香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力

Screening of Endophytic Antagonistic Bacteria from‘Kuerlexiangli’Pear and Their Biocontrol Potential Against Fire Blight Disease

该研究以健康香梨树的新鲜花器、当年生新生枝条、叶片和果实为试验材料,采用植物组织内生细菌分离法获得内生细菌菌株,培养菌落,并采用平板对峙法初筛和发酵液复筛对梨火疫病菌(Erwinia amylovora)、梢枯病菌(Pseudomonas syringae pv.syringae)和腐烂病菌(Valsa mali var.pyri)具有抑菌作用的细菌;将筛选获得的拮抗梨火疫病菌的菌株通过喷施香梨离体花序和盆栽杜梨苗测定其防病效果,以发掘内生细菌的生防资源,为探索梨树病害的生物防治途径奠定基础。结果显示:(1)从香梨枝条、叶、花和果实组织中共分离出337个内生细菌菌株,其中有14株具有明显的抑菌作用;进一步筛选出对梨火疫病菌、梢枯病菌和腐烂病菌具有抑菌作用的细菌分别有8株、9株和4株,其中ZN5、SN19和HN9菌株对梨梢枯病菌和腐烂病菌均有拮抗作用,HN89菌株对3种病原菌都具有较强的抑菌作用;共筛选出对梨火疫病菌具有抑菌作用的7个菌株(SN37、HN89、HN98、HN143、HN126、TN68、TN16)和具有竞争作用的TN50菌株。(2)内生菌株TN50预处理(喷施)对预防香梨花腐的效果显著,其防效(52.36%)与农用链霉素(60.67%)相近;HN89的防效(39.66%)其次;内生菌株TN50、HN89和SN37预处理(喷施)能够显著降低盆栽杜梨苗嫩枝的枝枯率和病情指数(P<0.05),7~15 d的平均保护性防效分别为67.20%、54.32%和45.91%,治疗性防效分别为63.88%、52.10%和36.17%。(3)结合形态特征和16S DNA序列分析,TN50、HN89和SN37菌株分别鉴定为克雷伯氏菌属(Klebsiella sp.)、类芽孢杆菌属(Paenibacillus sp.)和假单胞杆菌属(Pseudomonas sp.)。

In this study,the fresh flower apparatus,new branches,leaves and fruits of healthy‘Kuerle-xiangli’pear trees were used as experimental materials,we obtained endophytic bacteria strains and culture colonies with the method of endophytic bacteria isolation in plant tissues.The bacteria with bacteriostatic effect on Erwinia amylovora,Pseudomonas syringae pv.syringae and Valsa mali var.pyri were screened by plate confrontation culture and re-screening of fermentation broth.We tested the control effect of antagonistic strains against pear fire blight by spraying‘Kuerlexiangli’pear inflorescences in vitro and potted birch pear seedlings,in order to explore the biocontrol resources of endophytic bacteria and lay a foundation for exploring the biological control ways of pear diseases.The results showed the following:(1)a total of 337 endophytic bacterial strains were isolated from branches,leaves,flowers and fruit tissues of‘Kuerlexiangli’pear,of which 14 strains had obvious bacteriostatic activity.8,9 and 4 strains of bacteria with bacteriostatic effect on E.amylovora,P.syringae pv.syringae and V.mali var.pyri were further screened.Among them,ZN5,SN19 and HN9 strains had antagonistic effect on P.syringae pv.syringae and V.mali var.pyri pathogen,while HN89 strain showed potent bacteriostatic effect on three kinds of pathogens.A total of 7 strains(SN37,HN89,HN98,HN143,HN126,TN68 and TN16)with bacteriostatic effect against E.amylovora and competitive strains TN50 were screened.(2)The pretreatment(spraying)of endophytic strain TN50 had a significant effect on preventing flower rot of‘Kuerlexiangli’pear,and its control effect(52.36%)was similar in agricultural streptomycin(60.67%);followed by HN89(39.66%).The pretreatment(spraying)of endophytic strains TN50,HN89 and SN37 could significantly reduce the branch withering rate and disease index of potted birch pear seedlings(P<0.05).The average protective control effect of 7~15 d was 67.20%,54.32%and 45.91%,respectively,and the therapeutic control effect was 63.88%,52.10%and 36.17%,respectively.(3)Based on the morphological characteristics and 16S DNA sequence,TN50,HN89 and SN37 were identified as Klebsiella sp.,Paenibacillus sp.and Pseudomonas sp.,respectively.