免套袋栽培苹果采后真菌病害鉴定及精油抑菌研究

Fungal pathogens identification of postharvest apples in bagging-free cultivation pattern and antifungal activity of essential oil

【目的】苹果免套袋栽培是我国苹果产业发展的必然趋势,而在此模式下,苹果的病虫害发生较为严重。探究免套袋苹果采后真菌的发病情况以及精油对其抑菌效果,为免套袋苹果病虫害的绿色防控奠定基础。【方法】分别调查山东苹果主产区的免套袋、套袋2种栽培模式下苹果的采后发病情况;采用传统形态鉴定结合ITS序列比对对免套袋苹果致病真菌进行了分类统计及种属鉴定;熏蒸处理评价了肉桂精油对致病菌的离体抑菌效果,并通过电子显微镜分析了其抑菌机制。【结果】免套袋苹果的采后发病率显著高于套袋苹果(p<0.05),免套袋苹果果实上共分离鉴定出6种病原真菌,分别为葡萄座腔菌(Botryosphaeria dothidea)、链格孢属(Alternaria)、粉红单端孢菌(Trichothecium roseum)、韦司梅拟盘多毛孢菌(Pestalotiopsis vismiae)、胶孢炭疽菌(Colletotrichum gloeosporioides)及产核枝顶孢菌(Acremonium sclerotigenum)。80μL·L^(-1)的肉桂精油对以上6种真菌的抑菌率均超过80%。精油对苹果的活体防治试验表明,80μL·L^(-1)肉桂精油可显著降低苹果果实的发病率(p<0.05)。经显微镜分析可知,精油抑制了孢子萌发,改变了病原菌的正常菌丝形态。【结论】肉桂精油作为免袋栽培苹果采后防腐抑菌剂有较好的应用前景。

【Objective】Apple occupies a dominant position in fruit production of China.Growing interests exist in examining the potential of non-bagging-fruit cultivation pattern because of the decline of internal quality of bagging-apple fruits and the increase of labor cost as well as the environmental pollution.It is recognized that apple-bagging free cultivation is an inevitable trend in the development of China’s apple industry.However,with non-bagging-fruit cultivation pattern,some problems still occur such as serious pathogens and insects.In order to identify pathogens types and illuminate occurrence regularity of postharvest fungal diseases of non-bagging apples,we aimed to investigate the incidence of postharvest decay of bagging and non-bagging apple fruits in the main apple production areas of Shandong Province,and evaluate the efficacy of essential oils as a green antibacterial method.【Methods】Traditional morphological identification method combined with ITS analysis were performed for identification of the pathogenic fungi strains isolated from rotten non-bagging apples.Then,based on the previous results,we optimally selected cinnamon essential oil to assess its antifungal activity by in vitro and in vivo fumigation against such strains.Then,antifungal mechanism of cinnamon essential oil was assessed through determining the main chemical components by gas chromatography/mass spectrometry(GC/MS)analysis,and by observing spore germination,configuration of mycelia cells and ultrastructural alterations through scanning electron microscope(SEM)and transmission electron microscopy(TEM),respectively.【Results】The postharvest infection incidence of non-bagging apples was significantly higher than that of the bagging apples(p<0.05).Six strains were isolated and identified as Botryosphaeria dothidea,Alternaria,Trichothecium roseum,Pestalotiopsis vismiae,Colletotrichum gloeosporioides and Acremonium sclerotigenum according to the analysis on morphology and molecular biology.The results of GC/MS analysis showed that there were two major kinds of compounds in cinnamon essential oil we used,respectively,benzaldehyde(4.577 min)and trans-cinnamaldehyde(9.229 min),and the lateraccounted for 69.5%.Cinnamon essential oil exhibited obvious antifungal effect on all of the six strains,and the inhibitory rates could reach to 80%at 80μL·L^(-1) concentration and 100%at 100μL·L^(-1) concentration except for C.gloeosporioides.For T.roseum and Alternaria,the inhibitory rates of cinnamon essential oil were more than 90%at 20μL·L^(-1) concentration.Based on the radial growth of mycelium on the medium,the fumgitoxic ability of cinnamon essential oil was observed in a dose-dependent manner,which was not as dramatic as that under in vivo conditions.In the concentration range of 20-80μL·L^(-1),with the increase of concentration,the decay rates showed a downward trend.The decay rate of 80μL·L^(-1) treatment decreased by 33%compared with the control,while at 100μL·L^(-1),the decay rate was increased significantly compared with that at 80μL·L^(-1)(p<0.05),and we speculatde that the excessively high concentration of cinnamon essential oil would cause certain phytotoxicity to the fruits.We choosed B.dothidea,which is a serious hazard for bagging-free apples before and after harvesting,to analyze the effects of cinnamon essential oil treatments on its spore germination,hyphae morphology and cell internal structures.Cinnamon essential oil effectively inhibited the germination of B.dothidea spores and the elongation of thebud tubes.The germination rate of spores with 400μL·L^(-1) treatment decreased to 6%,and the length of the bud tube was reduced by 94.51%at 600μL·L^(-1) compared with the control group.SEM of B.dothidea exposed to cinnamon essential oil showed irreversible deleterious morphological such as lack loss of integrity and rigidity of the cell wall,folding of the cell,and shrink in appearance on the surface of mycelium compared to those of the control group.【Conclusions】In the non-bagging cultivation mode,the incidence of postharvest pathogenic fungi was significantly higher than that of bagging apples.Cinnamon essential oil exhibited excellent antibacterial effect both in vitro and in vivo,and was proved to be a promising candidate as safe and effective antifungal agent for postharvest fungal spoilage of non-bagging apples.It can be considered to further study the comprehensive control efficiency of the cinnamonon the diseases during the postharvest storage of bagging-free apples combining with cloves or other essential oils.In practical applications,it is recommended to innovate the load form or embedding technology to further improve the stability of the essential oils.