Optimizing inoculation methods of pest-suppressing root-endophytic fungi for mass application in a commercial banana tissue culture
Two in vivo studies were conducted at Namulonge, Uganda, to 1) To optimize inoculation methods, spore concentration, age of the plantlets and time of spore exposure facilitating the mass application and colonization of TC derived banana plantlets with mutualistic fungal endophytes, 2) To evaluate the effect of endophyte enhancement of the tissue culture bananas on plant-parasitic nematodes and banana weevils infestations in the field. In both studies, four endophytic fungal isolates were used: Fusarium oxysporum V5w2, Trichoderma asperellum TRC 900 and Beauveria bassiana 1IMI and G41. In order to optimize inoculation methods of fungal endophytes into commercial TC banana plantlets, screenhouse bioassays were conducted in which plantlets were inoculated using soil drenching, pouring (adding spore supension into the soil above plant-soil layer), and root and corm dip inoculation methods. For both soil drenching and pouring methods, TC banana plantlets were grown in multi-cell plug trays, while for the root and corm dip, plantlets were first grown in a water-soluble fertilizer for four weeks before fungal inoculation. All bioassays were evaluated at varying spore concentrations (1.0 × 106, 1.0 × 107, 0.4 × 108, 1.5 × 106, 1.5 × 107 and 1.5 × 108 conidia/ml) and exposure times (5, 10, 30, 60 and 120 min) to determine the optimal spore concentration and exposure time at deflasking and transplanting stages. For both F. oxysporum V5w2 and T. asperellum TRC 900, soil drenching was the best inoculation method, resulting in the highest tissue colonization without negatively affecting plant growth. For B. bassiana, root and corm dip offered the best inoculation method. Furthermore, inoculation of 1.5 × 107 conidia/ml for 30 min were the optimal conditions for F. oxysporum V5w2 and T. asperellum TRC 900, while inoculation of 1.5 × 107 conidia/ml for 2 h were the optimal conditions for B. bassiana. To evaluate the effect of endophyte enhancement of the tissue culture bananas on plant-parasitic nematodes and banana weevils’ infestations in the field. Two East African highland banana cultivars (cv. Kisansa and Kibuzi, AAA-EA genome) were subjected to one of five treatments: three fungal endophytes (F. oxysporum V5w2, T. asperellum TRC 900 and B. bassiana 1IMI), one nematicide (Nemacur/5GR, Fenamiphos, (Bayer Crop science, Bonn, Germany) and a negative control. The plantlets were drenched in 1.5 × 107 conidia/ml for 30 min (F. oxysporum V5w2 and T. asperellum TRC 900) or dipped in 1.5 × 107 conidia/ml and 2 h (B. bassiana 1IMI). The field comprised of six blocks, each having ten (2 cultivars × 5 non-plant treatments) treatments (plots) and twenty five plantlets per treatment, with plots arranged randomly per block. Sixty grams of the nematicide was applied twice (immediately at planting around each mat and after a 4 months interval), and 12.5 grams of chopped root segments containing mixed populations of Helicotylenchus multicinctus, Radopholus similis and Meloidogyne spp. were applied around each mat after three months post field planting. Evaluation was conducted at flowering and harvest stages and at 3, 6, 9 and 12 month intervals. Population densities of R. similis and H. multicinctus were not significantly different across treatments after 3, 6 and 12 months. At flowering, girth at zero cm, and number of standing leaves were significantly different between Kibuzi and Kisansa. Population densities of R. similis and H. multicinctus were significantly different across treatments. Within Kibuzi, nematicide treated plants produced the lowest numbers of H. multicinctus/100g root than F. oxysporum treated plants. Furthermore, within Kisansa, T. asperellum treated plants showed lowest numbers of H. multicinctus/100g root than Kibuzi. On the other hand, the population densities of H. multicinctus and Meloidogyne spp. were significantly different between Kisansa and Kibuzi. Within Kibuzi, F. oxysporum treated plants showed higher population densities of H. multicinctus root than the nematicide treated plants. Within Kisansa, T. asperellum-treated plants showed lower population densities of H. multicinctus /100g root than Kibuzi. Nematode root damage was not significantly different between treatments or cultivars. Bunch weight, time to flower and time to harvest, number of clusters and number of fingers per bunch were significantly different between Kisansa and Kibuzi, but did not significantly differ across treatments. Cv. Kibuzi produced the highest number of clusters (hands) and fingers per bunch than Kisansa. However banana weevil damages significantly differed across treatments and between cultivars. Trichoderma asperellum and F. oxysporum-inoculated plants demonstrated both the highest peripheral and inner corm damages within Kibuzi, while within Kisansa, B. bassiana-treated plants showed highest peripheral and inner corm banana weevil damages. From the current study, the optimal inoculation methods and conditions (spore concentration, exposure time and plant stages of inoculation were achieved. There were less significant effects of fungal endophytes in the field possibly because the plantlets were dipped in the fungicide post flasking. Therefore since it was the first attempt to study the effects of fungal endophytes (T. asperellum TRC 900 and B. bassiana 1IMI) in Uganda, more research will be needed to evaluate their field performance ( effects on pests and persistence with in plants).