Identification of pathogen causing leaf spot disease of Parthenocissus tricuspidata and establishment of LAMP rapid detection system
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摘要:目的
明确引起云南玉龙县地锦叶斑病的病原种类,并建立快速检测方法。
方法采用组织分离法分离病原菌,并依据科赫氏法则确定地锦叶斑病病原,通过形态学特征及多基因联合构建系统发育树,明确病原菌的分类地位;以病原菌的Alt a1基因序列为靶标设计特异性引物建立环介导等温扩增(Loop-mediated isothermal amplification,LAMP)检测方法。
结果地锦叶斑病病原菌鉴定为链格孢菌Alternaria alternata。建立的LAMP反应体系可特异有效地检测出地锦叶斑病菌,该体系最佳反应温度为65 ℃、最佳反应时间为50 min、最低检测灵敏度为1 pg/μL。使用该体系对人工接种病原菌不同时间的地锦叶片进行链格孢LAMP检测,检出时间为接种12 h及以上。
结论本研究可为地锦叶斑病的早期检测和科学防控提供理论依据。
Abstract:ObjectiveTo identify the pathogens causing leaf spot disease of Parthenocissus tricuspidata in Yunnan Yulong County, and establish a rapid detection method.
MethodThe pathogens were isolated by tissue separation method and verified by Koch’s postulate. Through morphological characteristics and multi-gene joint construction of phylogenetic tree, the taxonomic status of pathogenic bacteria was clarified. A loop-mediated isothermal amplification (LAMP) assay was established by designing specific primers based on the Alt a1 gene sequence of the pathogen.
ResultThe pathogen causing P. tricuspidata leaf spot was identified as Alternaria alternata. The established LAMP reaction system could specifically and effectively detect A. alternata. The optimal reaction temperature of the system was 65 ℃, the optimal reaction time was 50 min, and the minimum detection sensitivity was 1 pg/μL. The system was used to detect A. alternata in the leaves of the P. tricuspidata artificially inoculated with pathogens at different time, and the detection time was 12 h or more.
ConclusionThis study provides a theoretical basis for early detection, scientific prevention and control of the leaf spot of P. tricuspidata.
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图 5 LAMP反应体系的优化
A、B:LAMP反应温度优化;C、D: LAMP反应时间优化;A、C:以SYBR Green I为显色剂的LAMP反应;B、D: LAMP检测的电泳结果。在图A、B中,M:DL2000 DNA Marker;1~7:分别为60、61、62、63、64、65、66 ℃;8:阴性对照;在图C、D中,M:DL2000 DNA Marker;1~5:分别为20、30、40、50、60 min,6:阴性对照。
Figure 5. Optimization of LAMP reaction system
A, B: Optimization of LAMP reaction temperature; C, D: Optimization of LAMP reaction time; A, C: LAMP reaction with SYBR Green I as color developer; B,D: Electrophoresis results detected by LAMP. In figure A and B, M: DL2000 DNA Marker; 1−7 represent 60, 61, 62, 63, 64, 65, 66 ℃ respectively; 8: Negative control; In figure C and D, M: DL2000 DNA Marker; 1−5 represent 20, 30, 40, 50, 60 min respectively, 6: Negative control.
图 6 LAMP反应的特异性
M:DL2000 DNA Marker;1:链格孢菌Alternaria alternaria;2:小孢拟盘多毛孢Pestalotiopsis microspora;3:高粱附球菌Epicoccum sorghinum;4:暹罗炭疽菌Colletotrichum siamense;5:尖孢镰刀菌Fusarium oxysporum;6:葡萄座腔菌Botryosphaeria dothidea;7:平脐蠕孢Bipolaris sp.;8:草茎点霉Phoma herbarum;9:越橘间座壳Diaporthe vaccinii;10:木贼镰刀菌Fusarium equisetum;11:阴性对照Negative control。
Figure 6. Specificity of LAMP reaction
图 7 LAMP反应的灵敏性
M:DL2000 DNA Marker;1:1 ng/μL;2~4:分别为100、10和1 pg/μL;5~7:分别为100、10和1 fg/μL;8:100 ag/μL;9:阴性对照。
Figure 7. Sensitivity of LAMP reaction
M: DL2000 DNA Marker, 1: 1 ng/μL; 2−4 represent 100, 10 and 1 pg/μL respectively; 5−7 represent 100, 10 and 1 fg/μL, respectively; 8: 100 ag/μL; 9: Negative control.
表 1 本研究PCR采用的引物信息
Table 1 Primers for PCR used in this study
基因/序列
Gene/Sequence引物名称
Primer name引物序列(5′→3′)
Primer sequenceITS ITS1
ITS4TCCGTAGGTGAACCTGCGG
TCCTCCGCTTATTGATATGCgpd gpd1
gpd2CAACGGCTTCGGTCGCATTG
GCCAAGGAGTTGGTTGTGCAlt a1 Alt-for
Alt-revATGCAGTTCACCACCATCGC
ACGAGGGTGAYGTAGGCGTC -
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