Establishment and Application of Detection System for Millet Seed with Aphelenchoides besseyi

doi:10.7668/hbnxb.20194844

Abstract

Abstract:

In order to better solve the problem of false positive in the detection of Aphelenchoides besseyi Christie and establish a stable,efficient and low cost detection system for millet seed of parasitic nematodes,using millet seed of nematodes Jigu 20 as experimental materials,the number of nematode eluted by medical cotton gauze,coarse nylon gauze and thin nylon gauze was compared.Three effect methods of DNA extraction,namely,cutting,freeze-thaw,enzymatic lysis were designed and compared in order to check their efficiency of DNA extraction from singlenematode.A total of 109 millet seed samples from different seed companies in 2018,2020 and 2021 were detected by the system.The final detection system was as follows:1.5 g seeds were wrapped in thin nylon gauze and soaked for 4 h,then the elution was mixed and counted under microscope for nematode quantification; the single nematode DNA was extracted by enzymatic lysis and characterized by PCR amplification with specific primers.The pathogenic nematodes were molecular-identified as Aphelenchoides besseyi.A total of 4 seed samples from 109 millet seed samples with 5 nematodes per 1.5 g were detected.The system with nematode quantification and qualification can be used for rapid detection of nematodes in large quantities of millet seed,providing technical support for the early diagnosis,monitoring,and prevention and control of millet nematode disease.

Key words: Millet nematode disease, Aphelenchoides besseyi, DNA extraction, Specific PCR, Rapid diagnosis

CLC Number:

ZHANG Mengya, GONG Keke, DONG Zhiping, LIU Jia, MA Jifang, WANG Yongfang, LIU Lei, QUAN Jianzhang, LI Zhiyong, BAI Hui. Establishment and Application of Detection System for Millet Seed with Aphelenchoides besseyi[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(S1): 248-255. doi: 10.7668/hbnxb.20194844.

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Figures/Tables 7

Fig.1 Comparison of the cleaning effect of Aphelenchoides besseyi by different elution media A.Number of Aphelenchoides besseyi and liquid volume by different elution media; B.Number of impurities by different elution media.

Fig.2 Electrophoretogram of the Aphelenchoides besseyi DNA based on different cutting tools M.Marker; CK^-.Negative control(ddH₂O);1.Cutting 2 min with scalpel+freeze-thaw+enzymatic lysis;2.Cutting 2 min with plastic pestle+freeze-thaw+enzymatic lysis;3.No cutting+freeze-thaw+enzymatic lysis;4.Cutting 2 min with scalpel;5.Cutting 2 min with plastic pestle;6.No cutting.

Fig.3 Electrophoretogram of the Aphelenchoides besseyi DNA based on different cutting time with scalpel under freeze-thaw and enzymatic lysis M.Marker; CK^-.Negative control(ddH₂O);1.Cutting 2 min with scalpel+freeze-thaw+enzymatic lysis;2.Cutting 1 min with scalpel+freeze-thaw+enzymatic lysis;3.Cutting 30 s with scalpel+freeze-thaw+enzymatic lysis;4.No cutting+freeze-thaw+enzymatic lysis.

Fig.4 Electrophoretogram of the Aphelenchoides besseyi DNA based on freeze-thaw and enzymatic lysis M.Marker; CK^-.Negative control(ddH₂O);1.Cutting 30 s with scalpel+freeze-thaw+enzymatic lysis;2.Cutting 30 s with scalpel+freeze-thaw;3.Cutting 30 s with scalpel+enzymatic lysis;4.Cutting 30 s with scalpel;5.No cutting+freeze-thaw+enzymatic lysis;6.No cutting+freeze-thaw;7.No cutting+enzymatic lysis;8.No cutting+no freeze-thaw+no enzymatic lysis.The same as Fig.5.

Fig.5 Electrophoretogram of the stability of Aphelenchoides besseyi DNA based on different methods

Tab.1 Statistical of Aphelenchoides besseyi of different millet varieties examinated from microscope

采集年份 Sampling year	采集省份 Sampling province	来源公司 Origin company	采集地区 Sampling site	样本类型 Sample type	样品数量 Sample size	阳性样本数量 Positive sample size	1.5 g种子带线虫量/条 Number of nematodes from 1.5 g seeds
2018	内蒙古	内蒙古蒙龙种业	松山	春谷	17	0	0
			喀喇沁旗	春谷	3	0	0
			敖汉旗	春谷	1	0	0
		巴林左旗鑫达种业有限公司	巴林左旗	春谷	3	0	0
		内蒙古禾为贵种业有限公司	敖汉旗	春谷	2	0	0
	山东	鲁岩公司	章丘	夏谷	1	0	0
	河北	东昌种业	邯郸	夏谷	9	0	0
		河北巡天农业科技有限公司	张家口	春谷	12	1	5
	山西	山西省汾都香种业科技公司	石楼县	春谷	1	0	0
			平遥县	春谷	1	0	0
			汾阳市	春谷	1	0	0
			兴县	春谷	1	0	0
2020	吉林	吉林省乾安县天丰米业有限公司	乾安县	春谷	4	0	0
		松原市金禾农业科技开发有限公司	松原	春谷	5	1	5
	内蒙古	内蒙古蒙龙种业	松山	春谷	4	0	0
		内蒙古蒙清农业科技开发有限责任公司	呼和浩特	春谷	1	0	0
		巴林左旗鑫达种业有限公司	巴林左旗	春谷	12	0	0
	河北	平泉翠芳种植专业合作社	平泉	春谷	4	0	0
		刘杖子有机专业合作社	承德县	春谷	1	0	0
		承德县熙野产销合作社		春谷	1	0	0
		承德丰宁禾林小米种植专业合作社	丰宁	春谷	2	1	5
		黄旗皇翁公司		春谷	3	0	0
2021	吉林	松原市金禾农业科技开发有限公司	松原	春谷	5	0	0
	河北	河北巡天农业科技有限公司	张家口	春谷	15	1	5

Fig.6 PCR detection of positive samples by using specific primers GU-F/R for Aphelenchoides besseyi M.Marker; CK^-.Negative control (ddH₂O); CK⁺.Positive control (Aphelenchoides besseyi ); 1-4.Positive samples.

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