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Study on the Identification Methods for Effective Microorganisms in Commercially Available Organic Agriculture Materials
Microorganisms ( IF 4.5 ) Pub Date : 2020-10-12 , DOI: 10.3390/microorganisms8101568 Ashutosh Bahuguna , Ah-ryeong Joe , Vishal Kumar , Jong Suk Lee , Sung-Youn Kim , Ji-Young Moon , Soon-Kil Cho , Hyunjeong Cho , Myunghee Kim
Microorganisms ( IF 4.5 ) Pub Date : 2020-10-12 , DOI: 10.3390/microorganisms8101568 Ashutosh Bahuguna , Ah-ryeong Joe , Vishal Kumar , Jong Suk Lee , Sung-Youn Kim , Ji-Young Moon , Soon-Kil Cho , Hyunjeong Cho , Myunghee Kim
The identification of microorganisms in closely related groups is challenging. The present work focused on the different molecular methodology for the accurate microbial identification in the five commercially available organic agriculture materials enriched with effective microorganisms. From the tested five organic agricultural materials, a total of seven distinct bacterial colonies (A-1, B-1, C-1, D-1, E-1, E-2, and E-3) were isolated and processed for sequential identification utilizing HiCrome™ Bacillus agar, biochemical tests with API CHB50, 16S rRNA gene analysis, random amplified polymorphic DNA (RAPD), and species-specific PCR analysis. All the isolated microorganisms were Gram-positive rods and spore former belonging to Bacillus group and appeared as a differential characteristic feature on HiCrome™ Bacillus agar. All isolates showed high-percentage similarities with the different members of Bacillus species in biochemical testing and 16S rRNA gene analysis. The collective identification results revealed isolates, A-1, B-1, and C-1, close to B. velezensis. Further RAPD-PCR and species-specific PCR discriminated and provided confirmatory evidence for D-1 as B. thuringiensis and E-1, E-2, and E-3 as B. licheniformis, respectively. In addition, presence of B. thuringiensis was also confirmed by toxin crystal protein staining. In conclusion, the species-specific primers could be used as a rapid and accurate identification tool to discriminate closely related Bacillus species such as B. subtilis, B. licheniformis, and B. thuringiensis.
中文翻译:
市售有机农业原料中有效微生物的鉴定方法研究
在密切相关的群体中鉴定微生物具有挑战性。目前的工作集中于在五种富含有效微生物的可商购获得的有机农业材料中,用于准确鉴定微生物的不同分子方法。从测试的五种有机农业材料中,总共分离出七个细菌菌落(A-1,B-1,C-1,D-1,E-1,E-2和E-3)并进行处理。利用HiCrome™顺序识别芽孢杆菌琼脂,与API CHB50,16S rRNA基因分析,随机扩增多态性DNA(RAPD),和种特异性PCR分析的生化测试。所有分离出的微生物均为芽孢杆菌属的革兰氏阳性棒和孢子形成物。并在HiCrome™芽孢杆菌琼脂上表现出不同的特征。在生化测试和16S rRNA基因分析中,所有分离株均与芽孢杆菌属的不同成员具有很高的相似性。集体鉴定结果表明,分离株A-1,B-1和C-1接近vel.ensis。进一步的RAPD-PCR和物种特异性PCR分别为D-1(苏云金芽孢杆菌)和E-1,E-2和E-3(地衣芽孢杆菌)提供了鉴别证据。此外,苏云金芽孢杆菌的存在毒素晶体蛋白染色也证实了这一点。总之,物种特异性引物可以用作快速,准确的鉴定工具,以区分密切相关的芽孢杆菌属物种,例如枯草芽孢杆菌,地衣芽孢杆菌和苏云金芽孢杆菌。
更新日期:2020-10-12
中文翻译:
市售有机农业原料中有效微生物的鉴定方法研究
在密切相关的群体中鉴定微生物具有挑战性。目前的工作集中于在五种富含有效微生物的可商购获得的有机农业材料中,用于准确鉴定微生物的不同分子方法。从测试的五种有机农业材料中,总共分离出七个细菌菌落(A-1,B-1,C-1,D-1,E-1,E-2和E-3)并进行处理。利用HiCrome™顺序识别芽孢杆菌琼脂,与API CHB50,16S rRNA基因分析,随机扩增多态性DNA(RAPD),和种特异性PCR分析的生化测试。所有分离出的微生物均为芽孢杆菌属的革兰氏阳性棒和孢子形成物。并在HiCrome™芽孢杆菌琼脂上表现出不同的特征。在生化测试和16S rRNA基因分析中,所有分离株均与芽孢杆菌属的不同成员具有很高的相似性。集体鉴定结果表明,分离株A-1,B-1和C-1接近vel.ensis。进一步的RAPD-PCR和物种特异性PCR分别为D-1(苏云金芽孢杆菌)和E-1,E-2和E-3(地衣芽孢杆菌)提供了鉴别证据。此外,苏云金芽孢杆菌的存在毒素晶体蛋白染色也证实了这一点。总之,物种特异性引物可以用作快速,准确的鉴定工具,以区分密切相关的芽孢杆菌属物种,例如枯草芽孢杆菌,地衣芽孢杆菌和苏云金芽孢杆菌。