Preliminary Study On The Migration Patterns Of Penicillium Expansum And Its Toxins In Apples At Different Temperatures

Blue mold caused by Penicillium expansum is one of the diseases of apple during storage and transportation,which brings a lot of economic losses to the apple industry.Patulin,one of the secondary metabolites of P.expansum,is toxic in many ways and is widely found in apples and their products,and long-term consumption of food contaminated with patulin is hazardous to human health.In daily life,apple storage is usually normal temperature or cold storage,but P.expansum can be grown at a wide range of temperatures,from-6℃ to 35℃.At present,the rule of P.expansum infestation and migration in apples at different temperatures and the metabolic changes after P.expansum infestation in apples are not yet clear.Therefore,in this study,P.expansum and apple were used as test materials.Firstly,Propidium Monoazide（PMA）and Quantitative real-time Polymerase Chain Reaction（qPCR）were developed to rapidly detect viable mould of P.expansum to investigate the migration rule of P.expansum.Then,we investigated P.expansum and toxin migration rules at different temperatures by detecting lesion diameter,toxins and related to patulin synthesis genes,and conducted a preliminary study on the metabolic changes of P.expansum-infested apples at different temperatures by using metabolomics techniques.The results of this study are as follows:（1）By optimizing PMA concentration,dark incubation and exposure time,screening specific primers of P.expansum,combined with qPCR technology,a rapid detection method of live mould of P.expansum,based on PMA-qPCR was established.Finally,it was found that PMA treatment concentration of 10μg/mL,dark incubation for 5 min,exposure for 10 min were the best conditions for PMA treatment.Among the 4 primers,Pexp-pat F showed strong specificity to P.expansum,which could be used as primers for PMA-qPCR detection.The R²of the constructed quantitative standard curve was 0.9948,and the minimum detection limit was 10^2.6 CFU/mL.There was no significant difference between the detection result of the method and the plate colony count,and P.expansum could be detected in the no rot part of the apple,it suggests that P.expansum migrates towards healthy apple tissue.（2）The apples were artificially inoculated with P.expansum and cultured at room temperature or refrigerated（4℃～8℃）.The content of patulin was determined by high performance liquid chromatography.It was found that the diameter of the lesions in cold storage for 31 d was the same as that in room temperature for 9 d,but the content of patulin in cold storage at the 31 d was 16 times higher than that in room temperature at the 9 d.At the same time,patulin was detected 5 mm away from the lesions,and the content reached 10%of the lesions,is 7.99μg/g,which beyond the minimum national standard limit;The relative expression levels of PatK and PatJ of related to patulin synthesis genes were detected at room temperature and their correlation with patulin content was analyzed.It was found that the expression of PatK and PatJ of related to patulin synthesis genes was significantly correlated with the content of patulin.Expression of the related to patulin synthesis genes PatK and PatJ was found at sites other than the lesion at 11 d incubation at room temperature and was inversely proportional to the distance from the lesion.The results showed that cold storage culture could delay the growth rate of P.expansum,but could not prevent the synthesis of patulin.It could only delay and increase the accumulation of patulin content and the migration of patulin to healthy tissue of apple.（3）Metabolomics was used to study the apples stored at room temperature for 7 d after no treatment,simulated inoculation and inoculation of P.expansum.It was found that 75metabolite differences were detected in the lesions of the inoculation group,and 12 metabolic pathways were significantly changed.The overall trend of flavonoid biosynthesis pathway at the lesions in the inoculation group was upregulated,ABC transporter protein pathway confusion.This may be related to the impaired cell membrane transport in fruit and related to plant defence responses after P.expansum infestation;The difference metabolite quantity of apple tissue 0.5,1,2,3 cm away from the lesion was inversely proportional to the lesion distance,and the more differential metabolites are upregulated.Among them,the overall trend of flavonoid and flavonol biosynthesis pathway in the tissues within 2 cm from the lesion was up-regulated.Instruction that after 7 d of P.expansum infestation of apples in culture,flavonoids may be secreted in fruit tissue within 2 cm of the spot to defend against P.expansum infestation.（4）Metabolomics were also used to detect and compare the metabolites in infected apples with the same lesions diameter at different temperatures（incubated at room temperature for 7d and refrigerated for 24 d）.Compared with that cultured at room temperature for 7 d,83different metabolites were detected after 24 d of cold storage.In cold storage culture.66metabolites,including organic acid metabolites,lipid metabolites,flavonoid metabolites,were down-regulated,and 17 metabolites such as patulin,gluconic acid and betaine were up-regulated.Differential metabolites were involved in 31 metabolic pathways,of which 7metabolic pathways were significantly different,including flavonoid biosynthesis,pyruvate metabolism,pentose phosphate pathway,phenylalanine,tyrosine and tryptophan biosynthesis,and flavone and flavonol biosynthesis,et al.The contents of gluconolactone and gluconic acid metabolites in cold storage culture were significantly higher than those in room temperature culture,which was speculated that the accumulation of gluconin content was related to the metabolism of gluconic acid and other substances.