福建番石榴环斑病菌的分离鉴定、生物学特性及抑制剂筛选

Isolation,identification,biological characteristics and inhibitors screening of pathogen causing ring rot disease of guava fruit in Fujian province

【目的】明确番石榴果实采后环斑病发生的致病病原菌及其对番石榴果实采后品质的影响,筛选能抑制该病原菌的有效抑制剂。【方法】病原菌分离于发生环斑病的番石榴果皮病健交界处,并对该致病菌进行形态学鉴定、分子鉴定与系统发育树分析。另外,初步研究番石榴环斑病菌的生物学特性、环斑病菌侵染对果实采后品质的影响和评价不同抑制剂(ε-聚赖氨酸、水杨酸和褪黑素)对环斑病菌的体外抑菌效果。【结果】根据环斑病菌的菌丝与分生孢子的形态特征及基于rDNA-ITS、TUB和TEF-1α测序结果构建的系统发育树,将福建番石榴环斑病菌鉴定为棒状新拟盘多毛孢(Neopestalotiopsis clavispora)。葡萄糖和D-果糖、蛋白胨、7、25℃分别作为番石榴N. clavispora菌丝生长的最适碳源、氮源、pH、温度。此外,与未接种N. clavispora番石榴果实相比,接种N. clavispora果实具有较高的病斑直径和细胞膜透性,较低的果实硬度和色调角h值。体外试验表明,适当浓度的ε-聚赖氨酸、水杨酸和褪黑素处理对番石榴N. clavispora的菌丝生长有明显的抑制作用,可作为抑制N. clavispora侵染所致番石榴果实采后环斑病的抗菌剂。【结论】引起福建番石榴果实环斑病的病原菌为棒状新拟盘多毛孢(N. clavispora),且4.000 mg·mL^(-1)ε-聚赖氨酸对病原菌具有很好的抑制效果,可为后续番石榴环斑病的防治研究提供科学依据。

【Objective】Guava,acting as a characteristic subtropical fresh fruit in southern China,is rich in vitamin C,dietary fiber and other nutrients,which has the good nutritional value and medicinal ef-fects.However,in the harvest season at high temperature and with high humidity,the postharvest guava fruit is prone to the fungal diseases,resulting in the disease infection and fruit quality degradation.The occurrence of ring spot disease is a serious postharvest disease for guava fruit,which has seriously re-stricted the development of guava industry in Fujian province.The symptom of ring spot disease of gua-va fruit generally occurs in one week of storage at normal temperature,and the disease incidence of gua-va fruit Xiguahong is higher(about 30%),which has seriously restricted the maintenance of guava fruit quality after harvest.Therefore,the aim of this study was to isolate and identify the pathogen causing ring spot disease of guava fruit cv.Xiguahong in Fujian province and its biological characteristics,to study the effects of the infection of the pathogen on the postharvest quality attributes of guava fruit,and to explore the effects of key inhibitors ofε-poly-L-lysine(ε-PL),SA(salicylic acid)and melatonin(MT)on the inhibition rate of pathogen in vitro,so as to provide the theoretical references for control-ling the ring spot disease and stabilizing the quality attributes of postharvest guava fruit.【Methods】(1)The pathogen was isolated from the rotten guava fruit,and identified via using the morphological char-acteristics,like mycelial morphology and conidium morphology,molecular identification and phyloge-netic tree analysis based on sequences of rDNA-ITS,TUB and TEF-1α.(2)The biological characteris-tics of mycelia growth,including the different conditions of carbon source,nitrogen source,pH value and temperature,of the pathogen causing ring spot disease of guava fruit were studied.(3)The Xigua-hong guava fruit was treated in the following two methods after washing and disinfection:Guava fruit was inoculated by 20 microliters of pathogen spore suspension at the concentration of 1×105 spores·mL-1 and treated with sterile distilled water(control group),severally.After inoculation,the treated guavas were stored at 28℃with 90%relative humidity for 6 days.During storage,the effects of pathogen causing ring spot disease on the quality attributes(e.g.,lesion diameter,fruit firmness,cell membrane permeability or hue angle h value)of harvested guava fruit were measured.(4)The antifungal effects of different inhibitors containingε-PL(0,0.125,0.250,0.500,1.000,2.000 and 4.000 mg·mL-1),SA(0,0.500,1.000,1.500 and 2.000 mg·mL^(-1))and MT(0,1.000,2.000,4.000 and 8.000 mg·mL-1)on the my-celia growth and inhibition rate of pathogen causing ring spot disease of guava fruit in vitro were evalu-ated,and thus the optimal inhibitor was screened out.【Results】(1)According to the morphological characteristics of mycelium and conidium,and the homology of more than 98%with similar strains based on phylogenetic tree by rDNA-ITS,TUB and TEF-1αsequences,the pathogen causing ring rot disease of harvested guava fruit in Fujian province was identified as Neopestalotiopsis clavispora.(2)The results of biological characteristics displayed that the glucose and D-fructose,peptone,7,and 25℃were the optimum conditions of carbon source,nitrogen source,pH value and temperature for mycelia growth of N.clavispora in guava fruit,separately.(3)Compared with non-N.clavispora-inoculated gua-va fruit,N.clavispora-inoculated guava fruit revealed the higher levels of lesion diameter and cell mem-brane permeability,but the lower values of fruit firmness and hue angle h.Specially,on the sixth day of storage,the lesion diameter and cell membrane permeability in N.clavispora-inoculated guava fruit were 29.41 times and 1.39 times more than those of non-N.clavispora-inoculated guava fruit,respective-ly,while the fruit firmness or hue angle h value of N.clavispora-inoculated guava fruit were 66.96%or 91.10%of non-N.clavispora-inoculated guava fruit,severally.(4)The treatments of appropriate concen-trations of different inhibitors,such asε-PL,SA and MT,showed the obvious inhibitory effects on the mycelia growth of N.clavispora in vitro,which could be used as the potential technologies for inhibiting N.clavispora-induced ring spot disease of postharvest guava fruit.Further comparison showed that the inhibitory effect of 4.000 mg·mL-1ε-PL was the best,which could basically inhibit the mycelia growth of N.clavispora of guava fruit.【Conclusion】N.clavispora was the pathogen causing the ring spot dis-ease of harvested guava fruit in Fujian province.The biological characteristics of N.clavispora of gua-va fruit were also obtained.The infection of N.clavispora could accelerate the occurrence of fruit dis-ease,and seriously reduce the quality properties in guava fruit after harvest during the entire storage.The key inhibitors such asε-PL,SA and MT could be used as the potential technologies for inhibiting N.clavispora-induced ring spot disease of postharvest guava fruit.Therefore,these results might pro-vide a scientific basis for controlling the postharvest decay and extending the storage time for guava fruit.