基于传统PCR检测轴索黄单胞菌的简易快速DNA释放方案的建立。石榴中引起细菌性枯萎病的石榴

IF 1 Q3 PLANT SCIENCES

Archives of Phytopathology and Plant Protection Pub Date : 2023-06-22 DOI:10.1080/03235408.2023.2228004

N. Manjunatha, J. Sharma, S. Pokhare, Mansi G. Chakranarayan, R. Agarrwal, Bhagyashri Suresh Gavande, B. Raigond, M. Mallikarjun, R. Marathe

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引用次数: 0

摘要

建立了一种经济、简单、快速、不依赖培养的单胞菌常规分析检测方法。引起石榴细菌性枯萎病的一种真菌(Xap)。优化了5种DNA释放缓冲液(B1-B5)，用于直接从(a)有症状的石榴叶中提取细菌基因组DNA (gDNA)，然后进行基于常规聚合酶链反应(PCR)的Xap检测。发现B1、B3和B4适合释放gDNA，用16S rRNA和rpsL基因通用引物和病原体特异性xopQ引物进行PCR。从B1和B4释放的DNA成功地产生了预期大小的扩增子。进一步的分析发现，使用B4缓冲液释放的DNA在- 20°C/ -80°C条件下稳定45天，在4°C条件下稳定35天，并且可以通过基于pcr的检测检测到8-800 pg的DNA。通过提取引起柑橘溃疡病和高粱茎条病的黄单胞菌的DNA，进一步验证了B4的通用性。

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Development of simple and quick DNA release protocol for conventional PCR based detection of Xanthomonas axonopodis pv. punicae causing bacterial blight in pomegranate

Abstract An economical, simple, rapid, and culture independent method was developed for routine analyses and detection of Xanthomonas axonopodis pv. punicae (Xap) that causes bacterial blight in pomegranate. Five DNA release buffers (B1-B5) were optimized for extracting bacterial genomic DNA (gDNA) directly from (a)symptomatic pomegranate leaves followed by conventional polymerase chain reaction (PCR)-based detection of Xap. B1, B3, and B4 were found suitable to release gDNA, which was subjected to PCR using universal primers for 16S rRNA and rpsL genes, and pathogen specific xopQ primers. DNA released from B1 and B4 successfully produced amplicons of expected sizes. Additional analyses found that DNA released using B4 buffer was stable up to 45 days at −20 °C/−80 °C and 35 days at 4 °C and 8-800 pg DNA could be detected by the PCR-based assay. B4 was further validated for versatility by extracting DNA of Xanthomonas spp. causing citrus canker and sorghum shoot stripe disease.

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来源期刊

Archives of Phytopathology and Plant Protection Agricultural and Biological Sciences-Agronomy and Crop Science

CiteScore

2.20

自引率

0.00%

发文量

100

期刊介绍： Archives of Phytopathology and Plant Protection publishes original papers and reviews covering all scientific aspects of modern plant protection. Subjects include phytopathological virology, bacteriology, mycology, herbal studies and applied nematology and entomology as well as strategies and tactics of protecting crop plants and stocks of crop products against diseases. The journal provides a permanent forum for discussion of questions relating to the influence of plant protection measures on soil, water and air quality and on the fauna and flora, as well as to their interdependence in ecosystems of cultivated and neighbouring areas.