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1 Chinese Journal of Eco-Agriculture, Oct. 2012, 20(10): DOI: /SP.J * 林 1 森武 1** 侠 2 王凤龙 2 杨金广 ( ; ) 机会真菌对根结线虫的生物防治具有巨大潜力本研究于年针对来源于中国 7 个省份的 156 份烟草根结线虫样本, 分别分离根结线虫游离卵卵块和雌虫上定殖的机会真菌应用 18S rdna-its 片段测序结果结合菌株的形态学观察和培养性状鉴定定殖游离卵卵块和雌虫上的真菌根据分离到菌株的培养性状形态学和 rdna-its 序列分析, 对定殖于南方根结线虫或北方根结线虫的个游离卵 408 个卵块和 284 个雌虫的真菌菌株, 明确鉴定出 9 属 13 个种, 其中定殖根结线虫新记录种 5 个, 即长梗木霉芬芳镰刀菌渐狭蜡蚧菌虫草棒束孢和交枝顶孢淡紫紫孢菌 Purpureocillium lilacinum( 原名 : 淡紫拟青霉 ) 为优势种, 分布广泛, 在云南安徽湖北贵州和山东省均被分离到该菌在南方根结线虫和北方根结线虫的游离卵卵块和雌虫平均分离率分别为 0.49% 24.00% 和 16.90%, 说明其地理和生态适应性广泛不同地区来源的烟草根结线虫卵和雌虫上定殖的机会真菌种类存在明显差异目前尚无渐狭蜡蚧菌和虫草棒束孢作为线虫病原物的报道, 有待于进一步深入研究其对根结线虫的致病性机会真菌根结线虫烟草 18S rdna-its 多样性 : S432.1 : A : (2012) Diversity of opportunistic fungi colonizing egg and female root-knot nematodes of tobacco LIN Sen 1, WU Xia 1, WANG Feng-Long 2, YANG Jin-Guang 2 (1. College of Agronomy and Plant Protection, Qingdao Agricultural University; Key Lab of Integrated Crop Pest Management in Shandong Province, Qingdao , China; 2. Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao , China) Abstract Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is a more eco-friendly approach than using nematicides. Opportunistic fungi have been investigated as potential biological control agents for root-knot nematodes. In this study, sequence data for the 18S rdna-its region were used in conjunction with morphological observations to resolve opportunistic fungi taxonomy associated with eggs and female root-knot nematodes of tobacco. Fungal colonization was determined for eggs, egg masses and females of 156 specimens of tobacco root-knot nematodes from 7 provinces in China during A total of eggs, 408 egg masses and 284 females of Meloidogyne incognita and M. hapla were examined. Also 13 species, belonging to 9 genera of fungi, were isolated and identified. The common fungi associated with eggs, egg masses and females were Purpureocillium lilacinum, Fusarium oxysporum, F. solani, Penicillium purpurogenum, Alternaria alternata, Aspergillus unguis, A. niger and Acremonium strictum. The new strains of root-knot nematodes were Isaria farinose, Trichoderma longibrachiatum, Lecanicillium attenuatum, F. redolens and A. implicatum. P. lilacinum was the dominant species, which predominated in egg, egg masses and female root-knot nematodes with average frequencies of 0.49%, 24.00% and 16.90%, respectively. It occupied a wide range of niches in the study area, isolated from Yunnan, Anhui, Hubei, Guizhou, Shandong Provinces. Significant differences existed among fungal species communities isolated from eggs and females from various locations. As there hardly existed any information on the potential importance of I. farinose and L. attenuatum as nematode pathogens, pathogenicity tests were recommended in future studies. * ( ) ** : (1963 ),,,, wuxia3897@163.com (1987 ),,, linsen910@yahoo.com.cn : :

2 Key words Opportunistic fungi, Root-knot nematode, Tobacco, 18S rdna-its, Diversity (Received Apr. 28, 2012; accepted Jun. 28, 2012),,, [1],, [2], Drechsler (1933), 400 [3 4], 130 (Pochonia chlamydosporia) (Paecilomyces lilacinus) [5], [6] 9, [7] [8] [9] 6 14,,,, rdna- ITS,, 1 材料与方法 , : 10 10, 20, 20, 20, 20, 20, (WA) (LCMA) [5] (PDA) (CMA) :,, 2% 3 min, WA :, 1%, , 500, ml 1, 2%, LCMA, 50, 25,, PDA rdna-its DNA 9 cm PD,,,,,, 20 CTAB DNA [10] 5 μl 1 μl 6 Ⅰ (Golden View) 1% DNA PCR : ITS1-F 5 -CTTGGTCATTTAGAGGAAGT-3 ITS4-R 5 -C- CTCCGCTTATTGATATGC PCR (20 μl) DNA 1 μl PCR Master Mix(Thermo Fisher Scientific)10 μl 1 μl, 20 μl : 94 4 min ; 94 1 min, s, s, 35 ; min PCR 1% PCR, PCR Blast GenBank18S rdna,, MEGA 4.0 (neighbor-joining, NJ) [11] 2 结果与分析 S rdna-its

3 10 : 1355 GenBank ( 1) 18S rdna-its, 2, 42 4,, 3 100%, 50%, 100%,, 100%, Beauvenia brassiana Lecanicillium attenuatum L. muscarim, ITS (>98%), 100%, ; 83%,, 18S rdna-its,, 图 1 分离自烟草根结线虫真菌的 18S rdna-its 系统发育树 Fig. 1 Phylogenetic tree based on the nucleotide sequence of the 18S rdna-its region in the fungal species isolated from root-knot nematodes on tobacco 1 000, 5% Numbers above the branches represent bootstrap values based on replicates. Bar means the 5% sequence divergence.

1356 2012 20 2.2 2.2.1 Purpureocillium lilacinum CMA 25 7 d, 2.5~3.8 cm,,, 13.75~46.25 2.5~3.5 μm, 2~4 8.75~10.75 2.

4 cm,,,, 10 d,, 3~5,,,, 15.79~25.75 1.16~1.68 µm, ( 2B), 4.20~5.26 1.41~1.73 µm 2.2.3 Isaria farinosa,,, 4~5,, ( 2C) 2.

00% 0.49%, 图 2 烟草根结线虫主要机会真菌形态 (A: 淡紫紫孢菌 ; B: 渐狭蜡蚧 ; C: 虫草棒束孢 ; D: 长梗木霉 ) Fig.

Lecanicillium attenuatum; C: Isaria farinosa; D: Trichoderma longibrachiatum), 1 100% 90.0% F. oxysporum A. alternata A.

4 Purpureocillium lilacinum CMA 25 7 d, 2.5~3.8 cm,,, 13.75~ ~3.5 μm, 2~4 8.75~ ~3.75 μm,, 1~2 μm,,, 2.5~ ~3.0 μm( 2A) Lecanicillium attenuatum CMA, 12 d, 3.3~3.4 cm,,,, 10 d,, 3~5,,,, 15.79~ ~1.68 µm, ( 2B), 4.20~ ~1.73 µm Isaria farinosa,,, 4~5,, ( 2C) Trichoderma longibrachiatum PDA,,,,,, 2~5,,,,,, ( 2D) 2.3 rdna-its, 9 13,, 5, ( 1) 156, P. lilacinum,, 16.90% 24.00% 0.49%, 图 2 烟草根结线虫主要机会真菌形态 (A: 淡紫紫孢菌 ; B: 渐狭蜡蚧 ; C: 虫草棒束孢 ; D: 长梗木霉 ) Fig. 2 Morphology of main opportunistic fungi isolated from root-knot nematodes on tobacco (A: Purpureocillium lilacinum; B: Lecanicillium attenuatum; C: Isaria farinosa; D: Trichoderma longibrachiatum), 1 100% 90.0% F. oxysporum A. alternata A. niger, 3, T. longibrachiatum 36.0%, I. farinosa L. attenuatum [12], 3 讨论和结论 18S rdna-its, rdna-its GenBank, Blast GenBank, HF1 rdna-its T. longibrachiatum(jf694937) 96%; YCF2 GF1 FLF1 HBE6 HNE3

5 10 : 1357 Table 1 表 1 中国不同地区烟草根结线虫卵卵块和雌虫定殖真菌分离结果 Frequency of fungal species colonized in eggs, egg masses and females of root-knot nematodes on tobacco collected in various parts of China Fungi Nematode hosts Isolation rate (%) Areas of sampling Eggs Egg masses Females P. lilacinum Mh Mi F. oxysporum Mh Mi * F. redolens Mh 0.25 F. solani Mi Mh Mi Penicillium purpurogenum * L. attenuatum Mi 0.66 * Mi, Mh T. longibrachiatum Mh Mi Alternaria alternate * I. farinosa Mi 0.10 Mh Mi, Mi, Aspergillus unguis Mh A. niger Mh Mi, Mh * Mh Mi 0.03 Acremonium implicatum A. strictum Mi 0.02 * * shows new record species colonized on root-knot nematodes; Mi: Meloidogyne incognita; Mh: Meloidogyne hapla [13]. WLE1 rdna-its P. purpurogenum(hq839781) F. oxysporum (EU839377) A. niger(fj037755) L. attenuatum(ab378513) I. Farinosa (JF429897) A. implicatum(gu189520) 99% 9 96%~100%,, [14 15], 5, T. longibrachiatum F. redolens L. attenuatum I. farinosa A. implicatum,,, [16 19] Goswami [20 21] Singh [22] 1 A. strictum,, [20 22],, ( ) [23 24] Luangsa-Ard [12] rdna ITS TEF, P. lilacinus, P. lilacinum [25],, I. farinosa( : P. farinosa), Luangsa-ard [25] rdna β-tubulin ITS, Isaria, 10 Isaria, I. farinosa,, (Cordyceps) [26],,, 2,,

6 参考文献 [1],,,. [J]., 2003, 22(6): [2] Atkins S D, Hidalgo-Diaz L, Kalisz H, et al. Development of a new management strategy for the control of root-knot nematodes (Meloidogyne spp) in organic vegetable production[j]. Pest Manag Sci, 2003, 59(2): [3] Stirling G R. Biological control of plant parasitic nematodes[m]. Wallingford: CABI, 1991: [4],,. [M]. :, 2000: [5] Kerry B R. Exploitation of the nematophagous fungus Verticillium chlamydosporium Goddard for the biological control of root-knot nematodes (Meloidogyne spp.)[m]//butt T M, Jackson C, Magan N. Fungi as biocontrol agents: Progress problems and potential. Wallingford: CABI, 2001: [6],. [J]., 1987, 14(1): [7], S, T. [J]., 1993, 12(3): [8],,. [J]., 1994, 15(1): [9],,,. [J]., 2004, 47(6): [10] Graham G C, Mayser S P, Henry R J. A simplified method for the preparation of fungal genomic DNA for PCR and RAPD analysis[j]. Biotechniques, 1994, 16(1): [11] Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees[j]. Molecular Biology and Evolution, 1987, 4(4): [12] Luangsa-Ard J J, Hywel-Jones N L, Manoch L, et al. On the relationships of Paecilomyces sect. Isarioidea species[j]. Mycol Res, 2005, 109(5): [13] Eisenback J D. Diagnostic characters useful in the identification of the four most common species of root-knot nematodes (Meloidogyne spp.)[m]//sasser J N, Carter C C. An advanced treatise on Meloidogyne. USA, North Carolina State: North Carolina State University Graphics, 1985: [14] Sikora R A, Fernandez E. Nematode parasites of vegetable[m]// Luc M, Sikora R A, Bridge J. Plant parasitic nematodes in subtropical and tropical agriculture. UK: CABI, 2005: [15] Shaikhul A M, Tabreiz Ahmad K. Effect of opportunistic fungi on the life cycle of the root-knot nematode (Meloidogyne Javanica) on brinjal[j]. Archives of Phytopathology and Plant Protection, 2005, 38(2): [16] Pocasangre L, Sikora R A, Vilich V, et al. Survey of banana endophytic fungi from central America and screening for biological control of the burrowing nematode (Radopholus similis)[j]. Infomusa, 2009, 9(1): 3 5 [17] Sharon E, Bar-Eyal M, Chet I, et al. Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum[j]. Phytopathology, 2001, 39(6): [18] Siddiqui I A, Ali N I, Zaki M J, et al. Evaluation of Aspergillus species for the biocontrol of Meloidogyne javanica in mungbean[j]. Nematol Medit, 2001, 29(1): [19] Saifullah, Thomas B J. Studies on the parasitism of Globodera rostochiensis by Trichoderma harzianum using low temperature scanning electron microscopy[j]. Afro-Asian J Nematol, 1996, 6(1): [20] Goswami J, Pandey R K, Tiwari J P, et al. Management of root knot nematode on tomato through application of fungal antagonists, Acremonium strictum and Trichoderma harzianum[j]. Journal of Environmental Science and Health Part-B, Pesticide Food Contaminants and Agricultural Wastes, 2008, 43(2): [21] Goswami B K, Singh S. Fungal bioagents for management of root-knot nematode in tomato[j]. Pesticide Research Journal, 2004, 16(1): 9 12 [22] Singh S, Mathur N. In vitro studies of antagonistic fungi against the root-knot nematode, Meloidogyne incognita[j]. Biocontrol Science and Technology, 2010, 20(3): [23] Sharma H K, Singh S, Pankaj. Management of Meloidogyne incognita with Paecilomyces lilacinus and neem cake on Okra[J]. Pesticide Research Journal, 2007, 19(2): [24] Anastasiadis I A, Giannakou I O, Prophetou-Athanasiadou D A, et al. The combined effect of the application of a biocontrol agent Paecilomyces lilacinus, with various practices for the control of root-knot nematodes[j]. Crop Protection, 2008, 27(3/5): [25] Luangsa-ard J J, Houbraken J, van Doorn T, et al. Purpureocillium, a new genus for the medically important Paecilomyces lilacinus[j]. FEMS Microbiology Letters, 2011, 321(2): [26] Gams W, Zare R. A revision of Verticillium sect. Prostrata. Generic classification[j]. Nova Hedwigia, 2001, 72(3/4):

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis