中国孢囊线虫新记录种——野生豆孢囊线虫记述及其对豆科植物的寄生性测定

doi:10.3864/j.issn.0578-1752.2018.15.007

摘要/Abstract

摘要： 【目的】在江西婺源县孢囊线虫调查中，从大豆根及根际土壤中采集到一个孢囊线虫群体，其形态特征和分子特征与大豆孢囊线虫（Heterodera glycines）存在较大差异，因此对其形态学和分子特征以及寄生性等进行详细研究，以明确其种类及对豆科作物寄生性，为病害防控提供科学依据。【方法】从婺源县采集的大豆根及根际土样，用筛淘法分离孢囊，大豆根系上的孢囊用直接解剖法获取。用浅盘法分离根际土样中的2龄幼虫和雄虫。选取具典型特征的饱满孢囊制作阴门锥切片，浅盘法分离的2龄幼虫和雄虫制作永久玻片，在显微镜下观察记录孢囊、2龄幼虫和雄虫的形态特征，并进行形态特征测量。采用两对植物线虫通用引物AB28和TW81以及D2A和D3B分别扩增内转录间隔区（internal transcribed spacer，ITS)和28S大亚基D2-D3区段，经序列测定，用MEGA软件采用邻接法（neighbor-joining(NJ)method）构建该线虫群体与孢囊线虫属其他种群的ITS和LUS D2-D3系统进化树，分析其系统发育关系。在温室内采用盆栽人工接种的方法，测定江西孢囊线虫群体对11种豆科作物的寄生性，同时测定国内40个大豆栽培品种对该孢囊线虫的抗病性。【结果】形态学观察和特征值测量结果表明，从江西婺源大豆上采集的孢囊线虫群体，其孢囊形态、2龄幼虫以及雄虫的形态特征与野生豆孢囊线虫（Heterodera sojae）的形态特征基本一致；ITS序列（MG859982）比对发现其与野生豆孢囊线虫ITS序列（KU160510和KU160512）的同源性为99%和98%，而与大豆孢囊线虫ITS的同源性仅为81% (KY794762.1）。D2-D3序列（MG859981）与野生豆孢囊线虫（KU160511）相似度最高，为99%。序列系统进化树分析发现以ITS序列与D2-D3序列构建系统发育树的结果相一致，江西孢囊线虫群体与野生豆孢囊线虫分布在同一支，节点支持率为100%，而与大豆孢囊线虫聚在另一个分支中。结合形态学和分子生物学结果，将江西婺源大豆上的孢囊线虫群体鉴定为野生豆孢囊线虫，系中国新记录种。寄生性研究结果表明，在参鉴的11种豆科作物中，野生豆孢囊线虫的2龄幼虫能在大豆和相思豆（红豆）上侵染和繁殖，完成生活史；虽然能侵染豇豆、豌豆、扁豆、绿豆、赤豆、刀豆、菜豆和苜蓿8种作物根系，但不能完成生活史。测定的40个大豆栽培品种中，19个高感、11个中感、5个中抗、5个高抗。【结论】在我国江西婺源新发现的一种寄生于大豆的孢囊线虫为野生豆孢囊线虫, 系中国新记录种；人工接种条件下相思豆（红豆）也是野生豆孢囊线虫的寄主，40个大豆栽培品种中30个对该孢囊线虫表现感病。

关键词: 野生豆孢囊线虫, 中国新记录种, 形态学, 分子鉴定, 寄生性

Abstract: 【Objective】In a survey for cyst nematodes in Jiangxi Province, China, a cyst nematode population was collected and extracted from soybean roots and rhizosphere soil. The morphological characteristics of this population are different from that of the soybean cyst nematode (Heterodera glycines). The objective of this study is to research morphological and molecular characteristics and parasitism test of this population, identify their species and parasitism to legume crops, and to provide scientific basis for disease prevention and control.【Method】Soil samples were collected in Wuyuan County, Shangrao City, Jiangxi Province. The cysts were collected by sieving method or dissected from the roots. The 2nd stage juveniles (J2s) and males in soil were extracted by using the shallow disk method. The cysts with typical characteristics were selected to make a slice of the vulva cone, the J2s and males separated by shallow disk method were made into permanent slides. Morphological characteristics of cysts, J2s and males were observed and measured under microscope. For molecular studies, single cyst from soybean roots was picked and then ground to extract the genomic DNA. The ribosomal RNA-ITS region and the D2-D3 expansion segments of 28S large subunit rRNA gene were amplified with universal primers AB28, TW81 and D2A, D3B, respectively. The sequencing results were deposited in the GenBank database and compared with nematode sequences using the BLAST homology search program. The closest sequences were selected for phylogenetic analyses. The MEGA software was used to construct the ITS and LUS D2-D3 phylogenetic tree of cyst nematodes using neighbor-joining method. The parasitism of this cyst nematode to 11 legume crops and 40 soybean cultivars was tested by artificial inoculation in greenhouse. 【Result】 Morphological characteristics of cysts, J2s and males of population in this study agreed with Heterodera sojae reported in Korea. Sequence from the ITS-rDNA (MG859982) was 99% and 98% identical to those of H. sojae from Korea (KU160510 and KU160512) and only 81% identical to the soybean cyst nematode (H. glycines) (KY794762.1). The D2-D3 (MG859981) sequence was 99% identical to H. sojae from Korea (KU160511). In addition, analysis of phylogenetic trees constructed by the ITS and LUS D2-D3 both showed that the Jiangxi population and H. sojae were grouped in the same clade with a reliability of 100%. The cyst nematode population from Jiangxi was identified as H. sojae - a new record species in China. Inoculation of 11 legume crops showed that J2s of H. sojae could infect 10 crops (soybean, jequirity, cowpea, pea, lentil, mung bean, adzuki bean, sword bean, green beans and alfalfa), but could complete the life cycle only on soybean and jequirity. Soybean cultivars resistant identification test results showed that 19 cultivars of the 40 inoculated soybean cultivars were highly susceptible, 11 cultivars were moderately susceptible, 5 cultivars were moderate resistant and 5 cultivars were highly resistant.【Conclusion】The population of cyst nematode collected on soybean from Jiangxi, China was identified as H. sojae - a new cyst nematode record in China. Parasitism tests indicated that soybean and jequirity were suitable hosts for H. sojae, and 30 of the 40 soybean cultivars were susceptible to phenotypic disease of cyst nematodes.

Key words: Heterodera sojae, new record in China, morphology, molecular identification, parasitism

甄浩洋, 彭焕, 孔令安, 洪豹元, 朱桂兰, 汪锐辉, 彭德良, 文艳华. 中国孢囊线虫新记录种——野生豆孢囊线虫记述及其对豆科植物的寄生性测定[J]. 中国农业科学, 2018, 51(15): 2913-2924.

ZHEN HaoYang, PENG Huan, KONG LingAn, HONG BaoYuan, ZHU GuiLan, WANG RuiHui, PENG DeLiang, WEN YanHua. Heterodera sojae, a New Cyst Nematode Record in China and Its Parasitism to Legume Crops[J]. Scientia Agricultura Sinica, 2018, 51(15): 2913-2924.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.15.007

https://www.chinaagrisci.com/CN/Y2018/V51/I15/2913

参考文献

[1] MASUDA T, GOLDSMITH P D. World soybean production: area harvested, yield, and long-term projections. International Food and Agribusiness Management Review, 2009, 12(4): 143-161.

[2] 张俊立, 彭德良, 曹克强. 河北省大豆孢囊线虫分子鉴定及其分布. 植物保护, 2005, 31(1): 40-43.

ZHANG J L, PENG D L, CAO K Q. Molecular identification and distribution of Heterodera glycines in Hebei. Plant Protection, 2005, 31(1): 40-43. (in Chinese)

[3] OPPERMAN C H, BIRD D M. The soybean cyst nematode, Heterodera glycines: a genetic model system for the study of plant- parasitic nematodes. Current Opinion in Plant Biology, 1998, 1(4): 342-346.

[4] DUNCAN L W, MOENS M, PERRY R N, MOENS M. Migratory endoparasitic nematodes//Plant Nematology. CABI, 2013: 123-152.

[5] SUBBOTIN S A, MUNDOOCAMPO M, BALDWIN J G, SUBBOTIN S A, MUNDOOCAMPO M, BALDWIN J G. Systematics of cyst nematodes (Nematodes: Heteroderinae), Volume 8, Part B. Leiden, Netherlands: Brill Academic Publishers, 2010.

[6] 李惠霞, 柳永娥, 魏庄. 新疆和西藏发现禾谷孢囊线虫//中国线虫学研究: 第4卷. 北京: 中国农业科学技术出版社, 2012: 164-165.

LI H X, LIU Y E, WEI Z. The detection of Heterodera avenae from the cereal field in autonomous regions of Tibet and Xinjiang// Nematology Research in China: Vol.4. Beijing: China Agricultural Science and Technology Press, 2012: 164-165. (in Chinese)

[7] 陈品三, 王明祖, 彭德良. 我国小麦禾谷孢囊线虫 (Heterodera avenae Wollenweber) 鉴定研究. 植物病理学报, 1992, 22(4): 339-343.

CHEN P S, WANG M Z, PENG D L. A study of cereal cyst-nematode (Heterodera avenae Wollenweber) in China. Acta Phytopathologica Sinica, 1992, 22(4): 339-343. (in Chinese)

[8] PENG D L, YE W X, PENG H, GU X C. First report of the cyst nematode (Heterodera filipjevi) on wheat in Henan Province, China. Plant Disease, 2010, 94(10): 1262.

[9] 陈品三, 郑经武, 彭德良. 中国异皮属线虫一新种 (线虫纲: 垫刃目: 异皮科). 动物分类学报, 1996, 21(1): 18-24.

CHEN P S, ZHENG J W, PENG D L. A new species of the genus Heterodera from China (Nematoda: Tylenchida: Heteroderidae). Acta Zootaxonomica Sinica, 1996, 21(1): 18-24. (in Chinese)

[10] WANG H H, ZHUO K, YE W M, ZHANG H L, PENG D L, LIAO J L. Heterodera fengi n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China: a new cyst nematode in the Cyperi group. Zootaxa, 2013, 3652(1): 179-192.

[11] ZHUO K, WANG H H, YE W M, PENG D L, LIAO J L. Heterodera hainanensis n. sp. (Nematoda: Heteroderinae) from bamboo in Hainan Province, China-a new cyst nematode in the Afenestrata group. Nematology, 2013, 15(3): 303-314.

[12] ZHUO K, WANG H, ZHANG H, LIAO J L. Heterodera guangdongensis n. sp. (Nematoda: Heteroderinae) from bamboo in Guangdong Province, China-a new cyst nematode in the Cyperi group. Zootaxa, 2014, 3881(5): 488-500.

[13] DING Z, NAMPHUENG J, HE X F, PENG D L, HUANG W K. First report of the cyst nematode (Heterodera elachista)on rice in Hunan Province, China. Plant Disease, 2012, 96(1): 151.

[14] 王宏洪, 卓侃, 张洪玲, 廖金玲. 孢囊线虫中国新纪录种——朝鲜孢囊线虫 (Heterodera koreana). 植物病理学报, 2012, 42(5): 551-555.

WANG H H, ZHUO K, ZHANG H L, LIAO J L. Heterodera koreana, a new record species from China. Acta Phytopathologica Sinica, 2012, 42(5): 551-555. (in Chinese)

[15] 朱红雪, 田忠玲, 蔡瑞杭, 李晓琳, 郑经武. 朝鲜孢囊线虫——浙江省孢囊线虫新记录种. 浙江大学学报 (农业与生命科学版), 2017, 43(1): 81-88.

ZHU H X, TIAN Z L, CAI R H, LI X L, ZHENG J W. Heterodera koreana, anew record of cyst-forming nematodes in Zhejiang Province, China. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(1): 81-88. (in Chinese)

[16] KANG H, EUN G, HA J, KIM Y, PARK N, KIM D, CHOI I. New cyst nematode, Heterodera sojae n. sp. (Nematoda: Heteroderidae) from soybean in Korea. Journal of Nematology, 2016, 48(4): 280-289.

[17] KONG L A, WU D Q, HUANG W K, PENG D L. Assaying the potential of twenty-one legume plants in Medicago truncatula and M. sativa for candidate model plants for investigation the interactions with Heterodera glycines. Journal of Integrative Agriculture, 2016, 15(3): 702-704.

[18] 冯志新. 植物线虫学. 北京: 中国农业出版社, 2001: 132-135.

FENG Z X. Plant Nematology. Beijing: China Agriculture Press, 2001: 132-135. (in Chinese)

[19] 谢辉. 植物线虫分类学. 2版. 北京: 高等教育出版社, 2005: 132-134.

XIE H. Taxonomy of Plant Nematodes. 2nd ed. Beijing: Higher Education Press, 2005: 132-134. (in Chinese)

[20] 亓晓莉, 彭德良, 彭焕, 龙海波, 黄文坤, 贺文婷. 基于SCAR标记的小麦禾谷孢囊线虫快速分子检测技术. 中国农业科学, 2012, 45(21): 4388-4395

QI X L, PENG D L, PENG H, LONG H B, HUANG W K, HE W T. Rapid molecular diagnosis based on SCAR marker system for cereal cyst nematode.Scientia Agricultura Sinica, 2012, 45(21): 4388-4395. (in Chinese)

[21] CUI J K, PENG H, LIU S M, Orakci G E, HUANG W K, Imren M, Dababat A A, PENG D L. Occurrence, identification and phylogenetic analyses of cereal cyst nematodes (Heterodera spp.) in Turkey. Journal of Integrative Agriculture, 2017, 16(8): 1767-1776.

[22] SUBBOTIN S A, WAEVENBERGE L, MOENS M. Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP. Nematology, 2000, 2(2): 153-164.

[23] DE LUCA F, VOVLAS N, LUCARRLLI G, TROCCOLI A, RADICCI V, FANELLI E, Cantalapiedra-Navarrete c, Palomares-Rius J E , Castillo P. Heterodera elachista the Japanese cyst nematode parasitizing corn in Northern Italy: integrative diagnosis and bionomics. European Journal of Plant Pathology, 2013, 136(4): 857-872.

[24] MUNDO-OCAMPO M, TROCCOLI A, SUBBOTIN S A, CID J D, BALDWIN J G, INSERRA R N. Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae). Nematology, 2008, 10(5): 611-632.

[25] BYBD D W, KIRKPATRICK T, BARKER K R. An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology, 1983, 15(1): 142-143.

[26] SCHMITT D P, SHANNON G. Differentiating soybean responses to Heterodera glycines races. Crop Science, 1992, 32(1): 275-277.

[27] NOBBS J M, IBRAHIM S K, ROWE J. A morphological and biochemical comparison of the four cyst nematode species, Heterodera elachista, H. oryzicola, H. oryzae and H. sacchari (Nematoda: Heteroderidae) known to attack rice (Oryza sativa). Fundamental & Applied Nematology, 1992, 15(6): 551-562.

[28] PERRY R N, MOENS M. Molecular taxonomy and phylogeny// SUBBOTIN S A, MOENS M. Plant nematology. London: British library, 2006: 56.

[29] 李惠霞, 彭焕, 彭德良, 朱锐东, 徐鹏刚, 李建荣. 甘肃省高寒草原牧草孢囊线虫的鉴定. 草业学报, 2015, 24(8): 174-180.

LI H X, PENG H, PENG D L, ZHU R D, XU P G, LI J R. Identification of cyst nematode in alpine meadow steppe, Gansu Province. Acta Pratacultural Sinica, 2015, 24 (8): 174-180. (in Chinese)

[30] 宛煜嵩, 王珍. 中国大豆孢囊线虫抗性研究进展. 分子植物育种, 2004, 2(5): 609-619.

WAN Y S, WANG Z. Research progress on resistance of soybean cyst nematode (Heterodera glycines) in China. Molecular Plant Breeding, 2004, 2(5): 609-619. (in Chinese)

[31] 孔祥超, 李红梅, 耿甜, 黄文坤, 彭德良. 大豆种质资源对大豆孢囊线虫3号和4号生理小种的抗性鉴定. 植物保护, 2012, 38(1): 146-150.

KONG X C, LI H M, GENG T, HUANG W K, PENG D L. Resistance evaluation of soybean varieties and germplasms to the races No.3 and No.4 of soybean cyst nematode Heterodera glycines. Plant Protection, 2012, 38(1): 146-150. (in Chinese)