贵长猕猴桃花腐病病原鉴定及防控药剂筛选

Identification of the pathogens of blossom blight and screening of fungicides(bactericides)in Guichang kiwifruit

【目的】明确猕猴桃花腐病的致病病原及其防控药剂。【方法】从贵长猕猴桃发病花蕾中分离获得病原菌,通过致病性测定、形态学观察、生物学特性测定,以及16S r DNA、rpo D、gyr B、glt A和dna A基因联合分析鉴定,同时测定11种杀菌剂和组合药剂对该病原菌的抑制效果。【结果】从病样中分离获得的菌株G-2为猕猴桃花腐病病原,根据形态学观察、生理生化检测及分子鉴定将其鉴定为绿黄假单胞菌Pseudomonas viridiflava;毒力测定结果显示,供试11种药剂中四霉素对该病原菌的抑菌活性最高,EC50为1.24 mg·kg^(-1),其次为丙硫唑,EC50为9.62 mg·kg^(-1);四霉素与丙硫唑以有效质量比4∶1、3∶1时,共毒系数(co-toxicity coefficient,CTC)分别为272.70、129.86,具有增效作用。【结论】猕猴桃花腐病的病原菌为绿黄假单胞菌Pseudomonas viridiflava,对四霉素和丙硫唑及组合药剂均有敏感性。

【Objective】The disease damages kiwifruit buds,which have been found in Xiuwen county of Guizhou province.It causes the calyx to show dark brown,the filaments and anthers to become rot,which is called blossom blight of kiwifruit.It has so significant effect on the opening and pollination of the flower buds that it can result in a decline in the fruiting set rate and economic losses.It is important to determine the pathogen of Guichang kiwifruit flower rot.To screen out fungicides(or bactericides)that possess high-efficiency and low-toxic properties for prevention and control for blossom blight of kiwifruit,the experiment was carried out,so that the economic loss caused by the disease could be reduced.【Methods】The diseased symptomatic tissues were surface-disinfected with 75%ethanol for 3-4s and rinsed three times in sterile distilled water,and then cultured on the beef extract peptone medium(NA)with 75%relative humidity at 25℃.The isolated pathogens were tested for the pathogenicity of kiwifruit buds by using the local inoculation method,and the pathogenicity of the pathogens to Nicotiana benthamiana,Lycopersicon esculentum and Apium graveolens L.were tested with another method.The morphology of the colony on NA medium was observed,after Gram staining,capsule staining,spore observation,and scanning electron microscopy as the basis for morphology.LOPAT test was undertaken by gelatin liquefaction,starch hydrolysis,glucose oxidation fermentation,malonic acid utilization,citrate utilization,esculin hydrolysis,hydrogen peroxide and phenylalanine deaminase,fluorescence,methyl red and V-P test,whether it can use sorbitol,mannitol,glucose,ribotide,erythritol and sucrose,acting as a strong basis for researches on physiological and biochemical characteristics.The PCR amplification used16S ribosomal DNA,DNA replication initiation protein(dna A),DNA gyrase B subunit(gyr B),RNA polymerase sigma-70 factor(rpo D)and citrate synthase gene(glt A)primers.NCBI’s BLASTn tools were used to obtain highly homologous DNA sequence in Gen Bank and molecular phylogenetic tree was constructed by MEGA 7.0.14.The bacteriostatic zone method was used to screen the virulence of 11 kinds of fungicides(bactericides)and their combination against blossom blight of kiwifruit,so as to obtaine the related toxicity regression equation.【Results】Four days after inoculation,artificially infected flowers showed symptoms alike those observed in the farm,whereas the control was asymptomatic.After the same measurement was used,which was described above with the original strains,the bacteria were isolated and purified from the infected tissues.Inversely,it was not isolated from the control,which fully conformed to the verification of Koch’s Postulates.After the fruits,stems and leaves were infected by Nicotiana benthamiana,Lycopersicon esculentum and Apium graveolens L.,the necrotic spots and pus produced.The pathogen was off-white,smooth and translucent with neat edges on NA medium.It was a gram-negative bacteria with the size of(2.32^(-1).77)μm×(0.497-0.663)μm.The strain belonging to the LOPAT II group(--+-+,L negative,O negative,P variable,A negative,and T positive)did not produce spores and had capsule.It was capable of producing fluorescent pigments,causing fermentation of glucose,using citrate and malonic acid,and slightly liquefying gelatin and hydrolyzing esculin.However,it could not hydrolyze starch.Catalase peroxide was positive and phenylalanine deaminase was negative.The results of methyl red and V-P showed that the former was positive and the latter was negative.It can utilize glucose,sucrose,erythritol and sorbitol as carbon source except ribonic acid and mannitol.BLAST on the NCBI official website was used for analysis of the sequence that used universal primer 27F/1492R.The results showed that strain G-2(Gen Bank accessions:MT950156)and Pseudomonas viridiflava(Gen Bank accessions:AY180972.1)were 100%,Pseudomonas graminis were out-of-group strains in the periphery.The primers rpo D-FP/RP,gyr B-F/R,cts F/R and M209 F/R were used for PCR amplification and comparative analysis of strain G-2.The phylogenetic tree showed that strain G-2(Gen Bank accessions:rpo D,MT975512;gyr B,MT994325;glt A,MT975511;dna A,MT975513)cannot be distinguished from other sources of Pseudomonas viridiflava,but they were all in the same group.The susceptibility of the bacteria to bactericides(fungicides)showed that tetramycin had the highest antibacterial activity against the pathogen among the 11 kinds of fungicides,with an EC50of 1.24 mg·kg^(-1),followed by prothioazole with an EC50of 9.62 mg·kg^(-1).Both bactericides(fungicides)were combined,the CTC values were 272.70 and 129.86 when the effective mass ratio was 4∶1 and 3∶1.This result suggested that it had a synergistic effect.【Conclusion】It was proved that the pathogen of blossom blight of kiwifruit in Guizhou province is P.viridiflava through morphology,combined with biological characteristics and molecular biological methods.Tetramycin,prothiazole and other effective prevention and control fungicides(bactericides)and combination agents were screened out by the test.But the final control effect of the agent still needs to be verified in the field.This study provides a theoretical basis for the development of rapid detection technology that prevents blossom blight of kiwifruit in advance.