Ting Yuan, Izhar Hyder Qazi, Xinpeng Huang, Jiping Liu
{"title":"利用多重 PCR 快速检测桑细菌性枯萎病五种致病菌的毒力相关基因","authors":"Ting Yuan, Izhar Hyder Qazi, Xinpeng Huang, Jiping Liu","doi":"10.1186/s40538-024-00583-z","DOIUrl":null,"url":null,"abstract":"<div><p>Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, <i>Ralstonia pseudosolanacearum</i>, <i>Pantoea ananatis</i>, <i>Enterobacter cloacae</i> complex (<i>ECC</i>), <i>Klebsiella pneumoniae</i> species complex (<i>KpSC</i>), and <i>K. oxytoca</i> complex (<i>KoC</i>). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: <i>pleD</i> (GGDF structural domain-containing protein), <i>yjfP</i> (esterase), <i>pelY</i> (peripheral pectate lyase), <i>ampD</i> (N-acetyl-anhydromuranmyl-L-alanine amidase), and <i>ripW</i> (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 × 10<sup>3</sup> CFU/mL (DNA = 2.45 pg/μL). From 46 natural mulberry wilt samples, the mPCR detection rates of <i>P. ananatis</i>, <i>ECC</i>, <i>KpSC</i>, <i>KoC</i> and <i>R. pseudosolanacearum</i> were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was ≥ 65.5%, while the morbidity of the undetected pathogenic bacteria was ≤ 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, <i>R. pseudosolanacearum</i>, <i>P. ananatis</i>, <i>ECC</i>, <i>KpSC</i>, and <i>KoC</i>.</p><h3>Graphical abstract</h3>\n<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":512,"journal":{"name":"Chemical and Biological Technologies in Agriculture","volume":"11 1","pages":""},"PeriodicalIF":5.2000,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://chembioagro.springeropen.com/counter/pdf/10.1186/s40538-024-00583-z","citationCount":"0","resultStr":"{\"title\":\"Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt\",\"authors\":\"Ting Yuan, Izhar Hyder Qazi, Xinpeng Huang, Jiping Liu\",\"doi\":\"10.1186/s40538-024-00583-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, <i>Ralstonia pseudosolanacearum</i>, <i>Pantoea ananatis</i>, <i>Enterobacter cloacae</i> complex (<i>ECC</i>), <i>Klebsiella pneumoniae</i> species complex (<i>KpSC</i>), and <i>K. oxytoca</i> complex (<i>KoC</i>). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: <i>pleD</i> (GGDF structural domain-containing protein), <i>yjfP</i> (esterase), <i>pelY</i> (peripheral pectate lyase), <i>ampD</i> (N-acetyl-anhydromuranmyl-L-alanine amidase), and <i>ripW</i> (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 × 10<sup>3</sup> CFU/mL (DNA = 2.45 pg/μL). From 46 natural mulberry wilt samples, the mPCR detection rates of <i>P. ananatis</i>, <i>ECC</i>, <i>KpSC</i>, <i>KoC</i> and <i>R. pseudosolanacearum</i> were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was ≥ 65.5%, while the morbidity of the undetected pathogenic bacteria was ≤ 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, <i>R. pseudosolanacearum</i>, <i>P. ananatis</i>, <i>ECC</i>, <i>KpSC</i>, and <i>KoC</i>.</p><h3>Graphical abstract</h3>\\n<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>\",\"PeriodicalId\":512,\"journal\":{\"name\":\"Chemical and Biological Technologies in Agriculture\",\"volume\":\"11 1\",\"pages\":\"\"},\"PeriodicalIF\":5.2000,\"publicationDate\":\"2024-05-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://chembioagro.springeropen.com/counter/pdf/10.1186/s40538-024-00583-z\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Chemical and Biological Technologies in Agriculture\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://link.springer.com/article/10.1186/s40538-024-00583-z\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"AGRICULTURE, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Chemical and Biological Technologies in Agriculture","FirstCategoryId":"97","ListUrlMain":"https://link.springer.com/article/10.1186/s40538-024-00583-z","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"AGRICULTURE, MULTIDISCIPLINARY","Score":null,"Total":0}
Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt
Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, Ralstonia pseudosolanacearum, Pantoea ananatis, Enterobacter cloacae complex (ECC), Klebsiella pneumoniae species complex (KpSC), and K. oxytoca complex (KoC). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: pleD (GGDF structural domain-containing protein), yjfP (esterase), pelY (peripheral pectate lyase), ampD (N-acetyl-anhydromuranmyl-L-alanine amidase), and ripW (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 × 103 CFU/mL (DNA = 2.45 pg/μL). From 46 natural mulberry wilt samples, the mPCR detection rates of P. ananatis, ECC, KpSC, KoC and R. pseudosolanacearum were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was ≥ 65.5%, while the morbidity of the undetected pathogenic bacteria was ≤ 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, R. pseudosolanacearum, P. ananatis, ECC, KpSC, and KoC.
期刊介绍：
Chemical and Biological Technologies in Agriculture is an international, interdisciplinary, peer-reviewed forum for the advancement and application to all fields of agriculture of modern chemical, biochemical and molecular technologies. The scope of this journal includes chemical and biochemical processes aimed to increase sustainable agricultural and food production, the evaluation of quality and origin of raw primary products and their transformation into foods and chemicals, as well as environmental monitoring and remediation. Of special interest are the effects of chemical and biochemical technologies, also at the nano and supramolecular scale, on the relationships between soil, plants, microorganisms and their environment, with the help of modern bioinformatics. Another special focus is the use of modern bioorganic and biological chemistry to develop new technologies for plant nutrition and bio-stimulation, advancement of biorefineries from biomasses, safe and traceable food products, carbon storage in soil and plants and restoration of contaminated soils to agriculture.
This journal presents the first opportunity to bring together researchers from a wide number of disciplines within the agricultural chemical and biological sciences, from both industry and academia. The principle aim of Chemical and Biological Technologies in Agriculture is to allow the exchange of the most advanced chemical and biochemical knowledge to develop technologies which address one of the most pressing challenges of our times - sustaining a growing world population.
Chemical and Biological Technologies in Agriculture publishes original research articles, short letters and invited reviews. Articles from scientists in industry, academia as well as private research institutes, non-governmental and environmental organizations are encouraged.
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