Establishment of multiplex PCR detection method for two mulberry pathogenic fungi

LUO Longhui; ZHANG Xingnan; MENG Fang; HUANG Yuxin; LIU Jiping

doi:10.7671/j.issn.1001-411X.202201019

Journal of South China Agricultural University > 2022 > 43(5): 54-60. > DOI: 10.7671/j.issn.1001-411X.202201019

LUO Longhui, ZHANG Xingnan, MENG Fang, et al. Establishment of multiplex PCR detection method for two mulberry pathogenic fungi[J]. Journal of South China Agricultural University, 2022, 43(5): 54-60. DOI: 10.7671/j.issn.1001-411X.202201019

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Establishment of multiplex PCR detection method for two mulberry pathogenic fungi

College of Animal Science, South China Agricultural University, Guangzhou 510642, China

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Received Date: January 12, 2022
Available Online: May 17, 2023

Graphical Abstract

Abstract

Abstract

Objective
Mulberry brown spot disease and mulberry ring leaf spot disease are two common fungal diseases of mulberry trees (Morus alba), which are serious and cause great losses in production. This study was aimed to quickly and accurately diagnose the main pathogens of the two diseases, Neophloeospora maculans and Gonatophragmium triuniae.

Method
Based on the principle of multiplex PCR, specific primers for multiplex PCR were designed for the ribosome internal transcribed spacer (ITS) sequences of two pathogenic fungi, and the conditions of multiplex PCR reaction were optimized. Tests were performed with 45 samples to verify the feasibility of the established multiplex PCR.

Result
The established multiplex PCR detection system had good operability and good specificity, and the sensitivities for simultaneously detecting DNA of two pathogens were 0.1 and 1.0 pg/μL, respectively. The detection of mulberry disease samples could clearly distinguish the two types of pathogenic fungi in different regions.

Conclusion
The established multiplex PCR technology can be used for rapid detection of the pathogens of mulberry brown spot and ring leaf spot diseases, and provides a foundation for the prevention and control of mulberry fungal disease.
- Mulberry,
- Brown spot disease,
- Ring leaf spot disease,
- Neophloeospora maculans,
- Gonatophragmium triuniae,
- Multiplex PCR

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References

[1]	DHANYALAKSHMI K H, NATARAJA K N. Mulberry (Morus spp. ) has the features to treat as a potential perennial model system[J]. Plant Signaling & Behavior, 2018, 13(8): e1491267.
[2]	廖森泰, 肖更生, 黄先智, 等. 国家蚕桑产业技术体系成果: 蚕桑资源多元化利用技术与产品(Ⅰ)[J]. 蚕业科学, 2017, 43(1): 169-176.
[3]	张波静, 孙孝龙. 蚕桑资源综合开发利用模式与应用探讨. 江苏蚕业, 2012, 34(3): 32-35.
[4]	余茂德, 楼程富. 桑树学[M]. 北京: 高等教育出版社, 2016: 356-357.
[5]	李月秋, 赵燕春, 赵丽章, 等. 云南大理州桑褐斑病发生、危害现状及防治[J]. 植物保护, 2009, 35(5): 165-167. doi: 10.3969/j.issn.0529-1542.2009.05.039
[6]	HONG S K, KIM W G, SUNG G B, et al. Occurrence of leaf spot on mulberry caused by Phloeospora maculans in Korea[J]. The Plant Pathology Journal, 2011, 27(2): 193. doi: 10.5423/PPJ.2011.27.2.193
[7]	VIDEIRA S, GROENEWALD J Z, NAKASHIMA C, et al. Mycosphaerellaceae: Chaos or clarity?[J]. Studies in Mycology, 2017, 87(1): 257-421.
[8]	COSTA C, VELOSO J S, OLIVEIRA B F, et al. First report of Neophloeospora maculans causing leaf spots in Morus nigra and M. alba in Brazil[J]. Journal of Plant Diseases and Protection, 2021, 128: 317-321. doi: 10.1007/s41348-020-00370-6
[9]	SOYLU S, KURT S, SOYLU E M. First report of Phloeospora leaf spot on mulberry caused by Phloeospora maculans ( = Cylindrosporium maculans) in the East Mediterranean region of Turkey[J]. Plant Pathology, 2003, 52(3): 415. doi: 10.1046/j.1365-3059.2003.00837.x
[10]	MUNSHI N A, TANKI T N, SAHAF K A, et al. Behaviour of some newly evolved mulberry genotypes against leaf spot and powdery mildew diseases in Kashmir under field conditions[J]. Indian Journal of Sericulture, 2007, 46(2): 183-185.
[11]	江秀均, 柴建萍, 陆鹏飞, 等. 桑褐斑病叶的营养成分变化及对家蚕的健康效应分析[J]. 西南农业学报, 2019, 32(7): 1682-1686.
[12]	王谢, 唐甜, 张建华. 一种桑轮纹病病叶发病程度的评价指数[J]. 中国蚕业, 2018, 39(4): 19-21.
[13]	白景彰, 周顺心, 雷扶生, 等. 百色蚕区发生的桑轮纹病调查初报[J]. 广西蚕业, 1994, 31(2): 36.
[14]	章一鸣, 赵雁, 吴庭观, 等. 桑树轮纹病病原菌的分离鉴定及其生物学特性研究[J]. 西部林业科学, 2014, 43(3): 51-56. doi: 10.3969/j.issn.1672-8246.2014.03.011
[15]	SAIKI R K, SCHARF S, FALOONA F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia[J]. Science, 1985, 230(4732): 1350-1354. doi: 10.1126/science.2999980
[16]	HEID C A, STEVENS J, LIVAK K J, et al. Real time quantitative PCR[J]. Genome Research, 1996, 6(10): 986-994. doi: 10.1101/gr.6.10.986
[17]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): e63. doi: 10.1093/nar/28.12.e63
[18]	姜星. 桑枯萎病病原细菌的分离、鉴定及分子检测方法的建立[D]. 镇江: 江苏科技大学, 2012.
[19]	杨宏宇, 周轶楠, 孙勋勋, 等. 桑源阴沟肠杆菌LAMP检测方法的建立和应用[J]. 蚕业科学, 2019, 45(3): 321-330.
[20]	陈明洁, 方倜, 柯涛, 等. 多重PCR: 一种高效快速的分子生物学技术[J]. 武汉理工大学学报, 2005, 27(10): 33-36. doi: 10.3321/j.issn:1671-4431.2005.10.011
[21]	WHITE T J, BRUNS T, LEE S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[M]. PCR Protocols. Amsterdam: Elsevier, 1990: 315-322.
[22]	EGEA M N A , PITERA J G , GONZÁLEZ J C M, et al. Utility of multiplex PCR syndromic panel for respiratory infections in the diagnosis of acute bacterial meningitis. Enfermedades Infecciosas y Microbiologia Clinica, 2021, 39(9): 481-482.
[23]	邢珍娟, 董立明, 龙丽坤, 等. 应用多重荧光PCR快速筛查作物中转基因成分研究[J]. 玉米科学, 2021, 29(4): 35-42.
[24]	杨怡华, 王明郧, 曹瑱艳, 等. 麦冬主要病害病原菌巢式多重PCR检测方法的建立[J]. 植物保护学报, 2021, 48(4): 742-747.
[25]	李明骏, 廖萌, 蒋开蓉, 等. 五种玉米病毒多重PCR检测体系的建立[J]. 植物保护学报, 2021, 48(2): 465-466.
[26]	XUE L, ZHANG L, YANG X X, et al. Characterization, phylogenetic analyses, and pathogenicity of Colletotrichum species on Morus alba in Sichuan Province, China[J]. Plant Disease, 2019, 103(10): 2624-2633. doi: 10.1094/PDIS-06-18-0938-RE

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