| Litchi(Litchi chinensis Sonn.)is one of the most commercially important fruit cultivated widely in subtropical areas with good flavor and rich nutrient.However,the high temperature and humidity during harvest provided suitable condition for pathogen infection,which might lead to pericarp browning and fruit decay,thus limit the transportation,storage and marketing of litchi fruit.The causal pathogen of litchi downy blight,Peronophythora litchii(P.litchii),caused about 20%waste of litchi production,which had led to great economic lose.In order to avoid the disadvantages of abuse of artificial chemical fungicides,biological control which had shown great potential for inhibition of harvested litchi fruit disease,attracted much concerns.Biocontrol technology could inhibit the growth of pathogens,regulate the physiological metabolism of fruits,therefore improve the postharvest quality of fruit and prolong the shelf life.Our research group has isolated an antagonist B.amyloliquefaciens strain LY-1 with wide antifungal spectrum which showed strong inhibitory effect on the postharvest disease of longan fruit.In this study,we focused on the inhibition of B.amyloliquefaciens LY-1 on P.litchii in vitro and the effects of B.amyloliquefaciens LY-1 culture broth(BLCB)on inhibition of litchi downy blight caused by P.litchii associated with different metabolisms of litchi pericarp in vivo.Also,the effects of 1.0×108 CFU mL-1 BLCB treatment on physiology and storability of harvested litchis at 25±1℃ and 85～90%RH had been investigated.Meanwhile,the functional genes related to its antifungal activity were analyzed based on genome sequencing.1.B.amyloliquefaciens LY-1 inhibited the growth of P.litchii mycelium and sporangium germination effectively and increased the membrane peroxidation and damage of P.litchii mycelium in vitro.The membrane permeability,the protein and nucleic acid leakage were increased.Moreover,the whole culture broth of B.amyloliquefaciens LY-1 showed optimal inhibitory effect.2.The result of pre-test showed that the whole culture broth,cell-free supernatant and bacterial suspension of B.amyloliquefaciens LY-1 could effectively reduce the disease index and pericarp browning index of P.litchii-inoculated litchi fruit.Moreover,the whole culture broth of 1.0×108 CFU mL-1 LY-1 showed optimal inhibitory effect in vivo.3.B.amyloliquefaciens LY-1 inhibited the disease development caused by P.litchii-inoculation by regulating the metabolisms of litchi fruit:(1)Compared with P.litchii-inoculated fruit,B.amyloliquefaciens LY1 could effectively decrease the disease index and browning index.The treatment could obviously reduce the O2-· production rate and malondialdehyde content,keep higher contents of ascorbic acid and reduced glutathione during storage,also,the treatment could also keep higher activities of reactive oxygen species(ROS)-scavenging enzymes(superoxide dismutase,catalase and ascorbate peroxidase)and 1,1-diphenyl-2picrylhydrazyl scavenging ability and reducing power in pericarp of litchi fruit in late stage of storage.It was suggested that the culture broth of B.amyloliquefaciens LY-1 could reduce the accumulation of reactive oxygen species by maintaining the activity of active oxygen scavenging in litchi fruit,which could decrease the membrane lipid peroxidation and keep the cell membrane structure of litchi fruit,therefore,the treatment could control the P.litchii induced disease.(2)Compared with P.litchii-inoculated fruit,B.amyloliquefaciens LY1 could effectively retard the rapid increase of membrane permeability,reduce the activities of lipoxygenase,lipase and phospholipase D in P.litchii-inoculated litchis,therefore keep higher contents of phosphatidylcholine and phosphatidylinositol,retarded the degradation of oleic acid(C18:1),linoleic acid(C18:2)and linolenic acid(C18:3),reduced the relative content of capric acid(C10:0),palmitic aicd(C16:0)and stearic acid(C18:0),consequently maintained higher ratio of unfatty acid to fatty acid and index of unsaturated fatty acid.It was suggested that the culture broth of B.amyloliquefaciens LY-1 could control the disease development by reduce the degradation of membrane lipid and alleviated the damage of integrity of membrane structure.(3)Compared with P.litchii-inoculated fruit,B.amyloliquefaciens LY1 could maintain higher ATP level and energy charge in pericarp of litchi fruit during late storage time,meanwhile the treatment could keep higher activities of Ca2+-ATPase,Mg2+-ATPase,and H+-ATPase in protoplasm,vacuole and mitochondria,so that manitained the balance of Ca2+,Mg2+ and H+,which might maintain normal osmotic pressure in protoplasm and maintain the integrity of membrane structure,therefore,the treatment could effectively control the disease development caused by P.litchii-inoculation.(4)Compared with P.litchii-inoculated fruit,B.amyloliquefaciens LY1 could obviously decrease the respiration rate of fruit and lowered the activities of respiratory terminal oxidases in pericarp of P.litchii-inoculated fruit.Also,the treatment decreased the activities of phosphohexose isomerase(PGI)and succinate dehydrogenase(SDH),increasedthe total activities of glucose-6-phosphate dehydrogenase(G-6-PDH)and 6phosphogluconate dehydrogenas(6-PGDH).Meanwhile,the treatment kept higher activity of nicotinamide adenine dinucleotide kinase(NADK),lowered the contents of oxidized form of nicotinamide adenine dinucleotide(NAD)and reduced form of nicotinamide adenine dinucleotid(NADH),while increased the contents of oxidized form of nicotinamide adenine dinucleotide phosphate(NADP)and nicotinamide adenine dinucleotide phosphate(NADPH),enhanced pentose phosphate pathway(PPP)of respiration.It was suggested that the treatment could control the disease development by change the pathway of respiration.(5)Compared with P.litchii-inoculated fruit,B.amyloliquefaciens LY1 could keep higher contents of flavonoids and total phenolics during whole storage time,maintain higher content of lignin and higher activities of peroxidase(POD),phenylalanine ammonia-lyase(PAL),chitinase(CHI)and β-1,3-glucanase(GLU)in late stage of storage.It was suggested that the B.amyloliquefaciens LY-1 could increase the disease resistance by maintain the contents of endogenous resistant substances and activities of disease resistance related enzymes.4.The application of B.amyloliquefaciens LY-1 obviously retarded the increase of respiration rate of litchis,delayed the increase of cell membrane permeability,kept higher contents of chlorophyll,carotenoid and subsequently delayed the change of apparent pericarp color of litchis.Moreover,it also significantly retarded the decreases of total soluble solids(TSS),titratable acid(TA),total soluble sugars and vitamin C contents in pulp,which led to better quality and flavor of litchi fruit.BLCB also maintained higher contents of anthocyanin and total phenolics in pericarp and subsequently reduce browning index,rate of commercially acceptable fruit and weight loss of litchi.Thus,1.0×108 CFU mL-1 BLCB treatment could significantly delay the fruit senescence and keep better fruit quality and storability of litchi fruit.5.The whole genome of strain LY-1 was determined based on the Illumina Hiseq 2500 sequencing platform.The amount of data obtained after low-quality filtration was 0.37 Gb,Q30 was 80.08%,and the genome size was about 4.4 Mb.A total of 3,999 genes were obtained,the length was 3,466,905 bp,the average length was 866 bp,and the GC content was 47.43%.The genome of LY-1 contained genes encoding various nonribosomal peptide synthetases(itu,fenD,srf and bmy),polyketide synthases(mln,bae and dif)and other proteins(tasA and lci),which are responsible for the biosynthesis of secondary metabolites with antifungal activity.In addition,a special gene,mrs,involved in the ribosomal pathway synthesis of lantibiotic mersacidin was found.In this paper,the possible mechanism of B.amyloliquefaciens LY-1 retarded the occurrence and development of postharvest diseases of litchi fruit was discussed from the perspectives of plant pathology,genomics and bioinformatics,and the findings might provided the theoretical basis for the postharvest biological control of litchi and other subtropical fruits. |
PDF Full Text Request