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大白菜软腐病新病原菌Pectobacterium aroidearum的鉴定及其生物学特性

花匠小妙招
2024-11-29 13:43

细菌性软腐病是大白菜生产上主要发生病害之一,其病原菌通常为胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp. carotovorum(Pcc)。从北京房山区大白菜软腐病样中分离获得一株菌株KC20,其在LB固体培养基上的菌落呈圆形、乳白色、半透明、表面光滑且边缘整齐,在半选择性培养基CVP上产生典型的杯状凹陷。致病性测定结果表明,该菌株侵染引发的大白菜软腐症状与田间大白菜软腐自然发病症状相同。利用果胶杆菌属特异引物Y1/Y2可扩增出预期大小为434 bp的目的片段;ITS-PCR和ITS-PCR-RFLP结果发现,KC20与P. carotovorum菌株带型均不相同。KC20的16S rDNA基因完整序列与已报道的标准菌株P. aroidearum SCRI 109T(JN600323)的相应序列相似性高达99%以上;该序列系统发育关系分析表明,KC20与已报道的P. aroidearum菌株聚集,并形成了明显的P. aroidearum类群,基于pmrA基因序列和基于果胶杆菌8个看家基因(acnA、icd、gapA、mdh、mtlD、pgi、proA和rpoS)的MLSA-多位点序列分析进一步支持了这一结果。以上结果表明,引发北京地区大白菜软腐病的病原菌株KC20为果胶杆菌P. aroidearum。人工接种条件下,KC20还可侵染马蹄莲、虎眼万年青、马铃薯、鳄梨、西葫芦、胡萝卜、生菜和芹菜等植物;该菌株具备在37℃以及含有7% NaCl培养基中生长的能力,能液化明胶;可利用棉籽糖、纤维素二糖、蜜二糖,不能利用异麦芽酮糖、D-麦芽糖、D-阿拉伯糖、D-山梨醇、菊糖等。大白菜软腐病新病原菌Pectobacterium aroidearum的发现加深了我们对该病害的了解,为该病害的有效防治提供了参考。

Abstract

The bacterial soft rot caused mainly by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most important diseases on Chinese cabbage. A new strain KC20 was isolated from the soft rotted sample of Chinese cabbage in Fangshan district, Beijing. The colonies on LB medium were circular, milky white, translucent with even edges, and produced pits on CVP medium. Pathogenicity test showed that the strain KC20 caused similar soft rot symptoms to those naturally occurred in Chinese cabbage fields. PCR amplification with Pectobacterium-specific primers Y1/Y2 produced an expected amplicon of 434 bp. However, ITS-PCR and ITS-PCR-RFLP differentiated KC20 from the P. carotovorum strains. The entire 16S rRNA gene sequence of KC20 had the highest identity of 99% with the type strain SCRI 109T of P. aroidearum (JN600323). Based on the phylogenetic analyses of the 16S rDNA, pmrA and MLSA of 8 housekeeping genes (acnA, icd, gapA, mdh, mtlD, pgi, proA and rpoS), the strain KC20 was clustered into the P. aroidearum group, indicating that the strain KC20 was belong to P. aroidearum. By artificial inoculation, the strain KC20 was able to cause soft rot symptoms on Cucurbita pepo, Zantedeschia aethiopica, Ornithogalum dubium, Daucus carota, Solanum tuberosum, Persea americana, Lactuca sativa and Apium graveolens. The physiological and biochemical tests showed that the P. aroidearum KC20 could grow at 37℃ and even on high salt medium (7% NaCl), and caused gelatin liquefaction. It was also able to utilize raffinose, cellobiose, and melibiose, instead of palatinose, D-maltose, D-arabitol, D-sorbitol, inulin, etc. As a new soft rot pathogen on Chinese cabbage, the identification and characterization of P. aroidearum strain KC20 help us better understand this disease and provide scienti-fic evidence for further disease management.

关键词

大白菜 /细菌性软腐病 /Pectobacterium aroidearum /病原鉴定 /生物学特性{{custom_keyword}} /

Key words

Chinese cabbage (Brassica rapa L. ssp. pekinensis) /bacterial soft rot /Pectobacterium aroidearum /identification /biological characteristics{{custom_keyword}} /

李晓颖, 田宇, 张瑾, 陈昌龙, 谢华.大白菜软腐病新病原菌Pectobacterium aroidearum的鉴定及其生物学特性[J]. 植物病理学报, 2018, 48(4): 455-465 https://doi.org/10.13926/j.cnki.apps.000136

LI Xiao-ying, TIAN Yu, ZHANG Jin, CHEN Chang-long, XIE Hua.Identification and characterization of a Pectobacterium aroidearum strain causing bacterial soft rot on Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. Acta Phytopathologica Sinica, 2018, 48(4): 455-465 https://doi.org/10.13926/j.cnki.apps.000136

参考文献

[1] Nabhan S, Boer S H D, Maiss E, et al. Pectobacterium aroidearum sp. nov., a soft rot pathogen with pre-ference for monocotyledonous plants [J]. International Journal of Systematic and Evolutionary Microbiology, 2013, 63(7): 2520-2525.
[2] Pritchard L, Glover R H, Humphris S, et al. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens [J]. Analytical Methods, 2015, 8: 12-24.
[3] Onkendi E M, Moleleki L N. Characterization of Pectobacterium carotovorum subsp. carotovorum and brasiliense from diseased potatoes in Kenya [J]. European Journal of Plant Pathology, 2014, 139(3): 557-566.
[4] Tsuda K, Tsuji G, Higashiyama M, et al. Biological control of bacterial soft rot in Chinese cabbage by Lactobacillus plantarum strain BY under field conditions [J]. Biological Control, 2016, 100: 63-69.
[5] Hu N N, Li C, Wang Q, et al. Identification of soft rot pathogens on Chinese cabbage [Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsenet Lee] in Beijing (in Chinese) [J]. Acta Microbiologica Sinica (微生物学报), 2015, 55(10): 1253-1263.
[6] Waleron M, Waleron K, Lojkowska E. First report of Pectobacterium carotovorum subsp. brasiliense causing soft rot on potato and other vegetables in Poland [J]. Plant Disease, 2015, 99(9): 1271.
[7] Zhu L, Xie H, Chen S, et al. Rapid isolation, identification and phylogenetic analysis of Pectobacterium carotovorum ssp. [J]. Journal of Plant Pathology, 2010, 92(2): 479-483.
[8] Fang Z D. Plant Pathology Research Methods (3rd Ed.) (in Chinese) [M]. Beijing: China Agriculture Press (北京:中国农业出版社), 1998: 165-211.
[9] Schaad N W, Jones J B, Chun W. Laboratory guide for identification of plant pathogenic bacteria (2nd Ed.) (in Chinese) [M]. Beijing: China Agricultural Science and Technology Press (北京:中国农业科学技术出版社), 2011: 43-50.
[10]Tian Y, Ma Y L, He F X, et al. Identification and pathogenicity analysis of soft rot bacteria on celery in Beijing (in Chinese) [J]. Acta Phytopathologica Sinica (植物病理学报), 2016, 46(4): 433-442.
[11]Mukherjee A, Cui Y, Ma W, et al. Hexa of Erwinia carotovora ssp. carotovora strain Ecc71 negatively re-gulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone [J]. Environmental Microbiology, 2000, 2(2): 203-215.
[12]Darrasse A, Priou S, Kotoujansky A, et al. PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato disease [J]. Applied and Environmental Microbiology, 1994, 60(5): 1437-1443.
[13]Duarte V, De Boer S H, Ward L J, et al. Characte-rization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil [J]. Journal of Applied Microbiology, 2004, 96(3): 535-545.
[14]Toth I, Avrova A, Hyman L. Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses [J]. Applied and Environmental Microbiology, 2001, 67(9): 4070-4076.
[15]Mohamed K H, Meriam T, Mohamed A, et al. An easy, simple inexpensive test for the specific detection of Pectobacterium carotovorum subsp. carotovorum based on sequence analysis of the pmrA gene [J]. BMC Microbiology, 2013, 13(1): 1-8.
[16]Ma B, Hibbing M E, Kim H S, et al. Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya [J]. Phytopathology, 2007, 97(9): 1150-1163.
[17]Jin Z W, Song X W, Xie X W, et al. Isolation and identification of the pathogen causing celery bacterial soft rot (in Chinese) [J]. Acta Phytopathologica Sinica (植物病理学报), 2016, 46(3): 304-312.
[18]Nabhan S, Boer S H D, Maiss E, et al. Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov. [J]. Journal of Applied Microbiology, 2012, 113(4): 904-913.
[19]Nabhan S, Wydra K, Linde M, et al. The use of two complementary DNA assays, AFLP and MLSA, for epidemic and phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum [J]. Plant Pathology, 2012, 61(3): 498-508.
[20]Gardan L, Gouy C, Christen R. Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. [J]. International Journal of Systematic and Evolutionary Microbiology, 2003, 53(2): 381-391.
[21]Perombelon M C, Wolf M J. Methods for the detection and quantification of Erwinia carotovora subsp. atroseptica on potatoes suitable for commercial use: a laboratory manual [M]. Scottish Crop Research Institute, 2002.
[22]Moretti C, Fakhr R, Cortese C, et al. Pectobacterium aroidearum and Pectobacterium carotovorum subsp. carotovorum as causal agents of potato soft rot in Lebanon [J]. European Journal of Plant Pathology, 2016, 144(1): 205-211.
[23]Moraes A J G, Silveira E B, Mariano R L R, et al. First report of Pectobacterium aroidearum and Pectobacterium carotovorum subsp. brasiliensis causing soft rot of Cucurbita pepo in Brazil [J]. Plant Disease, 2016, 101(2): 379.
[24]Yuan F, Qu S P, Cui C S, et al. A new strain of Erwinia carotovora subsp. carotovora isolated from soft-rotted Chinese cabbage (in Chinese) [J]. Acta Microbiologica Sinica (微生物学报), 2004, 44(2): 136-140.