Abstract:Fritillaria thunbergii is a genuine medicinal material which is one of the important cash crops in Jinhua city, Lishui city and other places in Zhejiang Province. In recent years, fungal diseases have occurred frequently, including root rot, which affected the yield and quality of F. thunbergii. In this study, pathogenic fungi of root rot disease of F. thunbergii were isolated and purified on potato dextrose agar (PDA) medium by diseased tissue isolation method. The pathogenic fungi were preliminarily identified by morphological observation of colony, mycelium and spore. Using the genomic DNA of fungi, the DNA fragments of internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (G3PDH), translation elongation factor 1 alpha (TEF-1α), RNA polymerase Ⅱ beta subunit (RPB2), heat shock protein (HSP60), and ITS-specific fragment of Fusarium solani (SOL) were amplified by PCR and sequenced, respectively. The homology of each DNA fragment was aligned in GenBank and the phylogenetic adjacent trees were constructed to identify the species of the fungi. The pathogenicity of the fungi was determined by inoculation of host plant with acupuncture and smear method. A total of 14 fungal isolates were isolated by PCR amplification and electrophoresis, and 4 fungi species causing the root rot disease of F. thunbergii were isolated and identified. By constructing a developmental tree and pathogenicity experiments, it was proved that the pathogenic bacteria were F. solani, F. oxysporum, F. incarnatum and F. equiseti, respectively. The partial sequence of the G3PDH gene of F. incarnatum (MN386064) and F. equiseti (MN386065) was obtained by PCR amplification for the first time. The results enrich the gene sequences of the 2 Fusarium species, and also provid a basis for subsequent related research.
[1] 窦红涛, 李若瑜, 万哲, 等. 2008. 临床常见镰刀菌的鉴别[J]. 中国真菌学杂志, 03(5): 276-279. (Dou H T, Li R Y, Wan Z, et al.2008. Identification of clinically important Fusarium spp[J]. Chinese Journal of Mycology, 03(5): 276-279.) [2] 郭静, 王超, 张宏彬, 等. 2013. 系统发生树构建方法综述[J]. 计算机应用研究, 30(3): 648-654. (Guo J, Wang C, Zhang H B, et al.2013. Review of phylogenetic tree construction[J]. Application Research of Computers, 30(3): 648-654.) [3] 赖宝春, 胡先奇, 罗文富. 2014. 云南灯盏花根腐病的2种新病原鉴定及防治药剂的室内筛选[J]. 植物保护, 40(2): 141-145. (Lai B C, Hu X Q, Luo W F.2014. Identification of the two new pathogens of root rot disease on Erigeron breviscapus and screening of fungicides[J]. Plant Protection, 40(2): 141-145.) [4] 雷娅红, 况卫刚, 郑春生, 等. 2016. 基于DNA条形码技术对镰刀菌属的检测鉴定[J]. 植物保护学报, 43(4): 544-551. (Lei Y H, Kuang W G, Zheng C S, et al.2016. Detection and identification of the genus Fusarium by DNA barcoding[J]. Acta Phytophylacica Sinica, 43(4): 544-551.) [5] 廖海兵, 李云霞, 邵晶晶, 等. 2011. 连作对浙贝母生长及土壤性质的影响[J]. 生态学杂志, (10): 2203-2208. (Liao H B, Li Y X, Shao J J, et al. 2011. Impacts of continuous cropping on Fritillaria thunbergia Miq. growth and rhizosphere soil properties[J]. Chinese Journal of Ecology, (10): 2203-2208.) [6] 柳凤, 詹儒林, 韦继光, 等. 2012. 现代生物技术在镰刀菌分类学中的应用[J]. 中国农学通报, 28(30): 166-170. (Liu F, Zhan R L, Wei J G, et al.2012. Application of modern biological technology on taxonomy of the genus Fusarium[J]. Chinese Agricultural Science Bulletin, 28(30): 166-170.) [7] 李荣峰, 徐秉良, 梁巧兰, 等. 2012. 白菜型冬油菜根腐病病原鉴定及室内毒力测定[J]. 甘肃农业大学学报, (5): 98-104. (Li R F, Xu B L, Liang Q L, et al. 2012. Identification of root rot pathogen of winter turnip rapeseed and toxicity measurement[J]. Journal of Gansu Agricultural University, (5): 98-104.) [8] 吕先真, 潘兰兰, 黄海叁, 等. 2005. 8种药剂对浙贝母灰霉病、黑斑病的防治效果试验[J]. 农药科学与管理, 26(2): 26-28. (Lv X Z, Pan L L, Huang H S, et al.2005. Control effects of 8 kinds of fungicide on Botrytis cinerea and black spot diseases of Fritillaria thunbergii[J]. Pesticide Science and Administration, 26(2): 26-28.) [9] 吕先真, 郑永利, 潘兰兰, 等. 2006. 浙贝母主要病害及其综合防治[J]. 安徽农学通报, 2006, (2): 85-86. (Lu X Z, Zheng Y L, Pan L L, et al. 2006. The main diseases of Fritillaria thunbergii and its integrated control[J]. Anhui Agricultural Science Bulletin, (2): 85-86.) [10] 穆向荣, 马逾英, 杨枝中, 等. 2014. 药用植物根腐病防治的研究进展[J]. 中药与临床, 5(2): 5-8. (Mu X R, Ma Y Y, Yang Z Z, et al.2014. Research advance on the control of root rot disease of medical plants[J]. Pharmacy and Clinics of Chinese Materia Medica, 5(2): 5-8.) [11] 王拱辰, 郑重, 叶琪明, 等. 1996. 常见镰刀菌鉴定指南[M]. 中国农业出版社, 北京, pp. 3-21. (Wang Z C, Zheng Z, Ye Q M, et al.1996. Guidelines for the Identification of Common Fusarium spp[M]. Chinese Agricultural Press, Beijing, pp. 3-21.) [12] 王志霞. 2013. 新疆红枣叶斑病病原鉴定及其防治药剂筛选[D]. 硕士学位论文, 石河子大学, 导师: 赵思峰, pp. 16. (Wang Z X.2013. Identification of pathogen of red jujube leaf spot in Xinjiang and screening of its control agents[D]. Thesis for M.S., Shihezi University, Supervisor: Zhao S F, pp. 16.) [13] 韦传宝, 刘林帅, 郑淼淼. 2011. 药用贝母栽培中病虫鼠害发生情况及防治措施[J]. 湖南农业科学, (23): 101-104. (Wei C B, Liu L S, Zheng M M. 2011. Occurrence situations of disease, insect pest and rats during cultivation of medicinal Fritillaria and its control measures[J]. Hunan Agricultural Sciences, (23): 101-104.) [14] 吴文嫱, 韦永选, 周鑫, 等. 2015. 大薯组培苗抗炭疽病接种方法的比较[J]. 江苏农业科学, 43(10): 174-175, 278. (Wu W Q, Wei Y X, Zhou X, et al.2015. Comparison of inoculation methods for anthracnose resistance of large potato tissue culture seedlings[J]. Jiangsu Agricultural Sciences, 43(10): 174-175, 278.) [15] 姚振生, 熊耀康. 2016. 浙江药用植物资源志要[M]. 上海科学技术出版社, 上海. pp. 430. (Yao Z S, Xiong Y K.2016. The Medicinal Plant Resources of Zhejiang[M]. Shanghai Scientific and Technical Publishers, Shanghai, pp. 430.) [16] 张宴瑜. 2014. 葫芦根腐病病原菌鉴定及抗病种质资源筛选[D]. 硕士学位论文, 浙江农林大学, 导师: 皇甫伟国, pp. 2. (Zhang Y Y.2014. Identification of pathogens causing root rot of Lagenaria siceraria and selection of resistant germplasm resources[D]. Thesis for M.S., Zhejiang Agricultural and Forest University, Supervisor: Huangfu W G, pp. 2.) [17] 周书军, 陈银宝, 徐文吉, 等. 2006. 浙贝母黄化矮缩枯萎病发生特点与防治技术研究[J]. 浙江万里学院学报, (5): 81-85. (Zhuo S J, Chen Y B, Xu W J, et al. 2006. Studies on occurrence characteristic and the prevention and treatment of stunt yellow wilt of Fritilllaria thunbergii Miq[J]. Journal of Zhejiang Wanli University, (5): 81-85.)