【作者】 吴一晶；

【导师】 林河通；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2017， 博士

【摘要】 荔枝(Litchi chinensis Sonn.)是重要的亚热带水果,风味独特、营养丰富,具有较高的经济价值。荔枝果实成熟期恰逢盛夏高温高湿季节,极易因病原菌侵染造成果实腐烂变质。荔枝霜疫霉菌(Peronophythora litchii)侵染所致霜疫霉病是造成果实采后腐烂变质的重要原因,每年因此造成的损失约占年产量的20%以上。为减少化学杀菌剂的滥用,开发安全环保的新型杀菌保鲜技术迫在眉睫。生物保鲜技术因具有高效安全、无污染等优点备受关注。生防菌可抑制病原微生物生长,调节果实生理代谢,从而提高果实采后品质、延长货架期。本课题组前期自主分离鉴定了1株具有广谱抑菌活性的生防菌解淀粉芽孢杆菌(Bacillus amyloliquefaciens LY-1。本文以福建省主栽品种“乌叶”荔枝(Litchi chinensis Sonn.cv.Wuye)果实为试验材料,研究了解淀粉芽孢杆菌LY-1对离体P.litchii的抑制作用、解淀粉芽孢杆菌LY-1对P.litchii侵染所致荔枝果实病害发生的控制及其作用机理、及解淀粉芽孢杆菌LY-1对荔枝果实采后生理、品质和贮藏特性的影响;同时利用现代基因组学技术分析了解淀粉芽孢杆菌LY-1菌株的抑菌关键功能基因。主要研究结果如下:1、离体试验表明,解淀粉芽孢杆菌LY-1全发酵液、无菌发酵上清液、菌体悬浮液均能有效抑制荔枝果实采后主要致病菌P.litchii菌丝生长和孢子囊萌发,促进菌丝膜脂过氧化作用增强和膜结构破坏,导致菌丝膜透性升高且蛋白质、核酸泄露量增加。其中以1.0×108 CFU mL-1解淀粉芽孢杆菌LY-1发酵液对离体P.litchii的抑制效果最佳。2、预试验结果发现,解淀粉芽孢杆菌LY-1全发酵液、无菌发酵上清液、菌体悬浮液都能有效降低P.litchii侵染荔枝果实的感病指数和果皮褐变指数。其中以1.0×108 CFU mL-1的解淀粉芽孢杆菌LY-1发酵液控制P.litchii侵染所致荔枝果实病害发生的效果最佳。3、系统研究了 1.0×108 CFU mL-1解淀粉芽孢杆菌LY-1发酵液对P.litchii侵染所致荔枝果实病害发生的控制及其与活性氧代谢、膜脂代谢、能量代谢、呼吸代谢及抗病物质代谢的关系。发现:(1)与P.litchii接种的荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液有效降低P.litchii接种的荔枝果实感病指数和果皮褐变指数,延缓果皮O2-产生速率和膜脂过氧化产物MDA含量的上升,保持较高的果皮AsA、GSH等内源抗氧化物质含量,维持贮藏后期较高的果皮SOD、CAT、APX等活性氧清除酶活性及DPPH自由基清除能力和还原力。据此认为,解淀粉芽孢杆菌LY-1发酵液通过维持较高的荔枝果实活性氧清除能力而减少活性氧积累,从而降低荔枝果实的膜脂过氧化作用、较好保持荔枝果皮细胞膜结构的完整性,进而控制P.litchii侵染所致荔枝果实病害的发生。(2)与P.litchii接种荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液能有效延缓P.litchii接种荔枝果实的果皮细胞膜透性的上升,降低果皮脂酶、磷脂酶D、脂氧合酶等膜脂降解相关酶活性,保持较高的果皮磷脂酰胆碱、磷脂酰肌醇等磷脂含量,及较高的果皮油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)等膜脂不饱和脂肪酸相对含量,较低的果皮癸酸(C10:0)、棕榈酸(C16:0)和硬脂酸(C18:0)等膜脂饱和脂肪酸相对含量,从而维持较高的果皮膜脂脂肪酸不饱和度(U/S)和膜脂脂肪酸不饱和指数(IUFA)。据此认为,解淀粉芽孢杆菌LY-1发酵液通过降低荔枝果实膜脂降解相关酶的活性而减少膜脂降解,较好维持细胞膜结构的完整性,从而控制P.litchii侵染所致荔枝果实病害的发生。(3)与P.litchii接种的荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液能保持P.litchii接种荔枝果实贮藏中后期较高的果皮ATP含量和能荷值,较高的果皮质膜、液泡膜、线粒体膜的Ca2+-ATPase、Mg2+-ATPase和H--ATPase等ATP酶活性。据此认为,解淀粉芽孢杆菌LY-1发酵液通过保持较高的荔枝果实能荷水平和ATP酶活性,而维持细胞膜内外Ca2+、Mg2+、H+平衡及稳定渗透压,从而较好保持细胞膜结构和功能的完整性,进而控制P.litchii侵染所致荔枝果实病害的发生。(4)与P.litchii接种的荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液能有效降低P.litchii接种荔枝果实的呼吸速率及果皮细胞色素C氧化酶(CCO)、抗坏血酸氧化酶(AAO)、多酚氧化酶(PPO)等呼吸末端氧化酶活性,同时,该处理降低了磷酸己糖异构酶(PGI)和琥珀酸脱氢酶(SDH)活性,提高了葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶(G-6-PDH与6-PGDH)总活性,提高果皮烟酰胺腺嘌呤二核苷酸激酶(NADK)活性,降低果皮烟酰胺腺嘌呤二核苷酸(NAD)和还原型烟酰胺腺嘌呤二核苷酸(NADH)含量,提高烟酰胺腺嘌呤二核苷酸磷酸(NADP)和还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)含量,增强磷酸戊糖(PPP)呼吸代谢途径。据此认为,解淀粉芽孢杆菌LY-1发酵液控制P.litchii侵染所致荔枝果实病害的发生与呼吸末端氧化酶活性变化及呼吸代谢途径的改变有关。(5)与P.litchii接种的荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液能保持P.litchii接种荔枝果实采后贮藏期间较高的果皮类黄酮和总酚含量,保持贮藏中后期较高的果皮木质素含量,及较高的果皮过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶(CHI)和β-1,3-葡聚糖酶(GLU)等抗病相关酶活性。据此认为,解淀粉芽孢杆菌LY-1发酵液通过维持荔枝果实较高的木质素、类黄酮和总酚含量及较高的抗病相关酶活性,从而提高荔枝果实对P.litchii的抗病性,进而控制P.litchii侵染所致荔枝果实病害的发生。4、与对照荔枝果实相比,解淀粉芽孢杆菌LY-1发酵液能有效降低采后荔枝果实呼吸强度,延缓果皮细胞膜透性提高,保持较高的果皮叶绿素、类胡萝卜素、花青素、总酚和类黄酮含量,延缓荔枝果肉可溶性固形物、可滴定酸、可溶性总糖、维生素C等营养物质含量减少,降低荔枝果实感病指数和果皮褐变指数,减少果实失重率,提高果实商品率。据此认为,解淀粉芽孢杆菌LY-1发酵液能有效延缓采后荔枝果实衰老,较好保持其果实品质,提高荔枝果实的耐贮性。5、通过Illumina Hiseq2500测序平台完成菌株LY-1的全基因组测定,经过低质量reads过滤后所得数据量为0.37 Gb,Q30为80.08%,基因组大小约为4.4 Mb。共得到编码基因3,999个,长度为3,466,905 bp,平均长度为866 bp,GC含量为47.43%。基因组注释得到抗菌脂肽基因(itu、fenD、srf和bmy)、抗菌聚酮化合物基因(mln、bae和dif)、抗菌蛋白基因(tasA和lci)以及参与核糖体途径合成lantibiotic mersacidin的mrs基因等抑菌关键功能基因。本论文从植物生理学、病理学、基因组学、生物信息学等多角度,阐明解淀粉芽孢杆菌LY-1控制荔枝采后果实病害发生发展的可能机制,为荔枝等亚热带水果采后生物防治提供理论依据。更多还原

【Abstract】 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.更多还原