The breeding of triploid banana cultivars with improved traits, such as yield and disease resistance, remains a major challenge for breeders. One reason is that the molecular study and functional gene analysis in bananas fall behind due to the difficulties of its genetic manipulation. The plant protoplast-based transient transformation has been documented and widely used as a versatile and convenient system for functional gene analysis in many plant species. However, an efficient high-quality protoplast isolation and transformation system is still lacking for bananas. Here, we established an efficient protoplast isolation and transformation method for bananas by selecting proper source materials, optimizing conditions for enzymatic hydrolysis and PEG-mediated transfection. We found the best source materials for banana protoplasts’ isolation are young suckers, which give a yield of protoplasts ranging from 2.5 × 106 to 10.1 × 107 g−1 fresh weight after 5 to 6 h of enzymolysis. The yield is sufficient for most assays that have been established in protoplasts-based systems, such as protein subcellular localization and protein interaction assays. Moreover, using the established transient gene expression system in banana protoplasts, we validated the subcellular localization of Arabidopsis VESICLE SORTING RECEPTOR 1 (VSR1) and the protein self-interaction of Arabidopsis CNGC20 on the cell membrane. The results indicated this system works well and could be routinely used for the functional characterization of banana genes.
Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f. sp. cubense (Foc), is the most important constraint of the banana industry globally. In Nepal, epidemics resembling FWB have been increasingly observed on the Malbhog cultivar in the past several years. However, the disease has not been officially reported yet, and consequently, little is known about the pathogen present across the country. In this study, we characterized 13 fungal strains isolated from banana plants of the Malbhog cultivar (Silk, AAB) showing symptoms similar to FWB in banana plantations in Nepal. All of the strains were typed as belonging to the F. oxysporum and caused FWB symptoms when inoculated in the Malbhog and Cachaco (Bluggoe, ABB) cultivars. No symptoms were observed in the Williams cultivar (Cavendish, AAA). Vegetative compatibility group (VCG) analysis classified the strains as VCG 0124 or VCG 0125. PCR analyses conducted with primers specific for Foc race 1 (Foc R1) or Foc tropical race 4 (TR4) revealed that all the strains reacted positively for Foc R1 and none for TR4. Altogether, our results demonstrated that the pathogen populations causing FWB of the Malbhog cultivar in Nepal were Foc R1. This work reported, for the first time, the occurrence of FWB in Nepal. Further studies with larger Foc populations are needed to better understand disease epidemiology to design sustainable disease management strategies.
Banana wilt caused by Fusarium oxysporum f. sp. cubense has devastated a large number of banana plantations worldwide. Biological control is a possible method to conquer this disease. However, the control effect was often low and unstable while a single biocontrol strain had been applied in the field. Therefore, this study aimed to construct an effective compound microbial agent to control Fusarium wilt of banana (FWB) in the field. In addition to it, the compounding strategy of combining single strains for improving the control effect was investigated. Based on the compatibility test, five representative biocontrol strains were selected for the combination of all possible permutations. The pot experiment indicated that every biocontrol strain and their 26 combinations could control FWB to varying degrees. The control effect of combinations on FWB was higher than that of a single strain. In terms of the number of combinatorial biocontrol strains, the control effect of the four-strain combinations was the highest. According to the taxonomic differences of the five biocontrol strains, 26 biocontrol strain combinations could be divided into four groups. Among the strains in the combination, the larger the taxonomic differences the more easily it was to obtain a higher control effect. To obtain stable and efficient combinations, eight combinations were selected out and evaluated for their effectiveness in controlling FWB in different type soil. Compared with the other seven combinations, the four-strain combination T28 (Pt05 + Bc11 + Ba62 + gz-2) got the highest and stablest control effect in the four types of soil in greenhouse. And then the control effect of combination T28 was evaluated in field conditions, compared with commercially agents Bacillus subtilis, Trichoderma harzianum, and carbendazim. After four consecutive applications in the field, the control effect of T28 against FWB was the highest, reaching 57.14%. The results showed that combination T28 had a good application prospect, and the finding provided a reference for the construction of compound microbial agents.
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