| The banana fusarium wilt is a vascular bundle of systemic diseases caused by Fusarium oxysporum f. sp. cubense, which led to devastating damage and serious economic losses. Race 4 of Fusarium oxysporum f. sp. cubense?Foc4? can infect almost all banana varieties. Currently, there is no effective method for control of the banana fusarium wilt. In recent years, the research on biocontrol with antagonistic bacteria has aroused people's concern. Bacillus subtilis subsp. subtilis G-7 was previously isolated by our lab. It can effectively inhibit the mycelial growth and spore germination of Foc4. It has high potential for biocontrol. This research attempts to analyze from cloning and expression of antagonist gene, in order to explore the mechanism of it antagonizing the Fusarium oxysporum f. sp. cubense.When B. subtilis interact with fungal pathogen, it will secrete lipopeptide antibiotics, iturin A. It can lead to the cell death by interfering the pathogenic fungal cell membrane function. This study analyzed the vitro inhibitory effect between strain G-7 to Foc4. We cloned and analyzed the sequences of synthesis-related gene of iturin A—itu D-1 and lpa-14. And we also studied the expression status of itu D-1 and lpa-14 after strain G-7 antagonizing Foc4.Confront culture results showed that strain G-7 has obvious antagonistic effect to Foc4. The relative inhibition rate is 61.24±0.39?%? and the width of inhibition zones is 5.90±0.10?mm?.In potted planting experiment, Musa AAA plants of comparison group inoculated with dd H2 O and Foc4 showed typical wilt symptoms after 30 d, while Musa AAA plants of treatment group inoculated with the G-7 culture and Foc4 almost remained healthy, the morbidity of treatment group is evidently lower than that of comparison group.The growth rate method showed that the broth, the cell suspension and the culture filtrate of strain G-7 can effectively inhibit the mycelium of Foc4. The inhibition rate of these 3 treating fluid to mycelium of Foc4 were 69.15±0.49?%?, 59.54±0.13?%? and 31.83±0.08?%? respectively, significantly higher than the CK treatments?P<0.05?. The germination spore method showed that the broth, the cell suspension and the culture filtrate of strain G-7 can effectively inhibit the spore germination of Foc4. The spore germination rate of these 3 treating fluid were 23.28±0.10?%?, 44.42±3.82?%?, and 32.01±0.17?%?, significantly lower than the CK treatments?P<0.05?.This study cloned two antagonistic related genes, itu D-1 and lpa-14 of strain G-7. These two genes are related to synthesis of lipopeptide antibiotics, iturin A. Their DNA are 1202 bp and 675 bp. According to the NCBI similarity analysis, we found that lpa-14 belongs to the Sfp type protein which is in ACPS family. It has 99% similarity compared to the Phosphopantetheinyl transferase?PPTase? in Bacillus amyloliquefaciens?WP0606578-11.1?. Itu D-1 belongs to the Acy1transf1 family. It's the antibiotic biosynthesis protein in Fab D type protein, which has 99% similarity compared to the malonyl coenzyme A acylase?MCAT? in Bacillus amyloliquefaciens?CBI43040.1?.Fluorogenic quantitative PCR: After 24 h of strain G-7 antagonizing Foc4, the relative expressions of itu D-1 and lpa-14 were higher than the CK treatments. The expression quantity of lpa-14 was 2.3 times before the antagonism. The expression quantity of itu D-1 was 14.62 times before the antagonism. The results indicated that itu D-1 and lpa-14 are antagonistic-related genes. |
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