四川省马铃薯孢囊线虫的形态和分子鉴定

顾建锋; 邵宝林; 方亦午; 马欣欣; 李星月; 郑经武

doi:10.19303/j.issn.1008-0384.2022.004.013

四川省马铃薯孢囊线虫的形态和分子鉴定

doi: 10.19303/j.issn.1008-0384.2022.004.013

1.
宁波海关技术中心/宁波检验检疫科学技术研究院，浙江　宁波　315100
2.
成都海关技术中心，四川　成都　610041
3.
中盛产品检测有限公司，浙江　宁波　315100
4.
四川省农业科学院植物保护研究所，四川　成都　610066
5.
浙江大学农业与生物技术学院，浙江　杭州　310012

基金项目: 四川省科技计划项目（2021YFN0009）；海关总署科技项目(2020HK161) ；宁波市公益类科技计划重点项目(2021S024) ；宁波中盛产品检测有限公司技术开发项目（2020ZS003）

详细信息

作者简介:
顾建锋（1972−），男，博士，主要从事线虫研究（E-mail：jeffgu00@qq.com）

中图分类号: Q 945.8
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出版历程
- 收稿日期: 2022-01-05
- 修回日期: 2022-03-08
- 网络出版日期: 2022-04-24
- 刊出日期: 2022-04-28

Morphological and Molecular Identification of Potato Cyst Nematode from Sichuan

1.
Technical Center of Ningbo Customs/Ningbo Inspection and Quarantine Science Technology Academy, Ningbo, Zhejiang　315100, China
2.
Technical center of Chengdu Customs, Chengdu, Sichuan　610041, China
3.
Ningbo Joysun Product Testing Service Company, Ningbo, Zhejiang　315100, China
4.
Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan　610066, China
5.
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang　310012, China

摘要

摘要: 目的四川省越西县马铃薯发现疑似线虫侵染症状，为查清病原，对分离获得的线虫进行形态学和分子生物学鉴定。方法采集马铃薯根系和根围土壤，对根系内部线虫进行染色观察，用简易漂浮法分离孢囊，并对发现的二龄幼虫、雌虫和孢囊进行显微摄影和形态学测计。提取孢囊和二龄幼虫的DNA，用种特异PCR方法进行鉴定，同时PCR扩增后获得核糖体rDNA部分18S基因、28S基因D2～D3区和ITS区DNA序列，在GenBank中下载已登录的各近似种（球孢囊线虫属）的部分序列，进行序列比对，并使用MrBayes 3.2.3软件进行贝叶斯法系统进化树的构建。结果该种线虫能入侵寄主根系内部，二龄幼虫口针基部球近圆形，向后倾斜；口针平均长＜23 μm；孢囊Granek比值≥3，与马铃薯金线虫高度一致。特异PCR扩增3个孢囊和4条二龄幼虫均得到了430 bp左右的明亮条带，进一步测序后得到的序列长度均为434 bp，与马铃薯金线虫一致。核糖体rDNA 18S和28S进化分析表明，该四川越西马铃薯上发现的孢囊线虫属于球孢囊属线虫，核糖体ITS基因序列进一步分析证实该群体确为马铃薯金线虫。结论四川省越西县马铃薯根系分离获得的线虫为马铃薯金线虫，该线虫为我国内检和外检均十分关注的检疫性线虫，一旦传入和定殖扩散，根除十分困难。建议尽快建立马铃薯金线虫疫情监测体系，加强马铃薯种薯的检疫监督管理，增加马铃薯金线虫防控技术的研发投入、研发检测和防控技术，为我国马铃薯主粮化产业健康可持续发展保驾护航。
- 马铃薯孢囊线虫 /
- 形态学 /
- 分子生物学 /
- 检疫 /
- 鉴定 /
- 监测
Abstract: Objective To isolate and identify the pathogen causing cyst nematode infection in Yuexi, Sichuan. Method Roots of the diseased potato plants as well as soil around them were collected to find nematodes by staining and examining under a microscope, while cysts separated by means of simple floatation. The second stage juveniles (J2s), female adults, and cysts found were photographed, and their morphological measurements recorded. DNA of the cysts and the J2s were extracted and identified by species-specific PCR. DNA sequences of 18S gene and 28s D2~D3 region, as well as the ITS location in ribosomal DNA, were obtained. Sequences of some cyst nematode species were downloaded from GenBank for alignment. MrBayes 3.2.3 software was used to construct the Bayesian phylogenetic tree. Result The isolated nematode could infect the host root system. The J2 stylet knobs rounded to flattened anteriorly; the mean stylet length was less than 23 μm; and the cyst Granek’s ratio was higher or equal to 3, which is similar to that for Globodera rostochiensis. The DNA templates of 3 cysts and 4 J2s were amplified by species-specific PCR to obtain a band of approximately 430 bp. Further sequencing determined the length to be precisely 434 bp, same as G. rostochiensis. The cyst nematode was shown to be a species of Globodera by an evolutionary analysis on the rDNA 18S and 28S and confirmed to be G. rostochiensis by another evolutionary analysis on ITS gene. Conclusion The isolated nematodes from Yuexi, Sichuan was identified as G. rostochiensis, which is of great concern for the domestic disease control and import quarantine, as it can be highly difficult to eradicate once being introduced and colonized in the country. Consequently, a stringent monitoring system must be implemented as soon as possible. Reinforced quarantine, supervision, and management to curtail the possible invasion are urgently needed. Increased efforts on the basic studies as well as developments of detection methodology and prevention and control technology on G. rostochiensis are recommended.
- Potato cyst nematode /
- morphology /
- molecular identification /
- quarantine /
- identification /
- monitoring

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图 1 四川越西马铃薯金线虫根部雌虫和孢囊

注：A，马铃薯根部雌虫和孢囊；B-D，雌虫。

Figure 1. Cyst and female adult of G. rostochiensis on potato roots from Yuexi, Sichuan

Note: A: Female adults and cysts from root of potato; B-D: female adults.

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图 2 四川越西马铃薯金线虫孢囊及阴门锥

注：A，B：孢囊； C，D：孢囊阴门锥区域。

Figure 2. Cyst and cyst vulval basin of G. rostochiensis from Yuexi, Sichuan

Note: A and B: cysts; C and D: cyst vulval basin.

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图 3 四川越西马铃薯金线虫卵和二龄幼虫

注：A：二龄幼虫整体； B，C，G：二龄幼虫头部；D，H：二龄幼虫尾部；E：二龄幼虫侧区； F：卵。

Figure 3. Eggs and J2s of G. rostochiensis from Yuexi population

Note: A: J2; B, C, G: head of J2; D, H: tail of J2; E: lateral lines of J2; F: egg.

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图 4 马铃薯根系内染色后的二龄幼虫

Figure 4. Stained J2s on potato roots

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图 5 四川越西马铃薯金线虫样品特异PCR电泳图

注：M，100 bp Marker； 1-3，孢囊；4-9，二龄幼虫；10，阴性对照；11，空白对照。

Figure 5. Specific PCR electrophoresis of G. rostochiensis from Yuexi population

Note: M: 100 bp marker; 1-3: cyst; 4-9: J2; 10: negative control; 11: blank control.

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图 6 基于四川越西马铃薯金线虫与近似种ITS区DNA序列的系统发育树

Figure 6. ITS phylogenetic tree of G. rostochiensis from Yuexi population and similar Globodera species

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表 1 四川越西群体孢囊线虫样品与国外马铃薯金线虫测计值比较

Table 1. Morphometrics of cyst and J2 of Yuexi and foreign G. rostochiensis

项目 Items	四川越西样品 Sample from Yuexi Sichuan	Germany, Golden & Ellington^[16]	Russia, Kazachenko^[17]	Slovenis, Libelice, Sirca & Urek^[18]
孢囊 Cyst (n)	15	50	25	10
长（不包括颈） Length (exclude neck)/μm	484±79.9(362–599)	680(450–990)	589(467–725)	—
宽 Width /μm	424±83.4(327–550)	540(250–810)	559(430–713)	—
长/宽 Length/width	1.2±0.1(1.0–1.3)	1.27(1.0–1.8)	1.04(0.9–1.3)	—
膜孔直径 Fenestra width /μm	16.7±2.9(13.0–23.0)	15.0(8–20)	14.1(10.4–19.5)	17.9±1.7(14.6–19.4)
脊数 Numer of ridges	18.3±2.4(15.0–24.0)	—	—	18.3±1.6(16–22)
肛门至膜孔距离 Anus-fenestra distance/μm	61.4±9.6(43–76)	68(29–116)	50.7(33.8–78)	64.8±4.7(58.1–73.3)
Granek值 Granek’s ratio	3.7±0.6(2.7–4.7)	4.5(2.0–7.0)	3.7(2.1–5.4)	3.6±0.4(3.0–4.3)
J2(n)	20	50	25	30
体长 Length/μm	422.5±11.8(405–444)	430(370–470)	392(366–406)	434.4±16.7(402.7–468.0)
a	22.9±0.8(21.9–25.3)	19(16–23)	21.3(18.6–25.4)	21.4±1.1(18.9–23.0)
b	2.5±0.1(2.3–2.6)	2.3(2.2–2.5)	3(2.7–3.4)	—
c	9.1±0.5(8.2–10.3)	8(7–9)	9(8.1–11)	9.0±0.6(7.8–11.0)
口针长 Stylet /μm	20.2±0.5(19.4–21.9)	22(21–23)	19.9(19.5–20.8)	20.7±0.6(18.9–22.4)
唇高 Labial region height/μm	3.8±0.3(3.3–4.7)	—	—	—
唇宽 Labial region diameter/μm	9.4±0.4(8.3–10.1)	—	—	—
口针基部球至背食道腺的距离 DGO/μm	4.3±0.6(3.0–5.2)	5.5(5.0–6.7)	5.0(3.9–5.2)	3.8±0.8(2.4–5.0)
头顶至排泄孔 Anterior end to excretory pore /μm	96.1±2.8(88.0–100.0)	—	84.7(75.6–91)	—
头顶至中食道球瓣门 Anterior end to median bulb valve /μm	61.9±3.0(52.0–65.6)	68(64–76)	61(54.6–66.3)	65.0±6.9(55.3–77.6)
最大体宽 Max Body diameter /μm	18.4±0.3(17.5–19.0)	23(19–26)	—	20.1±0.6(18.9–22.4)
肛门处体宽 Body diameter at anus level /μm	11.9±0.6(11.0–13.0)	—	10.9(10.4–11.7)	12.7±0.7(11.3–14.4)
尾透明区长 Hyaline region /μm	21.4±1.8(17.7–25.0)	24(18–30)	19.9(19.5–20.8)	25.1±1.6(22.5–29.9)
尾长 Tail /μm	46.8±2.9(40.0–51.0)	51(44–57)	43.5(36.4–46.8)	48.5±2.7(42.6–53.8)
注：a，体长/最大体宽；b，体长/体前端至食道与肠连接处的距离；c，体长/尾长。 Note: a, body length/maximum body width; b, body length/distance from the front of body to the junction of esophagus and intestine; c, body length/tail length.

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