapakhel, Lalitpur in RCBD with three replications and ten treatments under natural epiphytotic conditions. The treatments for management experiments included Bacillus subtilis (NPDA), Pseudomonas fluorescens (NPDA), Lactobacillus sp. (NPDA), Trichoderma sp. (NPDA), Trichoderma BKK (Trichoderma harzianum + T. asperellum) (Thailand/NPDA), P. fluorescens (Bactvipe), T. viride (Sanjeevni), Jaibik IP Agro Liquid (Lactobacillus + Azotobacter + Bacillus spp. + Rhizobium + Bifidobacterium), Mantox Forte (mancozeb + copper oxychloride and copper sulfate) and untreated control. Disease assessment was done by calculating percent disease incidence, intensity and AUDPC. In-vitro efficacy of biocontrol agents was studied in the NPDA laboratory using dual culture techniques and detached leaf assay. The chemical fungicide was evaluated by food poison technique. Among the fungi isolated and identified from both locations (average of Godawari and Hattiban), Pestalotia longisetula was the most frequently occurring (53.33%), followed by Fusarium solani (46.67%), Rhizoctonia solani (43.33%), Botrytis cinerea (36.67%) and Gloeosporium fragariae (30%). Significant variation was found in the percent disease incidence, intensity and AUDPC values for both varietal screening and management plots. Incidence and intensity of disease was the highest with cultivar Sweet Sensation followed by Ruby Gems and Florida Beauty. Cultivars; Sweet Sensation, Ruby Gems and Florida were found highly susceptible and cultivars; Winter Dawn, EMCO 33 and EMCO 32 as susceptible. Mean AUDPC values varied from 512.78 to 837.87. Detached leaf assay also gave similar results. Lower disease incidence, intensity and AUDPC were observed in the plots treated with Jaibik IP Agro Liquid, Trichoderma BKK and Trichoderma sp. Effect of biocontrol agents in growth and yield of strawberry plant was significant. Treatment with Jaibik IP Agro Liquid gave the highest yield (1195.67 g) per plot. Among the bacterial biocontrol agents, Bacillus subtilis showed the highest inhibition (51.11%) of P. longisetula in dual culture. Trichoderma sp. (NPDA) gave the highest inhibition percent (72.71%) among the Trichoderma. The present results showed that all six cultivars are susceptible to Pestalotia leaf spot disease. Jaibik IP Agro Liquid, Trichoderma sp. and Trichoderma BKK could be the effective and promising alternatives for the management of the disease. The efficacy of these biocontrol agents against Pestalotia leaf spot should be further studied under different environmental conditions.

Rice (Oryza sativa L.) is the most important staple food crop of Nepal and accounts for about 50% of the total agricultural area and production. The crop is affected by a number of pathogens, including Fusarium proliferatum (Matsushima) Nirenberg. With an objective of evaluating efficacy of native Trichoderma against some important fungal pathogens of rice, laboratory and polyhouse experiments were conducted at Nepal Plant Disease and Agro Associates (NPDA) during the year 2020-2021. Eighty-three soil samples were collected from different ecosystems of Likhu Rural Municipality, Nuwakot. Isolation of Trichoderma from the soils was done on Trichoderma selective medium. Overall, 79 Trichoderma isolates were obtained from 43 of 83 soil samples. All 79 isolates were evaluated for their mycelial growth rate and sporulation capacity on potato dextrose agar (PDA) at 25 °C and 40 of them were selected for further studies. The selected 40 isolates were dual cultured with the rice pathogens (F. proliferatum, Pyricularia oryzae and Rhizoctonia solani) on PDA for antagonism and evaluated by seed biopriming for their effect on seedling vigour. Finally, ten isolates were selected for seed health on cv. Taichung-176, and pot experiments against F. proliferatum under polyhouse conditions using rice cv. Khumal-4. Two pot experiments (seed and soil treatments) were conducted in a completely randomized design with 12 treatments and six replications. The treatments included 10 selected Trichoderma isolates, F. proliferatum-inoculated control and untreated control. The selected 40 isolates of Trichoderma grew and sporulated well covering the entire Petri plate (9 cm) within 5 days at 25 °C. In dual culture, isolate 36/2B was most effective (100% overgrowth) against all three rice pathogens. Isolates 48/2A, 50/2A, 56/2B, 73/2A and TL-81 exhibited 100% overgrowth against F. proliferatum while isolate 2/2A showed 100% overgrowth on R. solani. Similarly, isolates 2/2A, 48/2A, 50/2A, 56/2B, 73/2A and TL-81 overgrew P. oryzae completely. In seed biopriming, all the 40 isolates increased significantly radicle length, plumule length, germination percentage and thereby seedling vigour. Among them, isolate 2/2A found best in increasing radicle length (10.30 cm), plumule length (8.23 cm), germination percentage (99%) and seedling vigour (1835) compared with the untreated control (5.73 cm, 4.03 cm, 70.33% and 687, respectively). In seed health testing, all ten isolates of Trichoderma reduced infection of Alternaria padwickii, Bipolaris oryzae, Fusarium spp., Phoma sp. and P. oryzae significantly. Overall, isolates TL-81 and 2/2A reduced the incidence of total seed borne infection by 7.5% and 9.5%, respectively. In pot experiments with F. proliferatum-inoculated seed, all the isolates significantly increased root length, germination percentage, seedling vigor and decreased disease incidence and severity in both seed and soil treatments. However, the effect on shoot length was not significant. In seed inoculation, isolate 2/2A increased root length and germination percentage by 103.66% and 44.95%, and TL-81 increased by 84% and 36%, respectively. Isolate 2/2A reduced the disease incidence and disease severity by 47.71% and 74.55%, and isolate TL-81 by 48.12% and 68.4%, respectively. Also, in soil inoculation, both the isolates 2/2A and TL-81 had similar effect on root length, germination percentage, disease incidence and disease severity Overall, the isolates 2/2A and TL-81 performed better among the isolates in both seed and soil inoculation experiments for the management of F. proliferatum in rice.

Rice is the number one staple food crop in Nepal and contributes significantly to livelihood of majority of people and to the national economy. Fusarium proliferatum is one of the important fungal pathogens, which causes foot rot/bakanae in rice. Major rice growing areas in the world have been affected by this pathogen causing a significant decrease in yield and grain quality. At early stage, the fungus also causes severe seedling blight. With the aim to manage Fusarium seedling blight of rice through host resistance and bacterial antagonists, laboratory and screenhouse experiments were carried out at Nepal Plant Disease and Agro Associates (NPDA) during the year 2021-2022. Fusarium proliferatum was isolated from infected rice seeds (cv. Khumal-4) and grown in PDA culture. Twenty-one rice genotypes, mainly fine and aromatic, were screened against the fungus under inoculated conditions in a screenhouse. For biological control of the pathogen, isolation of antagonistic bacteria from rice seeds was done using King’s B medium. The bacteria isolated from rice seeds along with some bacterial isolates from the stock of NPDA were subsequently tested for their antagonistic activity in-vitro against F. proliferatum using dual culture technique. Bacteria showing antagonistic activity were selected for final in-vitro assay in a completely randomized design (CRD) with four replications. Promising antagonists were selected and they were evaluated in-vivo in a two factorial CRD with six replications under screenhouse conditions. The factors were time of use of antagonists (before, after and no inoculation with pathogen) and antagonists (Pseudomonas-36, Bacillus JP, Rice bacterium T-176, Rice bacterium K-10, Rice bacterium M-4) along with carbendazim as chemical control. Of the genotypes evaluated, ‘Khumal-11’ and ‘Chainung-242’ were found highly resistant; ‘NGRC 09032’ as resistant; ‘NGRC 08263’, ‘NGRC 08304’ and ‘NGRC 05869’ as moderately resistant; ‘NGRC 05715’ as moderately susceptible; and the rest were susceptible to highly susceptible. In the in-vitro experiment, all five bacterial antagonists significantly (P<0.001) inhibited mycelial growth of F. proliferatum compared with untreated control (without antagonist) and they were not significantly different with each other. In the in-vivo experiment, there was significant difference (P<0.001) between the methods of use of antagonists, among antagonists, and also the interaction between these two factors was significant. Use of antagonists or the chemical carbendazim before inoculation with the pathogen was found to be more effective compared to after inoculation with the pathogen. Among antagonists, Pseudomonas-36 and Bacillus JP gave better performance though the chemical carbendazim provided better control of the pathogen. The results showed that there is a potential of biocontrol agents for the management of seedling blight caused by F. proliferatum. Further testing is required to find effective bacterial antagonists.

Bacterial wilt caused by Ralstonia solanacearum has become a severe problem mainly on tomato, potato, and brinjal in Nepal and no effective control measure is available yet. To explore possibilities for the development of biological control of bacterial wilt of tomato, laboratory and pot experiments were conducted at the laboratory of Nepal Plant Disease and Agro Associates (NPDA) and at Ratnanagar-10, Chitwan, respectively, during the year 2021-2022. Ten rhizospheric soil samples were collected from different places of Chitwan and Kathmandu and 162 rhizospheric bacteria were isolated. All the isolated rhizobacteria were screened for antagonistic potential against R. solanacearum by disc-diffusion methods under in-vitro experiment. On the basis of in-vitro screening, seven isolates viz. SPP2 (19.17 mm) , SPY8 (14.27 mm), SPP120 (11.67 mm), SPZ72 (12.33 mm), SPX1 (11.67 mm), SPB8 (10.17), and SPB4 (11.67) with greater zone of inhibition were selected for in planta test on susceptible variety Pusa Ruby. On in planta test, the potting media was treated with the antagonistic and their effects on R. solanacearum were studied. The study showed that all the isolates significantly reduced disease incidence and severity as compared to untreated control, but the isolates SPP2, SPY8, SPP120, and SPZ72 greatly reduced disease severity and wilting incidence as compared to the other isolates. Incidence area under disease progress curve (AUDPC) values were reduced by 62.92, 59.55, 50.06% for isolates SPP2, SPP120, SPY8, respectively, while severity AUDPC values were reduced by 74.90, 65.88, and 53.14% for the respective isolates. In laboratory studies, a maximum germination percent of 87.33%, and a vigor index of 1530.82 was noted in seed bacterization with isolate SPZ72, followed by 85.33% germination and vigor index of 1354.72 in treatment with isolate SPY8 over control (seed germination percent 65.67% and seedling vigor index 809.05). Among the tested eight antagonistic bacterial isolates, all showed a positive response to ammonia production, auxin production, acetoin production, catalase reaction, KOH production. However, only isolates SPZ72, SPY8 and SPX1 were able to produce hydrogen cyanide production. Based on the in planta results and the biochemical test, the present study revealed that isolates SPP2, SPY8, SPZ72 and SPP120 are the most promising ones. However, their effectiveness should be evaluated under field conditions and the effective ones can be used for ecofriendly bioformulations as an alternative to synthetic fungicides.

Molds and contaminants have been identified as one of the major problems associated with mushroom cultivation, leading up to 80% of economic losses. In vitro and in vivo experiments were carried out at the Nepal Plant Disease and Agro Associates (NPDA) laboratory, Balaju-Chakrapath to evaluate the efficacy of aqueous extract of botanicals against molds and contaminants prevalent in the mushroom cultivation atmosphere. Visible fungal colonies with different morphologies and colors were isolated in potato dextrose agar (PDA) plates from the indoor air of three mushroom farms and two spawn storage rooms. After transferring different colonies onto new sterile PDA plates, a total of seventeen pure cultures of fungi were obtained, among which multiple isolates of Trichoderma, Aspergillus, Cladosporium, and Penicillium were identified; however, some fungi remained unidentified. The isolated fungi and edible mushroom mycelia were assayed in a dual culture experiment to determine the mushroom mycelia inhibition by fungi. The experiment was laid out in a completely randomized design with eighteen treatments and five replications. Slow-growing fungi such as Penicillium and Cladosporium did not have a significant effect on mushroom mycelia growth. However, two isolates of Trichoderma (T1 and T12), an isolate of Aspergillus (T7), and an unidentified fungus (T11) showed higher inhibition of mushroom mycelia; and were selected further for poison food assay. PDA containing aqueous extract of onion, garlic, ginger, turmeric, shallot, and mugwort at 2%, 4%, and 8% concentrations were used as poison media. This experiment also followed a completely randomized design with thirty-five treatments and four replications. Mycelial growth of fungi was recorded on the 5th and 7th days after inoculation. Among the botanical treatments, garlic and mugwort at 4% concentration reduced the growth of Trichoderma isolate 1 by 47.5% and 48.2% and Trichoderma isolate 2 by 61.9% and 45.8%, respectively. However, both botanicals at this concentration completely inhibited the mushroom mycelia growth. On the other hand, onion at 8% concentration completely inhibited Aspergillus growth. In in vivo, both Trichoderma isolates (1 and 2) and Aspergillus isolate could not induce disease when inoculated after the completion of the spawn run. Garlic at 10% and 15% concentrations and mugwort at 15% concentration showed a curative effect against green mold and yielded mushrooms. The botanicals (garlic and mugwort) at 20% concentration did not yield mushrooms though the green mold was cured.

A field survey was conducted on 20 nursery plots of kiwifruit in four different farms in Boch, Dolakha to assess the status of crown gall disease caused by Agrobacterium sp. Field research was conducted in a two-factorial randomized complete block design with three replications at the Temperate Fruit Rootstock Development Centre at Boch, Dolakha to find out the effective control measures for crown gall disease management in Kiwifruit nursery. The first factor included six treatments (Kasu B, Hydrogen peroxide, Cuprex, Streptocycline, Kasu B+Streptocycline and untreated control). The second factor included three varieties of Kiwifruit (Hayward, Monty and Allison). Laboratory work such as bacterial population count, identification (morphological, biochemical, microscopic and pathogenicity), and in-vitro assays were performed at the laboratory of Nepal Plant Disease and Agro Associates (NPDA), Balaju, Kathmandu. In the field experiment, observations were taken on plant height, root length, colony count before and after the treatments, number of galls, and size of galls. The interaction of varieties and treatments on the disease management was significant (p<0.05). The highest bacterial population reduction (100%) was found in the plots of Allison treated with Streptocycline+Kasu B and in the Streptocycline-treated plots of Hayward. The Streptocycline showed lowest gall numbers (2.67±0.69) in Allison followed by Kasu B (6.76±0.78). The Cuprex-treated Allison variety showed smallest gall size (0.23±0.40 cm) which was at par with Cuprex-treated Hayward (0.29±0.50 cm) and Monty (0.32±0.56 cm) varieties. In overall, Allison variety was found comparatively less susceptible to crown gall disease and Streptocycline was effective for the management of the disease in Kiwifruit.

Sweet orange [Citrus sinensis (L.) Osbeck] is an economically important crop in the mid-hills of Nepal. Postharvest diseases are among the major constraints hindering the shelf life of sweet orange. Research was conducted at the laboratory of Nepal Plant Disease and Agro Associates (NPDA), Kathmandu with the objective of developing effective management practices against postharvest diseases. Infected fruit samples were brought from five locations of Sindhuli and associated molds were identified as green mold (Penicillium digitatum Sacc.) and blue mold (Penicillium italicum Wehmer). Efficacy of five different botanicals – lantana (Lantana camara), eucalyptus (Eucalyptus spp.), garlic (Allium sativum), thyme (Thymus vulgaris) and mugwort (Artemesia vulgaris), all at 4% concentration, and chemicals – carbendazim (50 ppm) and potassium metabisulphite (200 ppm) were evaluated against green and blue molds using poisoned food technique under in-vitro conditions at 25 ± 2℃. The experiment was set up in a completely randomized design with nine treatments and five replications. Similarly, in-vivo experiments included botanicals – lantana, eucalyptus, garlic, bitter aloe (Aloe barbadensis), thyme, and mugwort at 10% concentration, and chemicals – carbendazim (1000 ppm), sodium bicarbonate (5%) and potassium metabisulphite (200 ppm). The experiments were conducted at approx. 12℃ and 60% RH in a room during January-February 2023. Based on the results, carbendazim was further evaluated at different concentrations (1000, 500, 250, and 125 ppm) against green mold under in-vivo conditions. The effect of treatments on the growth of green and blue molds under both in-vitro and in-vivo conditions was significant (p<0.001). Under in-vitro conditions, garlic and carbendazim inhibited the growth of green mold by 88.29% and 100%, respectively on the 13th day after inoculation. Similarly, both garlic and carbendazim inhibited the growth of blue mold by 100%. Under in-vivo conditions, the least growth of green mold was found in garlic-treated fruits (2.68 cm lesion diameter) among the botanicals and in carbendazim-treated fruits (0 cm, no lesion) among the chemicals on the 12th day after treatment. Similarly, the growth of blue mold was least in garlic (1.62 cm lesion diameter) and carbendazim (0 cm, no lesion). Carbendazim even at 125 ppm was found effective against green mold under in-vivo conditions. Considering the results from in-vitro and in-vivo experiments, garlic and carbendazim were the most effective botanical and chemical, respectively in suppressing both green and blue molds. Other botanical treatments (lantana, eucalyptus, bitter aloe, thyme, and mugwort) and chemicals (sodium bicarbonate and potassium metabisulphate) did not control the growth of the molds. From these findings, carbendazim at a low concentration (125 ppm) can be used for the control of the molds provided residue in treated fruits is within the permitted limit. Further research and development of garlic-based pesticides could be explored for the management of green and blue molds in sweet orange.

Root rot is an important disease of strawberry caused by fungi complex, which can cause up to 30% losses. Root rot samples from strawberry farm of Nepal Agrovine Pvt. Ltd., Sami Chowk, Chitwan were collected and processed for isolation and identification of fungi associated with the disease. Pathogenicity of the isolated fungi (Fusarium, Rhizoctonia, Pythium and Macrophomina) was assessed. In-vitro evaluation of fungicides against the fungi were performed by poison food technique. That include SAAF (carbendazim 12% + mancozeb 63%), Shield (cymoxanil 8% + mancozeb 64%) and All-care (dimethomorph 9% + mancozeb 60%) at 100, 200, and 400 ppm and Nativo (tebuconazole 50% + trifloxytrobin 25%) at 25, 50 and 100 ppm. The above experiments were carried out in a completely randomized design with five replications at the laboratory of Nepal Plant Disease and Agro Associates, Balaju, Kathmandu. Two field experiments were conducted at the farm of Nepal Agrovine Pvt. Ltd., Sami Chowk, Chitwan. In the first experiment, five strawberry varieties were evaluated against root rot under natural infection in a randomized complete block design (RCBD) with four replications. The test varieties were Florida Beauty, Ruby Gem, Sweet Sensation, Palmaritas and Winter Dawn. In the second experiment, four chemical fungicides tested in vitro were evaluated in RCBD with four replications for the management of root rot. Disease scoring was done at 1-9 scale and percent disease intensity was calculated. Ten fungi were isolated and identified from the root rot samples. They were Fusarium, Rhizoctonia, Pythium, Macrophomina, Verticillium, Acremonium, Pestalotia, Curvularia, Penicillium and Alternaria. Among them, Fusarium and Rhizoctonia were the most frequently occurring (100%) followed by Pythium and Macrophomina (50%). The above-mentioned four fungi Fusarium, Rhizoctonia, Pythium and Macrophomina were found pathogenic to strawberry showing both root rot and wilting symptoms in varying degrees. The effect of fungicides on radial growth of all four test fungi was found significant. SAAF completely inhibited Fusarium, Rhizoctonia and Pythium at all concentrations and Macrophomina at 200 ppm. Nativo completely inhibited Fusarium and Pythium at all concentrations. Shield inhibited Rhizoctonia, and Pythium at all concentrations and inhibited Macrophomina at 200 ppm. All-care inhibited the growth of Rhizoctonia, Pythium and Macrophomina at 200 ppm, but enhanced the growth of Fusarium at all concentrations. In the field, significant variations were found for percent root rot intensity among the varieties. Palmaritas had the highest disease intensity (97.22%) followed by Ruby Gem (80.00%), Florida Beauty (59.44%), Sweet Sensation (52.22%), and Winter Dawn (38.89%). Winter Dawn had significantly highest fruit yield (3132 g/plot) followed by Sweet Sensation (2468 g/plot), Ruby Gem (1903 g/plot), Florida Beauty (1040 g/plot) and Palmaritas (471 g/plot). The effect of fungicides on percent disease intensity was significant (p<0.001). SAAF had least disease intensity (45.55%) which was at par with Shield (50.00%), followed by All-care (58.87%) and Nativo (60.55%). Winter Dawn performed comparatively better with higher yield and the fungicides SAAF and Shield found effective in managing root rot complex of strawberry.

Rice (Oryza sativa L.) is the major crop of Nepal in terms of production and cultivated area with annual productivity of 3.8 t/ha. Rice blast (Pyricularia oryzae Cavara), is a major fungal threat to rice production in Nepal. Two in vitro experiments and one pot experiment were conducted at Nepal Plant Disease and Agro Associates, Balaju, Kathmandu. In the first experiment (in vitro 1), isolates of blast fungus (nine from rice and one from finger millet) from different parts of Kathmandu valley were evaluated on four different agar media (Potato carrot agar, Oat meal agar, Prune agar and V8-juice agar) for their growth and cultural behavior. In the second experiment (in vitro 2) contaminant fungi detected while culturing blast fungus were isolated and evaluated for their antagonistic effect against P. oryzae using dual culture technique. In the third experiment (pot experiment), reactions of twenty-nine IRRI-bred blast differential rice lines against blast was studied under natural infection in a plastic house. All the experiments were conducted in a completely randomized design with four replications. Disease scoring was done as percentage of leaf area infected using 0-9 scale for three times at a four days interval starting from 40 days after seeding. Disease severity and area under disease progress curve (AUDPC) was calculated. The blast symptom first appeared in 30 days, most probably initiated from seed borne infection in the line LTH and spread in the whole plastic house in 10 days. Based on disease severity, IRBLz5-CA was found to be resistant; IRBL12-M, IRBLkh-K3, IRBLta2-Re and IRBLta2-Pi were moderately susceptible and the others 24 were susceptible to leaf blast. The differential lines varied significantly for both disease severity and AUDPC values. The cultural behavior of Pyricularia isolates varied on different media. Potato carrot agar had highest growth rate while V8 juice agar recorded the highest colony growth after nine days of incubation at 25±2^oC. V8 juice agar was found to be the best medium for cultural characters (color, pigmentation and density). On dual culture, the contaminant fungus FA5 showed the highest inhibition of P. oryzae (56.21%) and all the fungal isolates varied significantly for inhibition percentage. Further studies using different isolates of P. oryzae on differential lines would generate information to identify compatible/incompatible R-genes against the pathogen. The fungal antagonists should be identified and investigated further for their biocontrol potentials.

Bacterial wilt caused by Ralstonia solanacearum is a major disease of solanaceous crops; mainly on tomato, potato, and brinjal in Nepal and no effective control measures are available yet. To find out potential of native biocontrol agents for management of bacterial wilt of tomato, laboratory and pot experiments were conducted at the laboratory of Nepal Plant Disease and Agro Associates (NPDA), during the year 2023/24. Sixteen pathogenic isolates of R. solanacearum infecting tomato and capsicum were collected and tested for their virulence in tomato seedlings in two experiments. Wilt incidences were highest in Jayamangla (82.86% and 76.67%) and Chitlang (80% and 76.67%) isolates in the first and the second experiments respectively, after six days of inoculation. Eight soil samples from the rhizosphere of healthy plants and five healthy plant samples were collected from different places of Kathmandu, Lalitpur, Bhaktapur and Makwanpur and 65 bacteria were isolated from them. All the isolated bacteria were screened for their antagonistic potential against R. solanacearum by disc-diffusion method under in vitro experiment. On the basis of in vitro screening, twelve bacterial isolates with higher zone of inhibition were selected for in vivo test on susceptible variety Pusa Ruby under screenhouse conditions. The study showed that the isolate EBRPW significantly reduced disease severity (40%) as compared to untreated control (100%). Severity area under disease progress curve (AUDPC) values were reduced significantly by 68.29% by isolate A4B1 as compared to untreated control. An experiment was conducted to evaluate growth promoting effects of antagonistic bacteria by seed treatment with antagonistic bacteria for 24 hours. Seed treated with antagonistic bacteria had significantly higher germination percentage, shoot length, root weight and shoot weight than untreated control. Highest germination percent (83.33%), plant height (30.39 cm) and shoot weight (5.52 g) were found in seed treatment with isolate A5P2, root weight (3.93 g) was highest in seed treatment with isolate A1B3. Among the bacterial antagonists, all were catalase positive; EBB1 and A4B1 were oxidase negative; EBRN and EBTP1 levan positive; EBB1 and EBRN induced hypersensitive response in tobacco leaves; and isolates A1B3, A8B1, A4B1, A6B5 and A8B5 were Gram positive and endospore forming. The present study revealed that isolates EBRPW and A4B1 were effective antagonists and isolates A5P2, A1B3, and EBTP1 were effective in plant growth promotion of tomato. Their effectiveness should be evaluated under field conditions.

Damping-off, caused by Rhizoctonia spp., is a devastating disease of tomato, which can cause 100% crop loss under favorable conditions. With an objective of evaluating efficacy of native Trichoderma in managing damping-off of tomato, laboratory and screenhouse experiments were carried out at Nepal Plant Disease and Agro Associates, Balaju, Kathmandu. Thirty soil samples from multiple elevations of Chhatrakot Rural Municipality, Gulmi were collected and 43 Trichoderma isolates were isolated using Trichoderma selective medium. Based on growth and sporulation capacity, 28 isolates were tested for mass multiplication on finger millet grain substrate. Among them, 10 best sporulating isolates were selected for dual culture on potato dextrose agar against virulent isolate S13 of Rhizoctonia sp. Fifteen tomato genotypes were evaluated against Rhizoctonia sp. (isolate S13) in pot culture. The experiment was laid out in a completely randomized design (CRD) with 16 treatments and 5 replications. The pots (300 g capacity) filled with sterilized soil + vermicompost (2:1) were inoculated with Rhizoctonia sp. @ 3 g per pot three days prior to transplantation of 26-day-old tomato seedlings (cv. Sirjana F1). Management trial was conducted using three best selected native isolates of Trichoderma (based on dual culture results) alone and in combination, one commercial Trichoderma (Raithane Trichoderma, Ilami Agro), and one chemical fungicide M-killer (azoxystrobin 20% and difenconazole 12.5% SC) (Megmani). Scoring began two days after transplantation and continued every other day until the seedlings completely died in the pathogen control pots. In pathogenicity test, isolates S13 and S2a were found most virulent causing 100 and 93.75% disease severity, respectively, whereas isolate S9 was found least virulent (43.75%). In tomato genotypes evaluation test, NCL 1 had least disease severity (33.33%) and Sirjana F1 had highest disease severity (86.66%) followed by NS 53 (80%). In an in-vitro test, Trichoderma isolate W2S1-2R1 showed highest inhibition (53.41%) of mycelial growth of Rhizoctonia sp. (isolate S13) at 72 hours of incubation followed by W6S4-2R1 (51.29%) whereas, least showed by isolate W6S5-3R2 (21.18%). In a management experiment, among Trichoderma isolates W5S2R2 gave least disease severity (43.75%) at 16 days after transplanting (DAT), whereas the disease severity was 100% in pathogen control. In terms of disease reduction, M-killer gave the highest reduction in disease severity (66.67%) at 16 DAT followed by combined treatment of Trichoderma (W2S-2R1+W4S5-2R2+W5S2R2) (58.33%) and W5S2R2 (56.25%). The isolates W5S2R2 and W2S1-2R1 were found potential both in the laboratory and pot experiments for the management of damping-off disease in tomato.

Rice (Oryza sativa L.) is one of the staple food crops cultivated in Nepal. It is affected by several diseases such as blast, bacterial blight, sheath blight, false smut, etc. Among them, false smut (Ustilaginoidea virens (Cooke) Takahashi) and sheath blight (Rhizoctonia solani (Kühn) diseases have become a major problem in the areas with increased cultivation of high yielding cultivars and higher application of nitrogenous fertilizers. Two field experiments (i) screening of rice cultivars against false smut and sheath blight, and (ii) evaluation of different treatments for the management of the diseases were conducted at Tupche, Nuwakot, whereas lab experiments were conducted at Nepal Plant Disease and Agro Associates (NPDA), Balaju, Kathmandu. A roving survey was conducted in five municipalities of Nuwakot district   to determine the status of false smut disease. Research results revealed that out of twenty-four cultivars tested, sixteen were found resistant to false smut and none was found resistant to sheath blight. All the fungicides tested in vitro were found to be equally effective in reducing mycelial growth of U. virens even at 25 ppm where Tilt (propiconazole 25 EC) was found effective (least disease incidence, 1.8%) under field conditions. Nativo (trifloxystrobin 50% + tebuconazole 25% WG) was found effective in reducing mycelial growth of R. solani in vitro and in reducing both the disease incidence (1.85%) and percent disease index (8.84%) under field conditions. The survey results showed that the highest disease incidence (5.95%) and severity (13.99 %) of false smut was found in Panchakanya rural municipality and lowest disease incidence (2.06%) and severity (2.87%) in Bidur municipality. Results of an independent t test revealed higher disease incidence and percent disease index in late-transplanted rice, hybrid cultivars and upland than early-transplanted, non-hybrid and low land, respectively.

Oyster mushroom [Pleurotus ostreatus (Jacq.) P. Kumm.] is the most commonly cultivated mushroom in Nepal. The use of supplements in combination with substrate in mushroom production is much less in practice in Nepal. Choosing a right substrate and supplement is still limited in mushroom production. This study was conducted in Baijanath Rural Municipality, Banke district from January 2024 to April 2024. The main objective of this study was to evaluate the effect of substrates, supplements and their combinations on growth and yield of oyster mushroom. Two basal substrates, namely, rice straw and wheat straw; and supplements, namely, rice bran, wheat bran, corn flour and gram flour were used. Each basal substrate was mixed with each supplement (2.5%) and two basal substrates without supplement were kept as control. Ten treatment combinations were arranged in a two-factorial completely randomized design with sixteen replications for each treatment. Observations were recorded on days to spawn run, primordial initiation, days to harvest, total fresh yield, biological efficiency, number of fruiting bodies, average fruit weight, cap diameter, stipe length and B:C ratio. Wheat straw supplemented with gram flour had the shortest days for mycelial colonization taking an average of 17.5 days, early primordial initiation taking an average of 5.25 days during first harvest. The shortest cropping duration (69.63 days) was also recorded in wheat straw supplemented with gram flour. The highest number of fruiting bodies (137.94) and largest mean cap diameter (7.76 cm) was obtained in wheat straw supplemented with wheat bran whereashighest average fruit weight (6.78 g) was obtained in wheat straw supplemented with gram flour. The total fresh yield (780.20 g/bag), biological efficiency (88.05%) and B:C ratio (1.87) were also highest in wheat straw supplemented with wheat bran. Considering, total yield and biological efficiency performance, wheat straw and wheat bran combination was found to be better.

Trichoderma is one of the important biocontrol agents and its presence may vary with differences in the land use patterns. To assess the diversity of Trichoderma from different land use patterns and their biocontrol potential against clubroot disease of rapeseed, five experiments were carried out during July 14, 2023 – July 4, 2024 at Nepal Plant Disease and Agro Associates, Balaju Kathmandu in a completely randomized design. Diversity and richness of Trichoderma at the isolate level from 11 land use patterns was carried out by assessing the cultural variations on potato dextrose agar. In total, 101 isolates of Trichoderma were isolated and the highest number of isolates (18) was found in forests and Napier fields, while none was found in playground and commercial vegetable farms. Different land use patterns exhibited varying levels of Trichoderma diversity which was strongly affected by the soil nitrogen content (r = 0.92). Screening of 101 Trichoderma isolates for mycelial growth and sporulation at 20 °C on potato dextrose showed that the nine isolates (MO8, MM3, NFO13, VMV1, VMV3,VMV10, MV1, MV10, and CR2) had higher mycelial growth (> 6 cm) even at 2 days after incubation whereas 4 isolates (NFO18, NF1, NF18, NF19) had the least growth (< 3 cm). The evaluation of 12 different substrates for mass multiplication of two selected Trichoderma isolates (CR1 and NF1), both isolates completely colonized rice, barley, and wheat grains in between 6 and 9 days. However, the substrate preference for maximum spore production varied for the isolates, i.e. CR1 isolate produced the highest number of spores (38.93 × 108 spores/g) on buckwheat, whereas isolate NF1 produced the highest number of spores (50.80 × 108 spores/g) on crushed maize cob. To study the effect of eight fungicides and six insecticides at three concentrations (10 ppm, 25 ppm, and 50 ppm), on the mycelial growth and spore production of tested Trichoderma isolates (MM7, MV4, and NFO10), poisoned food technique was carried out. Results showed that carbendazim at all three concentrations gave complete growth inhibition of all three isolates which was at par with thiophanate methyl and tebuconazole + trifloxystrobin at 50 ppm. Decreased spore production was found with increased concentrations. Thiophanate methyl and tebuconazole + trifloxystrobin at 25 ppm significantly (p<0.001) reduced the spore production of isolate MV4 by 73.84 % and 89.95 %, respectively whereas rest of the fungicides responded differently to the tested Trichoderma isolates. The none of the tested insecticides inhibited the mycelial growth of Trichoderma isolates however, the significant (p<0.001) effect was observed on the spore production, chlorpyriphos + cypermethrin being the most inhibitive. The evaluation of 25 isolates of Trichoderma against clubroot disease of rapeseed in pot culture under screenhouse conditions, disease severity in different treatments ranged from 0 to 57 %, the highest being in untreated control. Among the tested isolates, NFO4, MV8, and NF14 completely suppressed the clubroot disease in rapeseed. Additionally, all isolates promoted the plant height and number of leaves when measured at 15 days interval after inoculation. These findings suggest that the isolates NFO4, MV8, and NF14 possess significant potential as biocontrol agent for the management of clubroot disease in rapeseed.