A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i>
Fusarium wilt of banana, caused by <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (<i>Foc</i>), especially Tropical Race 4 (TR4), seriously threatens banana production worldwide. There is no single effective control measure, although certain <i>Bacillus<...
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| Format: | Article |
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MDPI AG
2021-04-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/5/353 |
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| author | Shu Li Ping He Huacai Fan Lina Liu Kesuo Yin Baoming Yang Yongping Li Su-Mei Huang Xundong Li Si-Jun Zheng |
| author_facet | Shu Li Ping He Huacai Fan Lina Liu Kesuo Yin Baoming Yang Yongping Li Su-Mei Huang Xundong Li Si-Jun Zheng |
| author_sort | Shu Li |
| collection | DOAJ |
| description | Fusarium wilt of banana, caused by <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (<i>Foc</i>), especially Tropical Race 4 (TR4), seriously threatens banana production worldwide. There is no single effective control measure, although certain <i>Bacillus</i> strains secrete antibiotics as promising disease-biocontrol agents. This study identified five <i>Bacillus</i> strains displaying strong antibiotic activity against TR4, using a systemic assessment for presence/absence of genetic markers at genome level, and expression profiles at transcriptome level. A conventional PCR with 13 specific primer pairs detected biocontrol-related genes. An accurate, quantitative real-time PCR protocol with novel designed specific primers was developed to characterise strain-specific gene expression, that optimises strain-culturing and RNA-isolation methodologies. Six genes responsible for synthesising non-ribosomal peptide synthetase biocontrol metabolites were detected in all five strains. Three genes were involved in synthesising three Polyketide synthetase metabolites in all five strains, but the macrolactin synthase gene <i>mln</i> was only detected in WBN06 and YN1282-2. All five <i>Bacillus</i> strains have the genes <i>dhb</i> and <i>bioA</i>, essential for synthesising bacillibactin and biotin. However, the gene <i>sboA,</i> involved in subtilisin synthesis, is absent in all five strains. These genes’ expression patterns were significantly different among these strains, suggesting different mechanisms involved in TR4 biocontrol. Results will help elucidate functional genes’ biocontrol mechanisms. |
| first_indexed | 2024-03-10T11:47:12Z |
| format | Article |
| id | doaj.art-668187e09a8740499c0296337c5da8fa |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T11:47:12Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-668187e09a8740499c0296337c5da8fa2023-11-21T17:58:20ZengMDPI AGJournal of Fungi2309-608X2021-04-017535310.3390/jof7050353A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i>Shu Li0Ping He1Huacai Fan2Lina Liu3Kesuo Yin4Baoming Yang5Yongping Li6Su-Mei Huang7Xundong Li8Si-Jun Zheng9Yunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaBiotechnology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaYunnan Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests, Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, ChinaFusarium wilt of banana, caused by <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (<i>Foc</i>), especially Tropical Race 4 (TR4), seriously threatens banana production worldwide. There is no single effective control measure, although certain <i>Bacillus</i> strains secrete antibiotics as promising disease-biocontrol agents. This study identified five <i>Bacillus</i> strains displaying strong antibiotic activity against TR4, using a systemic assessment for presence/absence of genetic markers at genome level, and expression profiles at transcriptome level. A conventional PCR with 13 specific primer pairs detected biocontrol-related genes. An accurate, quantitative real-time PCR protocol with novel designed specific primers was developed to characterise strain-specific gene expression, that optimises strain-culturing and RNA-isolation methodologies. Six genes responsible for synthesising non-ribosomal peptide synthetase biocontrol metabolites were detected in all five strains. Three genes were involved in synthesising three Polyketide synthetase metabolites in all five strains, but the macrolactin synthase gene <i>mln</i> was only detected in WBN06 and YN1282-2. All five <i>Bacillus</i> strains have the genes <i>dhb</i> and <i>bioA</i>, essential for synthesising bacillibactin and biotin. However, the gene <i>sboA,</i> involved in subtilisin synthesis, is absent in all five strains. These genes’ expression patterns were significantly different among these strains, suggesting different mechanisms involved in TR4 biocontrol. Results will help elucidate functional genes’ biocontrol mechanisms.https://www.mdpi.com/2309-608X/7/5/353<i>Bacillus</i>biocontrol<i>Foc</i> TR4biocontrol marker genesPCRRT-qPCR |
| spellingShingle | Shu Li Ping He Huacai Fan Lina Liu Kesuo Yin Baoming Yang Yongping Li Su-Mei Huang Xundong Li Si-Jun Zheng A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> Journal of Fungi <i>Bacillus</i> biocontrol <i>Foc</i> TR4 biocontrol marker genes PCR RT-qPCR |
| title | A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> |
| title_full | A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> |
| title_fullStr | A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> |
| title_full_unstemmed | A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> |
| title_short | A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in <i>Bacillus</i> |
| title_sort | real time fluorescent reverse transcription quantitative pcr assay for rapid detection of genetic markers expression associated with fusarium wilt of banana biocontrol activities in i bacillus i |
| topic | <i>Bacillus</i> biocontrol <i>Foc</i> TR4 biocontrol marker genes PCR RT-qPCR |
| url | https://www.mdpi.com/2309-608X/7/5/353 |
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