Action Effect And Mechanism Of Melaleuca Alternifolia Oil Against Aspergillus Niger And Aspergillus Ochraceus In Grape Berries

Aspergillus niger and Aspergillus ochraceus are pathogens that widely distributed in grape berries.Infection of A.niger and A.ochraceus mostly occurs after the harvest,and during storage and marketing,leading to tremendous economic loss in the grape industry.It is well known that physical preservatives and chemical fungicide treatments are the main traditional antifungal methods for controlling postharvest diseases.Nevertheless,due to their potential danger on human health,new alternative methods of controlling postharvest diseases with good efficacy,little or no toxicity,and low residues have become items of increasing focus and interest for researchers.Plant essential oils and their components are natural volatile liquids extracted from plant material.Melaleuca alternifolia oil(MAO)not only has good antifungal effects,but also has a mild smell that doesn't affect the sensory property of preserved products.Therefore,we hope to apply MAO to the postharvest preservation of grapes.In this paper,the inhibition effects of MAO against A.niger and A.ochraceus on grapes were studied,and explained from the perspective of metabonomics and molecular biology of grape berries.The chemical components of MAO were analyzed to further explore the antifungal mechanisms of MAO on A.niger and A.ochraceus.The research results include the following three aspects:1.MAO at 2.5 mL L-1 almost completely block the mycelial growth and spore germination of A.niger and A.ochraceus.2.0 mL L-1 MAO significantly inhibited decay in grapes caused by A.niger or A.ochraceus.The effects of Aspergillus and MAO on contents of sugars and stilbenoids,and metabolic pathways in grape berries were studied by metabolite analysis through high performance liquid chromatography quadrupole time of flight mass spectrometry(HPLC-QTOF-MS2)and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry(UHPLC-QQQ-MS2).The contents of sucrose,glucose,fructose,trans-resveratrol and pterostilbene,and tricarboxylic acid cycle pathway,glycolysis pathway,pentose phosphate pathway,and phenolic metabolic pathway in berries were down-regulated due to the infection of A.niger or A.ochraceus,which was alleviated by MAO.2.Quantitative Real-time PCR(RT-qPCR)was carried out to analyze expression of corresponding key genes on the metabolic pathways.Both A.niger and A.ochraceus infection resulted in down-regulation of HK,PFK-1 and PK in the glycolysis pathway,and G6PDH2 and 6PGDH2 in pentose phosphate pathway of grape berries.STS and ROMT were up-regulated by A.niger or A.ochraceus.In addition,A.niger infection also resulted in down-regulation of C4H,4CL,F3'H,DFR and LDOX in phenylpropanoid pathway.While A.ochraceus infection induced down-regulation of expression of the key genes GLTA,IDH,a-KDG,DLST and DLD in tricarboxylic acid cycle pathway,and key genes VviCHS1,VviCHll and VviF3H1 in phenolic metabolic pathway of grape berries.The down-regulation of these genes expression was reduced by MAO.The changes in genes expression were consistent with the changes in corresponding metabolic pathways.3.Chemical compositions of MAO were analyzed by gas chromatography-mass spectrometry,with terpene-4-ol,?-terpineol and 3-carene identified as its main components.The inhibitory effects of MAO and its main constituents against A.niger and A.ochraceus were compared.?-terpineol and terpene-4-ol exhibited the greatest effectiveness,with the minimum inhibitory concentration of 0.8 and 2.0 mL L-1,respectively.The most serious destruction to cytomembrane permeability,and interference in microscopic morphology of hyphae and spores,as well as metabolic pathways of A.niger and A.ochraceus,was strongest with these two components--even stronger than complete MAO treatments.Histidine degradation pathway,asparagine degradation pathway,cleavage pathway of 2-keto-3-deoxy-6-phosphogluconate,and tricarboxylic acid cycle in A.niger were significantly down-regulated by ?-terpineol,terpene-4-ol and MAO.The cleavage pathway of 2-keto-3-deoxy-6-phosphogluconate,galactose degradation pathway,D-fructofuranose-6-phosphate synthetic pathway,and pyrimidine deoxynucleoside degradation pathway in A.ochraceus were significantly down-regulated by ?-terpineol,terpene-4-ol and MAO.3-carene showed little inhibitory effect.?-terpineol and terpene-4-ol also possess the best preservative effects which combat against aspergillus disease caused by A.niger and A.ochraceus in postharvest grapes.Overall,a-terpineol and terpene-4-ol possess strong antimycotic effects which contribute largely to the antifungal activity of MAO.Further studies on evaluating the effectiveness of combined usage of purified a-terpineol and terpene-4-ol against aspergillus growth in different foods should be implemented to better control the risk of aspergillus-mediated diseases.