Identification and description of Ditylenchus arachis from Codonopsis pilosula in Gansu Province
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摘要:目的
明确甘肃省渭源县党参Codonopsis pilosula病样中的茎线虫种类。
方法采用形态学特征、ITS-rDNA与28S-rDNA序列系统发育分析、特异性引物PCR扩增、PCR-ITS-RFLP相结合的方法进行种类鉴定。
结果甘肃省党参茎线虫群体形态特征与花生茎线虫Ditylenchus arachis相似，形态测量均值虽存在差异，但是范围值基本一致；系统发育分析显示，该线虫与花生茎线虫聚为一支，特异性引物PCR扩增片段、PCR-ITS-RFLP图谱均与花生茎线虫相同。
结论结合形态特征与分子特征分析，将甘肃党参茎线虫群体鉴定为花生茎线虫，表明该线虫已在甘肃发生分布。
Abstract:ObjectiveTo clarify the species of stem nematode from diseased sample of Codonopsis pilosula in Gansu Province.
MethodSpecies identification of stem nematode was carried out by combing morphological analysis, phylogenetic analyses of ITS-rDNA and 28S-rDNA sequences, PCR with species-specific primers as well as PCR-ITS-RFLP.
ResultThe morphological characteristics of the stem nematode population from C. pilosula in Gansu was similar to those of Ditylenchus arachis. The ranges of measurement values were basically agreed with those of D. arachis although some average values were different. The phylogenetic analyses revealed that the tested population was clustered into the same branch withD. arachis. The fragment amplified with specific primers, and PCR-ITS-RFLP map of this population were consistent with those of D. arachi.
ConclusionThe stem nematode population from C. pilosula in Gansu was identified as D. arachis based on morphological and molecular characters. D. arachis has occurred and distributed in Gansu.
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Keywords:
- Ditylenchus arachis /
- Morphological character /
- Phylogeny /
- Specific primer /
- ITS-RFLP
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图 1 花生茎线虫的光学显微镜结果
A：雄虫虫体；Ｂ：线虫体前端；C：半月体和排泄孔；D：雌虫尾；E：雄虫尾；F：阴门；G：肛门
Figure 1. Light photomicrographs of Ditylenchus arachis
A: Male entire body; B: Anterior body; C: Hemizonid and excretory pore; D: Female tail; E: Male tail; F: Vulva; G: Anus
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图 2 甘肃党参茎线虫群体与腐烂茎线虫ITS特异性引物扩增电泳图
M：DNA分子量标准(75~500 bp)；1~3：甘肃党参茎线虫群体；4~6：腐烂茎线虫
Figure 2. Electrophoresis of PCR products amplified from Ditylenchus arachis from Codonopsis pilosula in Gansu and D. destructor using species-specific ITS primers
M：DNA marker (75−500 bp)；1−3：D. arachis population from Codonopsis pilosula in Gansu；4−6：D. destructor
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图 3 基于 ITS-rDNA (A) 和28s D2/D3 (B)的甘肃党参茎线虫(GSDA1、GSDA2)系统发育树
最佳模型均为GTR+I+G，分支上数字表示后验概率
Figure 3. Phylogenetic tree of stem nematode (GSDA1、GSDA2) from Codonopsis pilosula in Gansu Province based on ITS-rDNA (A) and 28s D2/D3 (B)
All best model were GTR+I+G; The posterior probability values exceeding 50% are given on appropriate clades
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图 4 腐烂茎线虫(A)与甘肃党参茎线虫(B)ITS-rDNA区段PCR-RFLP结果
Figure 4. Electrophoresis of ITS-rDNA amplified by PCR-RFLP for Ditylenchus destructor (A) and D. arachis population in Gansu (B)
M：DNA marker(100~2000 bp)；1：PCR；2：DdeⅠ；3：HinfⅠ；4：Tru9Ⅰ(MseⅠ)；5：SduⅠ
表 1 甘肃党参茎线虫群体与河北原始群体测量值比较1)
Table 1 Morphometrics comparison of Ditylenchus arachis population from Codonopsis pilosula in Gansu with original Hebei population of D. arachis
群体
Population来源
Originn L/μm a b c c′ V/% 口针/μm
Stylet体宽/μm
Body
width尾长/μm
Tail
length交合刺长/μm
Spiculum
length雌虫
Female甘肃 Gansu 15 909±44
(855~976)35±2.7
(30~39)6.9±0.6
(6.0~7.8)14±0.9
(13~16)3.8±0.5
(3.4~4.6)82±0.8
(80~83)9.1±0.5
(8.7~9.8)27±1.7
(23~29)66±4.6
(56~70)河北 Hebei 20 893±78
(680~1007)37±5.7
(28~48)7.1±0.9
(6.0~9.6)14±1.2
(11~16)4.5±0.6
(3.3~5.6)81±0.9
(80~83)8.9±0.4
(8.6~9.8)25±4.9
(16~33)63±5.7
(53~75)雄虫
Male甘肃 Gansu 15 844±30
(787~870)35±2.2
(32~38)6.5±0.4
(6.0~7.1)13±1.7
(12~16)3.7±0.5
(3.0~4.3)8.9±0.4
(8.2~9.5)24±1.8
(22~27)64±6.5
(50~69)19±1.8
(16~22)河北 Hebei 20 884±91
(730~1022)41±4.0
(39~48)6.8±0.6
(5.6~7.8)15±1.3
(12~17)4.4±0.5
(3.7~5.1)9.1±0.4
(8.5~9.6)22±3.0
(18~28)59±3.8
(49~63)21±2.2
(16~24)1)n 代表样本数；L 为体长；a 为体长与最大体宽的比值；b 为体长与头端至食道与肠连接处长度的比值；c 为体长与尾长的比值；c′为尾长与肛门处体宽的比值；V 为阴门至头端的长度与体长的比值×100；括号内数据表示范围
1)n: Number of specimens；L: Total body length；a: Body length/the maximum body width；b: Body length/distance from head end to junction of oesophagus and intestine；c: Body length/tail length；c': Tail length/body width at anus；V: Distance from vulva to head/body length ×100; The range is represented by the data in parentheses
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表 2 软件Wbcuter酶切甘肃党参茎线虫和腐烂茎线虫ITS序列结果
Table 2 Enzyme digestion results of Ditylenchus arachis population from Codonopsis pilosula in Gansu and D. destructor ITS sequences by Wbcuter
bp 处理
Treatment河北花生茎线虫群体
D. arachispopulation
in Hebei甘肃党参茎线虫群体
D. arachis population from
Codonopsis pilosula in Gansu腐烂茎线虫
D. destructorUnrestricted PCR 816 816 915 DdeI 485,151,103, 34, 33, 10 485, 151,103, 34, 33,10 551, 354, 10 HinfI 751,65 751, 65 649,174, 57, 35 Tru9 I (MseI ) 469, 307, 31, 9 469, 307, 31, 9 468, 215,192, 31, 9 Sdu I 347, 321,148 347, 321, 148 319, 256,191,149
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