湖南牡丹根腐病病原菌的分离鉴定及其拮抗菌的筛选

Isolation and identification of pathogen of peony root rot in Hunan and screening of antagonistic bacteria

【目的】引发牡丹根腐病的镰刀菌属真菌种类繁多,且病害症状类似,难以判断。为了探明引发湖南地区牡丹根腐病主要病原菌的种类,并为牡丹根腐病的诊断与生物防治提供理论依据。【方法】从湖南农业大学园林花卉基地采集患病牡丹植株,采用组织分离法分离纯化病原菌,采用柯赫氏法则检验病菌分离物的致病性;分别以形态学、分子生物学方法对致病菌进行鉴定;采用平板对峙法对14株前期从健康牡丹根茎部分离获得的内生细菌菌株进行抑菌性筛选。【结果】在对分离所获12株病原真菌分离物的致病性的检测中发现,其中的5株在PDA平板上的侧根变黑腐烂且其周围菌丝生长旺盛,其致病性均强;将此5株致病菌置于PDA平板上培养7 d后观察发现,其菌落呈圆形,颜色呈灰白色,气生菌丝较密集且呈革质胶状;致病菌菌丝中含有大量的大、小型分生孢子,大型分生孢子呈镰刀形,具有3~5个隔膜,小型分生孢子呈卵形或肾形,具有0~1个隔膜;厚垣孢子呈球形且多为间生状。结合运用分子生物学方法,基于ITS序列分析,构建了5株致病菌的系统发育树,5株分离物全部被鉴定为茄病镰刀菌Fusarium solani。平板对峙培养结果表明,14株内生细菌菌株中有9株能拮抗茄病镰刀菌,其中编号为MRX2-1的细菌菌株其拮抗效果最佳,达到了52.78%;基于16S rRNA基因序列对此株菌株的进一步分析结果表明,编号为MRX2-1的拮抗菌与Bacillus amyloliquefaciens(HM107808.1)聚于同一分支中,其相似度高达99%以上。【结论】初步鉴定结果表明,湖南地区牡丹根腐病的主要病原菌为茄病镰刀菌,最佳拮抗菌为解淀粉芽孢杆菌。

【Objective】There are various types of Fusarium fungi that cause peony root rot,and the symptoms of the disease are similar,making it difficult to determine.In order to identify the main pathogenic bacteria causing peony root rot in Hunan region and provide theoretical basis for the diagnosis and biological control of peony root rot.【Method】Collect diseased peony plants from the garden and flower base of Hunan Agricultural University,use tissue isolation method to isolate and purify pathogenic bacteria,and use Koch’s rule to test the pathogenicity of the bacterial isolates;Identify pathogenic bacteria using morphological and molecular biology methods;Antibacterial screening was conducted on 14 endophytic bacterial strains isolated from the roots and stems of healthy peonies using the plate confrontation method.【Result】In the detection of the pathogenicity of 12 isolated pathogenic fungi,it was found that 5 of them had black and decayed lateral roots on the PDA plate,and their surrounding hyphae grew vigorously,all of which had strong pathogenicity;After culturing these 5 pathogenic bacteria on a PDA plate for 7 days,it was observed that their colonies were circular,grayish white in color,and the aerial hyphae were dense and leathery in shape;The mycelium of pathogenic bacteria contained a large number of large and small conidia,which were sickle shaped and had 3-5 septa.Small conidia were oval or kidney shaped and had 0-1 septa;The chlamydospores were spherical in shape and mostly interstitial.Combining molecular biology methods and based on ITS sequence analysis,phylogenetic trees of 5 pathogenic bacteria were constructed,and all 5 isolates were identified as F.solani.The results of plate culture showed that 9 out of 14 endophytic bacterial strains were able to resist F.solani,among which the bacterial strain MRX2-1 had the best antagonistic effect,reaching 52.78%;Further analysis of this strain based on the 16S rRNA gene sequence showed that the antagonistic bacterium MRX2-1 and Bacillus amyloliquefaciens(HM107808.1)clustered in the same branch,with a similarity of over 99%.【Conclusion】The preliminary identification results indicate that the main pathogen of peony root rot in Hunan region is F.solani,and the best antagonistic bacterium is B.amyloliquefaciens.