酵母菌对果蔬采后病害防治的研究进展
水果采后致病菌及拮抗酵母防治机制研究进展
果树资源学报 2023,4(4):73-79水果采后致病菌及拮抗酵母防治机制研究进展蔺 楠,杜静婷,施俊凤*,张立新*(山西农业大学食品科学与工程学院,山西太原030031)收稿日期:2023-04-20基金项目:山西省自然科学基金项目(面上)(20210302123422);山西省重点研发计划项(202102140601017);山西省现代农业产业技术体系(果树)建设专项资金(S X F R S -2022)㊂第一作者简介:蔺 楠(1998-),女,硕士在读,主要从事果蔬采后病害和生物防治研究㊂*通信作者:施俊凤(1977-),研究员,主要从事果蔬采后病害和生物防治研究㊂E -m a i l :s j f t y @126.c o m 张立新(1966-),研究员,主要从事果蔬贮藏保鲜技术研究㊂E -m a i l :139********@139.c o m摘 要:水果由于其含有丰富的营养物质,成为人类饮食中不可或缺的重要资源㊂但是水果在采收后处理不当会造成腐烂,带来严重亏损㊂这些腐烂主要是由真菌导致,其包括扩展青霉㊁灰葡萄孢霉㊁链格孢菌㊁曲霉和毛霉等㊂真菌不仅侵染果实导致腐烂,而且可分泌毒素,引发食品安全问题㊂酵母菌防治由于不产生有害代谢物,对环境友好,成为水果采后病害防治及真菌毒素降解的新策略㊂综述了采后常见病原真菌及其对水果的侵染特性,果实采后致病菌的侵染机理,以及酵母菌的生防机制㊂关键词:水果;采后病害;真菌毒素;拮抗酵母;生物防治文章编号:2096-8108(2023)04-0073-07 中图分类号:S 436.611.1+6 文献标识码:AR e s e a r c h P r o g r e s s i n C o n t r o l M e c h a n i s m o f P o s t h a r v e s t F r u i t P a t h o g e n i c B a c t e r i a a n d A n t a go n i s t i c Y e a s t L I N N a n ,D U J i n g t i n g ,S H I J u n f e n g *,Z H A N G L i x i n *(C o l l e g e o f F o o d S c i e n c e a n d E n g i n e e r i n g ,S h a n x i A g r i c u l t u r a l U n i v e r s i t y ,T a i yu a n S h a n x i 030031,C h i n a ) A b s t r a c t :F r u i t h a s b e c o m e a n i n d i s p e n s a b l e a n d i m p o r t a n t r e s o u r c e i n h u m a n d i e t b e c a u s e o f i t s r i c h n u t r i e n t s .H o w e v e r ,f r u i t i s p r o n e t o d i s e a s e a f t e r h a r v e s t ,r e s u l t i n g i n d e c a y a n d s e r i o u s l o s s e s .F u n g i a r e t h e m a i n p a t h o g e n s c a u s i n g f r u i t d e c a y.T h e s e f u n g i m a i n l y i n c l u d e P e n i c i l l i u m e x p a n s u m ,B o t r y t i s c i n e r e a ,A l t e r n a r i a ,A s p e r g i l l u s a n d M u c o r .F u n g i n o t o n l yi n f e c t f r u i t a n d c a u s e r o t ,b u t a l s o s e c r e t e t o x i n s ,c a u s i n g f o o d s a f e t y p r o b l e m s .Y e a s t c o n t r o l h a s b e c o m e a n e w s t r a t e g yf o r f r u i t p o s t -h a r v e s t d i s e a s e c o n t r o l a n d m y c o t o x i n d eg r a d a t i o n b e c a u s e i t d o e s n o t p r o d u c eh a r m f u l m e t a b o li t e s a n d i s e n v i r o n m e n t a l l yf r i e n d l y .T h i s p a p e r s u m m a r i z e d t h e c o m m o n p a t h og e n i c f u n gi a f t e r h a r v e s t a n d t h e i r i n f e c t i o n c h a r a c t e r i s t i c s t o f r u i t s ,t h e i n -f e c t i o n m e c h a n i s m o f p a t h o g e n i c b a c t e r i a a f t e r h a r v e s t ,a n d t h e b i o l o g i c a l c o n t r o l m e c h a n i s m o f ye a s t .K e yw o r d s :f r u i t ;p o s t h a r v e s t d i s e a s e ;m y c o t o x i n ;a n t a g o n i s t i c y e a s t ;b i o l o g i c a l c o n t r o l 水果中含有丰富的营养成分,定期摄入水果可以预防肥胖㊁心血管㊁眼睛和骨骼等疾病[1]㊂我国是水果消费大国,消费量达到全球总量的60%以上㊂然而,水果在采后容易受到各种致病菌的入侵而引起变质,导致严重亏损[2]㊂其中采后发生病害引起的腐烂损失最严重,据统计,我国每年水果蔬菜因腐烂造成的经济损失占其总产值的30%以上,可达750亿元[3-4]㊂水果采后生理性衰老㊁病害的侵染以及外界环境因素的共同作用,引起了水果的腐烂变质㊂引起水果采后病害的病原菌主要为真菌[4]㊂这些真菌包括青霉属(P e n i c i i i i u m s p p .)㊁灰葡萄属(B o t r yt i s c i n e r e a )㊁交链孢霉属(A l t e r n a r i a s p p .)㊁曲霉属曲霉(A s p e r g i l l u s s p p .)和毛霉属(M u c o r s p p .)等㊂这些病原真菌能够在较低的温度条件下正常生长并产孢,它们繁殖速度快,对环境的适应能力强,因而可迅速侵染果实㊂这些病原真菌主要从果实的伤口部位或皮孔处入侵,引起伤口周围软化和水溃化,并长出菌丝形成霉斑,霉斑往往会分泌大量分生孢子,引起果实溃烂并发出腐败气味[5]㊂这些致病菌不仅导致果实腐烂,而且常常会分泌一些次生代谢产物如展青霉素,黄曲霉素,赭曲霉毒素等,对于人体产生较大的危害作用[6]㊂试验表明,这些毒素有些具有致癌㊁致突变和免疫等负面作用,有些是神经毒素可引起恶心㊁呕吐㊁便血㊁惊厥等㊂目前用于防治水果采后病害的方法主要包括化学防治㊁物理防治和生物防治㊂物理方法包括低温贮藏㊁热处理㊁辐照处理等㊂物理方法成本较高,实施难度较大,受设备条件限制较多㊂化37Copyright ©博看网. All Rights Reserved.学防治法是使用化学杀菌剂来进行防治[7],常见的化学杀虫剂有双胍盐㊁抑霉唑㊁苯菌灵等㊂这些化学杀菌剂不易分解,其在果实表面残留问题会带来食品安全隐患[8],同时,化学药剂常常会导致病原菌产生抗性,降低防治效果[9]㊂生物防治的方法是通过生防菌㊁病原菌以及环境之间相互影响,改善微环境,达到防病的目的㊂生物防治的方法也被认为是最有可能替代化学杀菌剂的方法之一[10]㊂迄今为止,人们已经筛选鉴定出许多有效的拮抗菌株[11],其中包括细菌(P s e u d o m o n a s s p p, B u r k h o l d e r i a s p p.等)㊁小型丝状真菌(T r i c h o d e r-m a s p p.,A s P e r g i l l u s s p p.等)和酵母菌(A u r e o-b a s i c h i u m s p p.,K l o e c k e r a s p p.,M e t s c h n i k o w i a s p p.,R h o d o t o r u l a s p p.)等㊂H a s s a n等[12]发现生防菌沙福芽孢杆菌(B a c i l l u s s a f e n s i s)B3可以通过水解病原菌的细胞壁,来抑制草莓采后灰霉病的发生㊂L u[13]从土壤中分离到洋葱伯克霍尔德氏菌(B u r k h o l d e r i a c o n t a m i n a n s)M S14,该菌对多种植物病原真菌表现了广泛的杀灭作用,可以抑制黑曲霉(A s p e r g i l l u s n i g e r)㊁青霉菌(P e n i c i l l i u m s p.)㊁互隔交链孢菌(A l t e r n a r i a a l t e r n a t a)㊁立枯丝核菌(R h i z o c t o n i a s o l a n i)等多种病原菌的生长,而且可使真菌的细胞内部和细胞形态发生畸变㊂在拮抗菌中,拮抗酵母由于对环境和人体健康友好,一般不产生抗菌素,成为了采后生物防治领域的一大亮点[14]㊂关于拮抗酵母应用于果蔬釆后保鲜的报道也较多㊂张奇儒等[15]从土壤中发现1株对梨果青霉病有防治效果的异常威克汉姆酵母(W i c k e r h a m o m y c e s a n o m a l u s),经检测证实该菌株无毒性,在浓度为1ˑ108个/m L时生防效果显著;王淑培[16]发现桔梅奇酵母(M e t s c h n i k o w i a c i t r i e n-s i s)能在果实伤口处快速定殖,形成生物膜,不仅能有效控制橘采后酸腐病,还能显著抑制柑橘白地霉(G e o t r i c h u m c i t r i-a u r a n t i i)孢子萌发及菌丝生长㊂1水果采后主要致病菌及其毒素1.1扩展青霉扩展青霉(P e n i c i l l i u m e x p a n s u m)可引起苹果㊁梨㊁葡萄等果实采后青霉病[8]㊂该菌属子囊菌门(a s c o m y c o t a)散囊菌纲(E u r o t i o m y c e t e s)青霉属(P e n i c i l l i u m)[17]㊂果实主要在成熟时期容易受到其侵染,发病后病斑呈褐色圆形状,会使果实变软烂并逐渐从发病处扩散[4],在适宜环境下,8d左右便会完全腐烂㊂在温度和湿度较高的情况下,病斑处会出现霉块,初期为白色,后面逐渐变为青绿色,并伴有浓烈的腐烂味㊂菌落草绿色,小斑点状,中央一般呈凸起状,背面黄褐或肉桂色,菌丝无色透明,聚集时常呈白色,分子孢子梗呈不对称帚状分枝,且表面光滑,呈淡青绿色,一般近圆形或圆形,孢子大小为(1.8~2.2)μmˑ(1.8~2.2)μm[18]㊂扩展青霉分泌的毒素为展青霉素(P A T),又名棒曲霉素㊁珊瑚青霉素㊂该毒素还可由棒形青霉(P.c l a v i-f o r m e)㊁粒状青霉(P.g r a n u l a t u m)㊁产黄青霉(P.c h r y s o g e n u m)和曲霉属的棒曲霉(A.c l a v a l u s)等分泌[19]㊂展青霉素对胃具有刺激作用,可从口腔进入消化系统并入侵至肠道,还可引起肠炎㊁胃溃疡和上皮细胞恶化等疾病,当胃肠道黏膜受损,毒素可随着血液在人体内循环,引起免疫系统受损[20]㊂展青霉素分子的内酯结构和半缩醛结构是引起其毒性的主要机制,因此这些结构的消失被认为是解毒的标志[21]㊂展青霉素具有强的亲水性和稳定性,容易通过食物和饮用水积累在人体内[22]㊂1.2灰葡萄孢霉灰葡萄孢霉(B o t r y t i s c i n e r e a)属子囊菌门(a s-c o m y c o t a)锤舌菌纲(L e o t i o m y c e t e s)核盘菌科(S c l e r o t i n i a c e a e),可侵染葡萄㊁番茄㊁梨㊁桃㊁草莓等多种果蔬,引发灰霉腐烂病[23],该菌对低温有很好的适应性㊂果实发病初期病斑为水浸状,淡褐色,后病斑颜色逐渐加深,环境湿度大时可产生深灰色霉状菌丝㊂灰葡萄孢霉不仅可侵染果实,还可侵染植株,慈志娟等[24]研究表明灰葡萄孢霉可以在草莓生长期间侵染其叶片,叶柄,花瓣等部位,首先会从衰老受损部分开始入侵,感染后果实的颜色变为浅褐色或果肉坏死腐烂,病斑处出现灰色霉菌㊂灰葡萄孢霉不同的菌株常常致病性差异较大,K e r s s i e s 等[25]对30株具有不同致病性的灰葡萄孢霉进行随机扩增多态性D N A分析,发现菌株的侵染能力㊁采样时间㊁地点和R A P D模式之间并无关联㊂二环倍半萜烯(B o t r y d i a l)是灰葡萄孢霉产生的最主要的毒性,其毒性也最强,其他毒素大部分为其前体或衍生物㊂研究发现B o t r y d i a l并不会由植物本身分泌,而是植物组织被感染后产生,且宿主越衰败,B o t r y d i a l含量越高,因此灰葡萄孢霉主要通过该毒素致病[26]㊂1.3链格孢霉链格孢霉菌(A l t e r n a r i a s p p.)又称脉孢霉,属于半知菌亚门(D e u t e r o m y c o t i n a)丝孢纲(H y p h o-m y c e t e s)暗色孢科(D e m a t i a c e a e),易在低温环境下47果树资源学报2023,4(4)Copyright©博看网. All Rights Reserved.生存[27]㊂其菌丝体呈黑色,分生孢子呈倒棒状㊁顶部延长成喙状,有纵横格,多个为链状[28]㊂营养菌丝匍匐生长,菌落整体呈现黑色绒状[29],背部呈深褐色有同心螺纹[30]㊂链格菌不仅能够侵染农作物还能侵染多种果蔬,包括寄生㊁腐生和兼性寄生或腐生[31]㊂在低温避光潮湿的环境下,链格孢霉会产生毒素,这些毒素包括链格孢酚(a l t e r n a r i o l,A O H)㊁细链格孢菌酮酸(t e n u a z o n i c a c i d,T e A)㊁链格孢甲基醚(a l t e r n a r i o l m o n o m e t h y l e t h e r,A M E)㊁细格菌毒素Ⅰ㊁Ⅱ㊁Ⅲ㊁链格孢霉烯(a l t e n u e n e,A L T)[32]㊂它们具有基因毒性㊁细胞毒性㊁和致突变性[33]㊂根据其结构可分为5大类:二萘嵌苯及其衍生物㊁二苯并吡喃酮类及其衍生物㊁细交链格孢菌酮酸及其衍生物㊁丙三羧酸酯类化合物和混杂结构[34]㊂1.4黑曲霉菌黑曲霉菌(A s p e r g i l l u s n i g e r)属于半知菌类亚门(D e u t e r o m y c o t i n a)丝孢纲(H y p h o m y c e t e s)曲霉属(A s p e r g i l l u s)㊂分生孢子梗为球形顶囊,分生孢子直径约2.5~4.0μm㊂菌落由白色经黄色后变为黑色,厚绒状[35]㊂黑曲霉侵染果实后,会使果实腐烂皱缩,糖和酚类等营养物质流失,最终严重影响果实口感[36]㊂黑曲霉菌会产生赭曲霉毒素㊁黄曲霉毒素㊁奥卡那胺和阿库雷星等类似物,能损伤人体器官,同时还具有致癌等不良影响㊂且毒素不会高温灭活,稳定性较高[36]㊂1.5毛霉菌毛霉(m u c o r)又名白霉,属于接合菌亚门(Z y-g o m y c o t i n a)接合菌纲(Z y g o m y c o t i n a)毛霉属(M u-c o r a l e s)[37]㊂是多种果蔬采后病害的病原菌,其无法通过果皮侵染,只能感染果实伤口㊂霉菌菌丝为白色,菌丝体十分发达,有分枝,一般无假根及匍匐菌丝,个别菌种罕见假根㊂在室温下几天就会完全侵染果实,并在较短时间内侵染健康果实,侵染后果实腐烂软塌,内部汁液化较重㊂毛霉在6ħ以后生长繁殖会受到抑制[38]㊂毛霉菌会引发毛霉病,是一种深部真菌病,其广泛存在于自然界中,为土壤㊁植物㊁面包㊁水果㊁粮食等的常见腐生菌和空气中的气生菌㊂主要通过空气孢子吸入感染㊂2果实采后致病菌的侵染机理2.1分泌细胞壁降解酶细胞壁是致病菌入侵的首道防线㊂致病菌侵入植物时通过释放各种酶来降解蛋白质㊁多糖以及芳香族聚合物,使植物的细胞壁产生破裂,以此来降低植物的防御功能[39]㊂卢晶晶等[40]研究发现,病原菌释放各种酶类来使病原菌成功入侵宿主,同时酶降解产物可以促进致病菌生长,这一结论也在基因方面得到验证,如切除果胶裂解酶基因C c p e l A可以使炭疽病菌侵染绿色番茄果实的能力下降[41]㊂2.2分泌毒素致病菌分泌的毒素也能使果实发病㊂毒素会影响原有胞内构造,破坏膜通透性和果实防御系统,以此来降低果实抗性,干扰果实生理代谢,使宿主病变或衰亡[42]㊂比如,烟曲霉分泌的二羟基萘(D i-h y d r o x y n a p h t a l e n e,D H N)-黑色素能通过破坏宿主防御系统来增强其致病能力[43]㊂灰葡萄孢霉主要通过B o t r y d i a l毒素致病,使宿主细胞结构发生变化,引发宿主软腐,组织坏死[26]㊂马铃薯立枯丝核菌产生的毒素可使马铃薯幼苗细胞膜透性增加,电解质外渗[44]㊂莲子草假隔链格胞毒素可作用于空心莲子草细胞叶绿体基粒片层,使叶绿体基粒片层肿胀甚至发生紊乱[45]㊂2.3分泌辅助因子致病菌入侵果实时,果实通过激发自身的先天免疫系统来进行防御㊂一方面,果实可以识别致病菌的一些分子,然后通过信号传导来激活相关的分子模式(P A M P)触发免疫反应(P T I)[46]㊂另一方面,当致病菌入侵时会分泌一些效应因子来抑制宿主的防御[46]㊂比如,核盘菌可以分泌草酸降低宿主的p H,以确保其细胞壁降解酶P G等的活性表达,同时宿主抗病相关酶活性降低,加速入侵㊂此外,草酸还可与植物细胞壁的C a2+螯合,而C a2+可以参与信号传导和防御反应,从而阻碍宿主正常代谢㊂而螯合的产物草酸钙,可形成堵塞导管和维管束的晶体,宿主的水分代谢也受影响,从而加速发病[47]㊂2.4p H调节致病菌在入侵宿主过程中,会分泌一些酸碱物质来调整p H,以此来增强致病菌入侵能力,确保完成入侵[48]㊂比如,扩展青霉和指状青霉等产酸菌在入侵苹果时分泌葡萄糖酸和柠檬酸等有机酸改变环境p H,在适宜p H条件下会促进霉菌毒素和果胶酶的产生和表达[49-50]㊂互隔交链孢(A l t e n a r i a a l t e r-n a t a)通过分泌氨类物质将p H值由酸性转变为碱性,会使纤维素酶关键基因A a K l激活,加速了A.57蔺楠,等:水果采后致病菌及拮抗酵母防治机制研究进展Copyright©博看网. All Rights Reserved.a l t e r n a t a对在柿子体内的入侵[51]㊂3拮抗酵母的生防机制3.1营养和空间竞争拮抗酵母常以二糖和单糖的碳水化合物和氮源为养料[52],大量繁殖后在伤口处聚集形成生物膜,空间上阻断了致病菌的侵染[7,53]㊂K l e i n等[54]发现在培养基中加入0.5%蔗糖和1%硫酸铵有利于酵母菌生长,可刺激生物膜产生,提高对柑桔酸腐病的抑制效力㊂此外,酵母中抑制性化合物的合成通过原位或非原位吸收营养物质而增加,提高了它们对植物病害的生物防治能力㊂如李侨飞[55]等发现拟粉红锁掷孢酵母Y16(S p o r i d i o b o l u s p a r a r o s e u s)能在短时间内快速生长繁殖,以此来构成一个完整的菌膜,阻隔致病菌入侵,抑制葡萄黑曲霉(A s p e r-g i l l u s n i g e r)的生长㊂田亚琴[56]发现将葡萄糖作为外源营养物质加到伤口处时,与单独加美极梅奇酵母相比抑制率更高,且越早接种效果越好㊂3.2杀手毒素杀手毒素通常是由不同品种的酵母产生的糖基化蛋白质,可以破坏特定的细胞壁成分,包括β-1, 3-d-葡聚糖,β-1,6-d-葡聚糖,甘露蛋白和几丁质等,从而导致病原真菌细胞死亡[57]㊂所有毒素都是在酸性条件下产生的,它们的活性在一定范围内随着培养基的p H和温度的增加而降低[58]㊂杀手毒素附着在细胞膜上,在细胞膜上它们与导致细胞变化的次级受体相互作用㊁D N A合成抑制㊁细胞周期破坏和R N A片段化[59]㊂如膜醭毕赤酵母(P i c h i a m e m b r a n i f a c i e n s)可以产生两种杀手毒素P M K T1和P M K T2,这两种毒素对腐败酵母和病原菌均有杀伤作用㊂3.3溶解酶酵母可以通过产生葡聚糖酶㊁几丁质酶和蛋白酶等溶解酶对病原菌产生拮抗作用,其能够将致病菌细胞壁分解,破坏其结构,导致细胞溶解和死亡[60]㊂田亚琴[56]发现在特殊培养基上接种美极梅奇酵母(M e t s c h n i k o w i a p u l c h e r r i m a)后,该酵母能够分泌胞外几丁质酶和胞外葡聚糖酶㊂李侨飞[55]试验表明拟粉红锁掷孢酵母Y16(S.p a r a r o s e u s)能降解青霉菌的几丁质成分,会使霉菌菌丝发生坏死并抑制霉菌生长繁殖㊂杨其亚[61]发现胶红酵母(R h o d o t o r u l a m u c i l a g i n o s a)能提高β-1,3-葡聚糖酶和几丁质酶活性,从而延缓苹果青霉病㊂3.4诱导宿主抗性酵母细胞壁的一些物质可以诱导宿主产生抗性,加速果实伤口愈合过程,从而达到生防作用[62]㊂美极梅奇酵母(M.p u l c h e r r i m a)可以诱导苹果果实中的细胞壁相关酶类,并能够提高芒果P P O㊁P O D活性,降低了芒果炭疽病的发病率[63]㊂试验表明,将膜醭毕赤酵母(P.m e m b r a n i f a c i e n s)接种于桃果实时,可以使果实产生多种抗性蛋白酶,这些酶可以使果实由于氧化造成的伤害得到缓解,延缓果实腐烂[64]㊂诱导寄主抗性的两种基本形式包括系统获得性抗性(S A R)和系统诱导抗性(I S R)[65]㊂S A R是多种病程相关蛋白(P R)共同作用的结果,拮抗酵母作为一种生物刺激因子,可以激发果实中多种病程相关蛋白的表达,从而增强果实对病害的抗性㊂如梅奇酵母属(M e t s c h n i k o w i a s p.)可以有效控制采后柠檬的蓝色和绿色青霉病,而且酵母菌的浓度越高,柠檬的腐烂率和病斑直径就越低,同时能够显著提高柠檬果实中抗病相关酶几丁质酶(C H T)㊁苯丙氨酸解氨酶(P A L)和过氧化氢酶(C A T)的活性[66]㊂红东孢酵母(R h o d o s p o r i d i u m k r a t o c h v i l o v a e)可以增强果实病程中蛋白基因的表达,提高梨果体内多种防御酶活性,同时果实发病率也降低[67]㊂3.5挥发性代谢物质近年来,国内外许多研究表明,拮抗菌产生的挥发性物质可以用于防治果实病害[68]㊂由于挥发性物质分子小㊁易挥发扩散,杀菌范围广等优点得到广泛应用[69]㊂如郭虹娜等[70]发现有孢汉逊酵母(H a n s e n i a s p o r a u v a r u m)挥发代谢物的主要抑菌成分是桂皮醛,并通过研究发现使用62.5μL/L的桂皮醛对草莓进行熏蒸处理能有效保持草莓品质,延缓品质下降,对草莓采后灰霉病抑制效果明显,能抑制贮藏期间草莓M D A含量的升高,有效诱导草莓C A T㊁P P O㊁A P X相关抗性酶活,延缓S O D酶活的下降,对草莓相关抗性基因表达的影响与上述酶活类似㊂陈莹莹等[71]研究发现膜醭毕赤酵母(P. m e m b r a n i f a c i e n s)产生的挥发性物质可以有效抑制灰葡萄孢菌,且熏蒸第2天就抑菌率超过70%,同时验证了挥发性物质可破坏菌丝细胞膜使细胞内容物大量外泄,同时分生孢子数量也大大减少㊂3.6活性氧作用植物遭受致病菌胁迫时,首先会诱导产生活性氧(R O S),包括超氧阴离子(S u p e r o x i d e a n i o n,O2-)67果树资源学报2023,4(4)Copyright©博看网. All Rights Reserved.和过氧化氢(C h y d r o g e n p e r o x i d e,H2O2)㊂超氧化物歧化酶(S O D)可以催化超氧阴离子自由基歧化为H2O2和O2,并且在F e2+或C u2+离子存在下, H2O2通过F e n t o n反应进一步转化为㊃O H自由基[72],从而达到清除O2-的目的㊂活性氧的清除包括酶促反应和非酶促反应,其中酶促反应包括过氧化物酶(P O D)和抗坏血酸过氧化物酶(A P X)等,而非酶途经包括植物自身含有的的抗氧化物质如谷胱甘肽(G S H)和谷胱甘肽还原酶(G R)等㊂C a s t o r i a 等[73]发现罗伦隐球酵母(C.l a u r e n t i i)对果实病害的抑制能力与其对R O S的耐受能力呈正相关,也说明了氧化应激反应在生防机制中的作用㊂M a c a r i-s i n D[74]研究发现将两种拮抗酵母(梅奇酵母和丝酵母)接种于不含营养的培养基或苹果表面(置于无营养空间),会产生大量超氧阴离子,而接种于果实伤口处则导致果实组织中产生大量H2O2,在伤口处收集的酵母中也会含有许多H2O2,说明R O S是酵母提高其拮抗能力的其中一个原因㊂4结论这些病原菌入侵果实后会分泌细胞壁降解酶,毒素,辅助因子以及改变p H来使水果腐烂变质㊂同时拮抗酵母可以通过竞争空间和营养,分泌溶解酶,杀手毒素,诱导宿主抗性等方式来抵御致病菌,以维持果实正常品质并保障食品安全㊂拮抗酵母具有来源广泛㊁适应性强㊁繁殖能力强㊁代谢产物无毒且无致敏性的特点,逐步应用于有害微生物的防控领域㊂未来可继续从体内外两个方向筛选种类更多,效果更强的拮抗酵母菌株,并进行食品安全验证㊂同时对酵母菌在分子水平上的代谢机制进行深入的研究分析,系统性地总结和归纳拮抗酵母在分子水平上的最新研究进展,进一步研究拮抗酵母的拮抗机制并为基因工程改性高活性拮抗酵母提供科学参考㊂参考文献[1] V A L C K E M,B O U R G A U L T M H,R O C H E T T E L,e ta l.H u m a n h e a l t h r i s k a s s e s s m e n t o n t h e c o n s u m p t i o no f f r u i t s a n d v e g e t a b l e s c o n t a i n i n g r e s i d u a l p e s t i c i d e s:A c a n c e r a n d n o n-c a n c e r r i s k/b e n e f i t p e r s p e c t i v e[J].E n v i r o n m e n t i n t e r n a t i o n a l,2017,108:63-74.[2] G R A M I S C I B R,L U T Z M C,L O P E S C A,e t a l.E n-h a n c i n g t h e e f f i c a c y o f y e a s t b i o c o n t r o l a g e n t s a g a i n s tp o s t h a r v e s t p a t h o g e n s t h r o u g h n u t r i e n t p r o f i l i n g a n dt h e u s e o f o t h e r a d d i t i v e s[J].B i o l o g i c a l C o n t r o l,2018,121:151-158.[3]张红印,蒋益虹,郑晓冬,等.酵母菌对果蔬采后病害防治的研究进展[J].农业工程学报,2003,19(4):23-27.[4]罗杨.膜醭毕赤酵母对柑橘采后青绿霉病害的生物防治及机理研究[D].西南大学,2011.[5]卞文怡.微胶囊化水杨酸对苹果采后青霉病的诱导抗病研究[D].上海应用技术大学,2019.[6]王利平,王丽霞,刘保友,等.苹果及其制品中展青霉素研究进展[J].落叶果树,2022(54):39-43. 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《美极梅奇酵母T-2VOCs对蓝莓采后灰霉病的防效及对贮藏品质的影响》
《美极梅奇酵母T-2 VOCs对蓝莓采后灰霉病的防效及对贮藏品质的影响》一、引言随着农业科技的不断发展，果蔬采后保鲜技术日益受到关注。
蓝莓作为一种营养丰富的水果，其采后保鲜和病害防治显得尤为重要。
灰霉病是蓝莓采后常见的病害之一，严重影响了蓝莓的贮藏品质和经济效益。
近年来，美极梅奇酵母T-2 VOCs（以下简称T-2 VOCs）在果蔬保鲜方面的应用逐渐受到研究者的关注。
本文旨在研究T-2 VOCs对蓝莓采后灰霉病的防效及其对贮藏品质的影响。
二、材料与方法1. 材料实验材料包括蓝莓果实、美极梅奇酵母T-2 VOCs及灰霉菌。
实验蓝莓品种为XXX品种，健康、成熟、大小均匀的果实作为实验样本。
2. 方法（1）处理组与对照组设定：将蓝莓果实分为实验组和对照组，实验组用T-2 VOCs进行处理，对照组不作处理。
（2）灰霉病处理：对两组蓝莓进行灰霉菌接种，观察并记录发病情况。
（3）贮藏品质检测：在贮藏过程中，定期检测蓝莓的重量、硬度、色泽等指标。
（4）数据分析：采用SPSS软件进行数据分析，比较实验组与对照组在灰霉病发病率、贮藏品质等方面的差异。
三、实验结果1. 灰霉病防效实验结果显示，经过T-2 VOCs处理的蓝莓，其灰霉病的发病率明显低于对照组。
实验组在贮藏期间，灰霉菌的生长受到显著抑制，有效降低了蓝莓的病害发生率。
2. 贮藏品质影响（1）重量损失：实验组蓝莓在贮藏期间的重量损失低于对照组，表明T-2 VOCs有助于保持蓝莓的重量。
（2）硬度：实验组蓝莓果实的硬度在贮藏期间保持较好，硬度的下降速度较慢，说明T-2 VOCs能够减缓蓝莓果实的软化速度。
（3）色泽：实验组蓝莓在贮藏期间色泽保持较好，无明显褪色现象，而对照组蓝莓色泽变化较为明显。
这表明T-2 VOCs能够减缓蓝莓果实的色泽退化。
四、讨论实验结果表明，美极梅奇酵母T-2 VOCs对蓝莓采后灰霉病具有显著的防效，能够有效降低灰霉菌的生长速度和病害发生率。
果蔬采后病理及病害的控制研究现状及发展趋势
果蔬采后病理及病害的控制研究现状及发展趋势摘要：本文主要对果蔬采后主要病原菌及侵染过程做了介绍，主要介绍了酵母类抗菌剂防病害的生物防治方法，并对微生物种类、拮抗机理及生物防治应用前景进行了介绍。
关键词：采后病理；酵母菌类抗菌剂；病害侵染；微生物防治；1.前言影响食品食用安全性的最主要因素是化学农药残留。
天然植物成分(精油和植物提取物)、生物药剂( 酵母和细菌类拮抗菌)和非选择性生物杀菌剂(碳酸钠、碳酸氢钠、活性氯、山梨酸)等防治果蔬采后病害的技术已越来越受到关注。
本文重点介绍微生物抗菌剂防治果蔬采后病害的一些研究进展。
迄今为止，已从苹果、柑橘、梨、桃、猕猴桃等10余种水果中筛选出几十种拮抗微生物，目前商品化应用的主要有：丁香假单胞杆菌、枯草芽胞杆菌、酵母菌中的季也蒙毕赤酵母菌、哈氏木霉、白粉寄生菌[1~2]。
果蔬采后病害造成的腐烂损失十分巨大。
据统计报道, 发达国家为l0%～30%, 发展中国家则高达40%～50%。
长期以来防治果蔬采后病害主要依靠化学杀菌剂, 然而, 连续使用化学杀菌剂易使病原真菌产生抗药性, 易造成环境污染, 且危害公众健康。
上世纪80 年代中期开始, 在农作物大田病害生物防治蓬勃发展的带动下, 生物防治果蔬采后病害成为研究热点[3～4]。
果实采后病原性腐败的生物防治技术是近年来国外发展起来的极具前途的绿色防腐技术，主要原理是利用微生物之间的拮抗作用，通过改变果实表面微生态环境，促进病原微生物拮抗菌的繁殖，达到抑制病原微生物生长，减少腐败的目的[1]该技术安全环保性能优越，越来越受到普遍欢迎可以预见，采用生物防治将是今后果蔬防腐保鲜技术的发展方向果蔬采后病害的生物防治因其无毒无害不污染环境，深受人们的重视与欢迎。
2. 果蔬采后主要病原菌及侵染过程2.1主要病原菌引起蔬菜采后腐烂的病原菌主要有真菌、细菌、病毒和原生动物，其中以真菌和细菌性病原菌为主[5~6]。
2.1.1 真菌真菌是最主要和最流行的病原微生物，侵染广，危害大，是造成果菜类在贮藏运输期间损失的重要原因。
果蔬采后生理学结课论文
果蔬采后生理学结课论文拮抗酵母菌对苹果采后病害防治的研究进展学生：符利华(2021112870) 指导老师：魏长庆摘要: 果蔬采后病害在世界范围内一直造成较重大的经济损失,使用化学杀菌剂是控制其发生的主要手段。
但化学杀菌剂的大规模使用不仅会导致病原菌产生抗药性,而且造成食品中的农药残留问题，因此急需寻找新的方法用于果蔬采后病害防治。
近年来,生物防治已逐渐成为一种可替代化学杀菌剂的防治方法,其中拮抗微生物的应用,尤其是来自果蔬表面的拮抗酵母菌对控制采后病害已经取得了巨大作用关键词：苹果；采后病害；生物防治；拮抗酵母菌;防治机理Resistance is the virus to pick apples after the diseaseprevention and control researchStudent：Fu lihua(2021112870) Instructer: Wei ChangqingAbstract: Fruits from the disease in the world has caused a major economic losses, the use of chemical antiseptic is the main means of control. but the chemical fungicides of the large-scale use not only lead to a pathogen , and the cause of the pesticide residue problems of food, so urgently required for the new method is used to adopt disease prevention. In recent years,biological control has gradually become a substitute for chemical fungicides of control methods which is against the application, especially from thefruits of the surface of the population is to gather to control the disease has already made great use. Keywords:apple; Postharvest diseases; biological control; Antagonism yeast ;Control mechanism1果蔬采后生理学结课论文0 前言果蔬因采后腐烂而造成的严重损失早已经成为全球性的问题。
海洋酵母对樱桃番茄采后链格孢病害防治效果研究
Biocontrol of Alternaria alternata on Cherry Tomato Fruit by Use of marine yeast Rhodosporidium paludigenum Fell &Tallman1Wang Yifei a，Feng Wu b，Shen Danhong a，Yu Ting a，Zheng Xiaodong a,*a Department of Food Science and Nutrition，Zhejiang University，Hangzhou（310029）b College of Food Science and Technology，Huazhong Agricultrual University，Wuhan（430070）E-mail：xdzheng@AbstractThe vasidiomycetous yeast Rhodosporidium paludigenum Fell & Tallman, which was isolated from the south of East China Sea was evaluated for its activity in reducing postharvest decay of cherry tomatoes caused by Alternaria alternata in vitro and in vivo test. The result showed that washed cell suspensions of R. paludigenum provided better control of A. alternata than other treatments. Autoclaved cell culture failed to provide protection against the pathogen. The concentration of antagonist had significant effect on biocontrol effectiveness. When the washed yeast cell suspension reached the concentration of 1×109 CFU/ml and challenged with pathogen spore suspension at 5×104 spores/ml, the rate of decay was only℃R. paludigenum was obvious when it was applied 37% after 5-day incubation at 25. Efficacy ofsimultaneously or prior to inoculation with A. alternata. There was little efficacy when R. paludigenum was applied after A. alternata inoculation. The longer incubated time of R. paludigenum was, the lower disease incidence would be. When the incubation time was reached 54 h, the decay was only 33%. On the wounds of cherry tomato, the population of R. paludigenum increased 50-fold during the first 12 h ℃-fold over the original ones after 24 h.at 25. The maximum marine yeast population was nearly 100Keywords：Cherry tomato；Biological control；Alternaria alternata；Rhodosporidium paludigenum Fell & Tallman；Marine yeast1. IntroductionCherry tomato (Lycopersicon esculentum) is popular all over the world. And in China, it also suffers significant losses during the growth and after the harvest due to clime and diseases. The main postharvest disease of Cherry tomato caused by various pathogenic fungi are Alternaria alternata (Feng and Zheng, 2007), Botrytis cinerea (Lee et al., 2006) and Rhizopus stolonifer (Stevens et al., 1997; Leonardo Schena et al., 1999). A. alternata is a saprophytic pathogen of tomato causing postharvest losses at high frequency (Akhtar et al., 1994).Several kinds of synthetic fungicides have been used to control the postharvest decay of fruits and vegetables successfully. However, using of fungicides for long time will result in the development of resistant strains (Rosslenbroich and Stuebler, 2000). At the same time, many of these synthetic chemicals are gradually becoming ineffective (Spotts and Cervantes, 1986). As a result, rot caused by A. alternata on cherry tomato is difficult to control in the last 20 years. Meanwhile, there is an increasing consumer concern over pesticide residues on foods, shifting fungicides to other methods for disease control (Wisniewski and Wilson, 1992). Currently, several promising biological approaches that include essential oils (Tzortzakis, 2007; Feng and Zheng, 2007), chitosan (Liu et al., 2007), 1-MCP (Guillen et al., 2007) and microbial antagonist(Schena et al., 1999; Liu and Tian, 2005) have been advanced as potential alternatives to synthetic fungicides to control postharvest of cherry tomatoes.In recent years, some antagonists have been successfully applicated in biological controlling of postharvest diseases of cherry tomato. Aureobasidium pullulans, a cosmopolitan yeast-like fungus,1This research was supported by the grants from National Natural Science Foundation of China (30571301) and the Ph.D. Programs foundation of Ministry of Education of China (20040335025) and the National High-Tech Research and Development Program (863 Program) of China (06160807).have been found as an effective biological agent for controlling postharvest rots of cherry tomatoes (Schena et al., 1999). Crytococcus laurentii in combination with different concentrations of sodium bicarbonate also can reduce obviously the decay incidence of postharvest diseases of cherry tomatoes caused by B. cinerea and Pythium aphanidermatum (Liu and Tian, 2005).Up to now, most yeast antagonists were isolated from fruit surface, fruit wounds, fruit plant and soil (Janisiewicz, 1991, 1996; Wilson et al., 1993; Qi et al., 2006). The strain resource is limited and has restrained the development of biological control of postharvest. The oceans covering 71% of the planet represent an important bioresource for microorganism including yeasts (Chi and Liu, 2005). Our laboratory has tried to screen some marine yeast in controlling postharvest of fruits and vegetables and some isolates were significantly effective against postharvest rots (unpublished). One of the promising strains was a marine yeast Rhodosporidium paludigenum Fell & Tallman, which has been not reported previously as biological control agent.This study aimed to investigate the effects of R. paludigenum on the control of postharvest diseases caused by A. alternata in fresh cherry tomatoes.2. Materials and methods2.1 Plant material and fungal culturesCherry tomatoes were obtained from Shandong Province with a commercial level of maturity. Fruits℃-94% relative humidity for no longer than 48 were used immediately after harvest, or stored at 20, 92h before using during the Spring of 2005 (Brown et al., 2000). Before treatments, fruit were washed with tap water, then surfaced-disinfected with 0.1% sodium hypochlorite for 1 min, at last cleaned with tap water and air dried prior to wounding.A. alternata was obtained from IMCAS (Institute of Microbiology, Chinese Academy of Sciences) and maintained on potato dextrose agar (PDA: extrat of boiled potatoes, 200 ml; dextrose, 20 g and distilled water, 800 ml) at 4. Spore suspensions were obtained by flooding 7℃-14-day-old PDA cultures of pathogen with sterile distilled water containing 0.05% (v/v) Tween 20. Spore concentrations of the pathogen was determined by a haemocytometer and adjusted with sterile distilled water to meat the concerntration of 5×104 spores/ml (Zhang et al., 2003).2.2 Marine antagonistThe marine yeast strain R. paludigenum Fell & Tallman (1980) used in this study was isolated from the south of East China Sea. It is identified in CABI Bioscience Identification Services as Rhodosporidium paludigenum Fell & Tallman (1980) (IMI 394084). Yeast cells were prepared by growing cultures in nutrient yeast dextrose broth (NYDB: nutrient broth, 8g/l; yeast extract, 5g/l;℃dextrose, 10g/l; sodium chloride, 20g/l) for 24h at 28 with shaking at 200 rpm. The medium was centrifuged (KA1000, Shanghai Anke) at 3000 rpm (7000g) for 10 min and the cells were re-suspended in sterile-distilled water. The way of doing above was repeated twice, in order to wash the yeast cells from the nutritional compounds of the initial medium. The concentration of the yeast cell suspention was obtained with a hemocytometer, and adjusted to a concentration as required with sterile-distilled water fitting for different experiments (Wisniewski et al., 1995).2.3 In vitro antifungal assay2.3.1 In vitro contact assayTo evaluate the interactions between the antagonist and the pathogens in culture, 6 mm diameter plugs were cut from 7-days-old NYDA cultures of A. alternata and then placed on another potato-dextrose agar (PDA) plates (15 ml/plate) seeded with 1.0 ml of different treatments of R.℃paludigenum. Three replicates were used per treatment. Plates were incubated for 7 days at 28 .Fungal growth was recorded after 7 days. Growth inhibition was calculated as the percentage of inhibition of radial growth relative to the control. Experiments were repeated two times.2.3.2 Spore germination assayThe effect of R. paludigenum on spore germination and germ tube elongation of pathogen was tested in potato dextrose broth (PDB). Aliquots of 5 ml PDB were placed in glass tubes (150×10 mm). 100µl of spore suspension of A. alternata (about 107 spores/ml) and 100µl cells of R. paludigenum were added into the tube. All treated tubes were placed in rotary shaker(QYC 2102，Shanghai FUMA ) at ℃pores of pathogens were microscopically 150 rpm at 28 . After 20 h incubation, approximately 100 sfor germination rate and germ tube length (Droby et al., 1997). Each treatment was replicated three times and the experiment was repeated twice.2.4 Effect of R. paludigenum in controlling A. alternata20 cherry tomatoes were used for each treatment and one wound were made per cherry tomato. R.℃paludigenum were grown in 50 ml NYDB for 24 h at 28 . The treatments were as following: Control: sterile distilled water, A: culture filtrate, B: autoclaved culture. C: 1×108 CFU/ml unwashed cell culture mixture, D: 1×108 CFU/ml washed cell suspension. Every 20µl of the above mixture were inoculated into each wound and allowed to dry for about 4 h. Wounds were then inoculated with 10µl of 5×104 spores/ml suspension of A. alternata. The fruits were sealed in polyethylene-lined plastic boxes to℃determined after 5 days maintain high humidity and incubated at 25. Decay incidence wasinoculation. There were three replicates for each treatment, and each experiment was repeated twice. 2.5 Effects of different concentrations of R. paludigenum in controlling A. alternataThe suspension of washed cells were adjusted to concentrations of 1×106, 1×107, 1×108, 1×109 CFU/ml washed cell suspension with sterile distilled water by a hemacytometer, respectively (Fan and Tian, 2000). Aliquot of 20µl of above mentioned treatments was respectively inoculated into each wound. Wounds were inoculated with 10µl of 5×104 spores/ml suspension of A. alternata 4 h later.℃te were Treated fruits were stored at 25 for 5 days as the method described above. Infection ra recorded. There were three replicated trials of 20 fruits per treatment with complete randomization. The test was repeated two times.2.6 Effects of different incubated time of suspension of R. paludigenum in controlling A. alternataR. paludigenum were grown in a NYDB for 18, 24 30, 42 and 54 h, and then 20µl of 1×108 CFU/ml washed cell suspension was respectively inoculated into cherry tomato wounds. After 4 h, 10µl of 5×104 spores/ml suspension of A. alternata was inoculated into each wound. The fruits were stored at ℃rate as the method described above. Each treatment was 25 for 5 days to record the infectionreplicated three times and the experiment was repeated twice.2.7 Effect of interval between R. paludigenum application and A. alternata inoculation in controlling A. alternata20µl of washed cell suspension of R. paludigenum at 1×108 CFU/ml washed cell suspension was respectively inoculated to each wound at 12, 4, 2, and 0 h after or 2 and 4 h before inoculation of A. alternata at 5×104 spores/ml suspension. The fruits were sealed in polyethylene-lined plastic boxes to maintain high humidity and incubated at 25. Decay development was determined after 5 days. Each℃treatment consisted of three replicates with 20 fruits in each test, and the test was repeated three times.2.8 Population dynamics of the R. paludigenumOne wound was made by a cork-borer (0.5 cm diameter, 1cm deep) on each fruit. 20µl of 1×108CFU/ml washed cell suspensions of R. paludigenum was injected into each wound. The samples were taken at different times (0, 12, 24, 48, 72, 108 h) after treatment (Zheng et al., 2007). Wounded tissuesof cherry tomatoes were removed with another cork-borer (1.0 cm diameter, 1.5 cm deep) and groundwith a mortar and pestle in 10 ml of sterile water. The cells were counted using a haemacytometer. There were three replicates of six fruits for each treatment, and the experiment was repeated twice.2.9 Date analysisIn this study, all experiments were repeated twice and the results of two experiments were in close agreement. The data from two experiments were combined. All data were analyzed by one-way analysis of variance (ANOV A) in the statistical software SAS v. 8.0. Mean separations were performedby Duncan’s multiple range tests. Differences at P=0.05 were considered as significant.Data of antagonist populations (CFU/ml) were transformed to logarithms to improve the homogeneity of variances.3. Results3.1 In vitro antifungal assay3.1.1 In vitro contact assayIn the test on PDA plates, all treatments of R. paludigenum, which include culture filtrate, autoclaved culture, 108 CFU/ml unwashed cell suspension and 108 CFU/ml washed cell suspension, did not inhibite the growth of A. alternata, whose plugs completely developed all over the disk (Table 1).Table 1 Inhibitory effects of different treatments of R. paludigenum on A. alternateTreatments of R. paludigenum Percent of inhibition (%)Control －aculture filtrate －autoclaved culture －108 CFU/ml unwashed cell culture mixture －108 CFU/ml washed cell culture mixture －a Indicates ineffective on microorganism.3.1.2 Spore germination assayIn the assay of spore germination, all treatments of R. paludigenum, which include culture filtrate, autoclaved culture, 108 CFU/ml unwashed cell suspension and 108 CFU/ml washed cell suspension, didnot inhibite the spore germination of A. alternata in PDB. The germ tube length of A. alternata was entwined each other, which was too long to be determined (Table 2).Table 2. Effect of different treatments of R. paludigenum on spore germination of A. alternataTreatments of R. paludigenum Spore germination (%)a Germ tube length(um)100±0a b－cControl culture filtrate 100±0a －autoclaved culture 100±0a －108 CFU/ml unwashed cell suspension 100±0a －108 CFU/ml washed cell suspension 100±0a －020*********D i s e a s e i n c i d e n c e (%)Treatments aGermination rate were measured after 20 h incubation at 28 in PDB. ℃b Values are the mean of three replicates±SE. Where the letters are the same, there is no significant difference between the means of different concentrations.c The germ tubes were too long and entwined each other so that the germ tube length could not be determined. 3.2 Effect of R. paludigenum in controlling A. alternataBest control of A. alternata was got with washed yeast cell suspension (Figure 1). The infection rate of cherry tomatoes of control was 93%, while the treatment of 1×108 CFU/ml washed cell suspension was 13% after 5 days at 25, which was lower than other treatments. The rot rate of unwashed cell ℃culture mixture were 35%, which also showed remarkable control effects (p <0.05). But its control effect was significantly lower than that of the washed cell suspension at the same concentration of yeast cells (p <0.05). Autoclaved cell culture failed to provide protection against the pathogen.Figure 1 Effect of R. paludigenum on control effectiveness. (A) autoclaved culture; (B) culture filtrate; (C) 1×108 CFU/ml unwashed cell mixture; (D) 1×108 CFU/ml washed cell suspension. Control is sterile distilled water. Values followed by different letter were significantly different according to Duncan’s mutiple range test p =0.05.3.3 Effects of different concentrations of R. paludigenum in controlling A. alternataDifferent concentrations of R. paludigenum can control the rate of disease incidence efficiently on cherry tomato at 25 (℃p <0.05). The concentration of R. paludigenum influenced the control effectiveness dramaticallyon cherry tomato (Figure 2). The results showed that the higher concentration of R. paludigenum was, the lower disease incidence would be. When the concentration of the washed cell suspension of R. paludigenum reached 1×109 CFU/ml and spore suspension of A. alternata was at 5×104 CFU/ml, the rate of decay declined to 52%.020*********D i s e a s e i n c i d e n c e (%)Treatments020*********D i s e a s e i n c i d e n c e (%)Figure 2 Effects of different concentrations of R. paludigenum in controlling A. alternata . (A) 1×106 CFU/ml R. paludigenum ; (B) 1×107 CFU/ml R. paludigenum ; (C) 1×108 CFU/ml R. paludigenum ; (D) 1×109 CFU/ml R. paludigenum . Control is sterile distilled water. Values followed by different letter were significantly differentaccording to Duncan’s mutiple range test p =0.05.3.4 Effects of different incubated time of R. paludigenum in controlling A. alternataDifferent incubated time of R. paludigenum can significantly influence the control effectiveness on the disease incidence on cherry tometo at 25 (℃p <0.05). The results showed that the control effectiveness of longer incubated of R. paludigenum was better than the lower ones (Figure 3). The best control effect was obtained by the washed cell suspension of R. paludigenum which was incubated for 54 h.Figure 3 Effects of different incubated time of R. paludigenum in controlling A. alternata . Control is sterile distilled water. Values followed by different letter were significantly different according to Duncan’s mutiple rangetest p =0.05.20406080100D i s e a s e i n c i d e n c e (%)Treatment3.5 Effect of interval between R. paludigenum application and A. alternata inoculation in controlling A. alternataThe incidence rate of A. alternata was effected seriously by the interval between R. paludigenum application and A. alternata inoculation (p <0.05). The biocontrol efficacy of R. paludigenum treatments which was done before A. alternata inoculation was better than that of done after A. alternata inoculation or simultaneous inoculation (Figure 4). In general, the earlier the antagonist inoculated and the later the pathogen inoculated did, the lower the disease incidence would be. The disease incidence of cherry tomatoes declined to 12% when R. paludigenum application at 4 h before the A. alternata inoculation. The cherry tomato which was treated with A. alternata 12h before R. paludigenum was completely decayed.Figure 4 Effect of interval between R. paludigenum application and A. alternata inoculation in controlling A. alternata . (A) A. alternata + 12h + R. paludigenum ; (B) A. alternata +4 h + R. paludigenum ; (C) A. alternata +2h + R. paludigenum ; (D) A. alternata + R. paludigenum ; (E) R. paludigenum + 2 h + A. alternata ; (F) R. paludigenum + 4h + A. alternata , Control is sterile distilled water. Data in columns with different letters arestatistically different according to Duncan’s multiple range test at p =0.053.6 Population dynamics of the R. paludigenumPopulations of R. paludigenum in wounded fruits stored increased by approximately 50-fold during the first 12 h (Figure 5). And the maximum population of the yeast was increased to 100-fold after 24 h and then began to decline very slowly.4.55.05.56.06.5p o p u l a t i o n s (l o g c e l l s p e r w o u n d )hours after inoculationR. paludigenumFigure 5 Population dynamics of R. paludigenum in wounds of cherry tomato incubated at 25. Wounds were ℃treated with 20µl of 1×108 CFU/ml washed cell suspensions of R. paludigenum alone or plus 10µl of 5×104 spores/ml A. alternata . Bars represent standard deviations.4. DiscussionBiological control has be considered as one of most promising alternative to fungicides, which employs microorganisms to protect fruits and vegetables from infection by phytopathogens (Wilson et al., 1991; Janisiewicz and Korsten, 2002; Spadaro and Gullino, 2004).The basidiomycetous yeast Rhodosporidium sp. has been reported to have urease activity (Phillips et al., 1991) and produce microbial lipid (Li et al., 2007). The marine yeast R. paludigenum was isolated from the south of East China Sea, which has been isolated from sea water, marsh and mangrove swamp in previous (Fell and Tallman, 1984). The basidiomycetous yeast Rhodosporidium sp. has been reported to have urease activity (Phillips et al., 1991) and produce microbial lipid (Li et al., 2007). And marine yeast has not been reported for biological control so far. The result of this study demonstrated that the marine yeast R. paludigenum isolate showed great potential in controlling postharvest disease caused by A. alternata on cherry tomatoes for the first time.In the test on in vivo test, all treatments of R. paludigenum had no effect on the growth of the pathogen in culture. These results suggested that R. paludigenum does not inhibit the pathogens as well as B. subtilis due to antibiotics production (Pusey and Wilson, 1984). The result was also different from other reported antagonistic yeast such as C. laurentii (Zhang et al., 2005), Metschnikowia pulcherrima (Piano, 1997) and Pichia membranefaciens (Fan and Tian, 2000).Moreover, this study showed that autoclaved cell cultrate did not show distinct control effect on cherry tomatoes. Unwashed cell culture of R. paludigenum was not as efficiently as the washed cell suspension, which suggests the competition for space and nutrition maybe the main mode of action of the R. paludigenum . Biocontrol is competitive for space and nutrition concern, as previously demonstrated for other antagonistic yeasts (Maclaughlin et al., 1992; Filonow, 1998; Fan and Tian, 2001). At the same concentration, the effect of the washed cell suspension of R. paludigenum to controlA. alternata was significantly better than the unwashed one.At the same time, Effect of controlling A. alternata was also related to the concentration of the cell suspension. The results were as the same as other yeast antagonists such as R. glutinis (Zheng et al.,2005), C. albidus (Fan and Tian, 2001).The result from this study indicated that better control was obtained when the R. paludigenum was applied before inoculation, which is consistent with the result obtained by other authors (Spotts and Chand-Goyal, 1997; Fan and Tian, 2001). This result shows the best opportunity of applying biocontrol agents is pre-wound during harvest (Spotts et al., 1998).Although the 54 h of incubated time had the best control of A. alternate, it is not adapt to commercial production. R. paludigenum incubated in cultures for 24 h as well as 30 h was significantly effective in reducing the number of infected wounds.In conclusion, our data indicated that the marine yeast R. paludigenum could be an alternative for controlling postharvest diseases caused by A. alternata on cherry tomatoes. 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FEMS Microbiology Letters 15 (1), 21-25.Piano, S., Neyrotti,V., Migheli Q., Gullino, M. L., 1997. Biocontrol capability of Metschnikowia pulcherrima against B. cinerea postharvest rot of apple. Postharvest Biology and Technology 11, 131-140.Pusey, P. L. 1991. Integration of biocontrol agents with postharvest systems. In: Wilson, C. L., Chaltuz, E. (Ed.), Biological Control of Postharvest Diseases of Fruits and Vegetables. Proceedings of aWorkshop, ARS-92, Shepherdtown, USDA-WV, pp. 211-213.Schena, L., Ippolito, A., Zahavi, T., Cohen, L., Nigro, F., Droby, S., 1999. Genetic diversity and biocontrol activity of Aureobasidium pullulans isolates against postharvest rots. Postharvest Biology and Technology 17, 189-199. Spadaro D., Gullino M L., 2004. State of the art and future prospects of the biological control of postharvest fruit diseases. International Journal of Food Microbiology 91, 185-194Spotts, R. A., Cervantes, L. A., 1986. Populations, pathogenicity and benomyl resistance of Botrytis spp., Penicillium spp. and Mucor piriformis in Paking houses. Plant Disease 70, 106-108.Spotts, R. A., Sanderson, P. G., Lennox, C. L., Sugar, D., Cervantes, L. A., 1998. Wounding, wound healing and straining of mature pear fruit. Postharvest Biological Technology 13, 27-36.Stevens, C., Khan, V. A., Lu, J. Y., Wilson, C. L., Pusey, P. L., Igwegbe, C. K., Kabwe, K., Mafolo, Y., Liu, J., Chalutz, E., Droby, S. 1997. Integration of ultrabiolet (UV-C) light with yeast treatment for control of postharvest storage rots of fruits and vegetables. Biological Control. 10, 98-103.Tzortzakis, N. G.., 2007. Maintaining postharvest quality of fresh produce with wolatile compounds. Innovative food science and emerging technologies 8, 111-116.Wilson, C. L., Wisniewski, M. E., Biles, C. L., McLaughlin, R., Chalutz, E., Droby, E. 1991. Biological control of post-harvest diseases of fruits and vegetables: Alternatives to synthetic fungicides. Crop Protection 10, 172–177. Wilson, C. L., Wisniewski, M. E., Droby, E., Chalutz, E., 1993. A selection strategy for microbial antagonists to control postharvest diseases of fruits and vegetables. Scientia Horticulturae 53, 183-89.Wisniewski, M.E., Wilson, C.L., 1992. Biological control of postharvest diseases of fruits and vegetables: recent advances. HortScience 27, 94–98.Wisniewski, M. E., Droby, S., Chqlutz, E., Eilam, Y., 1995. Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila. Plant Pathology 44, 1016-1024.Xi, L., Tian, S., P., 2005. Control of postharvest diseases of tomato fruit by combining antagonistic yeast with sodium bicarbonate. Scientia Agriciultura Sinica, 38 (5), 950-955.Zhang, H. Y., Zheng, X. D., Xi, Y. F. 2003. Biocontrol of postharvest blue mold rot of pear by Cryptococcus laurentii. Journal of Horticultural science and Biotechnology，78, 888-893.Zhang, H. Y., Zheng, X. D., Xi, Y. F. 2005. Biological control of postharvest blue mould of oranges by Cryptococcus laurentii (Kufferath) Skinner. Biocontrol 50, 331-342.Zheng, X. D., Y u, T., Chen, R. L., Huang B., Wu, V. C., 2007. Inhibiting Penicillium expansum infection on pear fruit by Cryptococcus laurentii and cytokinin. Postharvest Biology and Technology 45, 221-227.Zheng, X. D., Zhang, H. Y., Sun, P., 2005. Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis. European Food Research and Technology 220, 353-357.。
几丁质对海洋酵母Rhodosporidium paludigenum果实采后病害防治效力的影响及相关机理研究
几丁质对海洋酵母Rhodosporidium paludigenum果实采后病害防治效力的影响及相关机理研究由真菌病害引起的果实采后腐烂变质是导致水果大量损失的主要原因之一,利用拮抗酵母生物防治的方法近几十年来已经取得了一定的进展,被认为是最有希望替代化学杀菌剂的方法之一。
海洋酵母Rhodosporidium paludigenum是本实验室从中国东海海域分离筛选到的一株生防酵母,对多种果实采后病害具有显著的生物防治效力。
但是目前拮抗酵母单独使用时的效力与化学杀菌剂相比仍有较大差距,因此如何有效提高其防治效果是影响商业化推广应用的关键性因素。
几丁质是由N-乙酰-D-葡萄糖胺通过p-1,4糖苷键连接而成的长链高分子多糖,已被美国环境保护署和食品药品管理局批准作为生物农药和食品添加剂使用,其开发利用深受国内外关注。
本论文主要研究了几丁质对海洋酵母R.paludigenum拮抗效力的影响,探讨了影响拮抗效力的因素,从体外试验、体内试验、酵母生理、果实生理等多个角度分析了相关机理,并通过同位素标记相对与绝对定量技术(iTRAQ)系统分析了几丁质对海洋酵母R. paludigenum蛋白表达的影响。
本论文主要获得了以下研究结果：(1)在NYDB培养基中添加几丁质可以提高海洋酵母R. paludigenum对多种果实的多种采后病原真菌的防治效力,与其他几种诱导培养物质(海藻糖、葡聚糖、壳聚糖)相比,几丁质对R. paludigenum拮抗效力的提高作用最为显著。
(2)几丁质诱导培养的最优添加浓度为1.0%(w/v),最优诱导培养时间为36小时,在最优条件诱导培养后,在各个使用浓度上,海洋酵母R. paludigenum的拮抗效力显著增强,且在冷藏条件和采前处理条件下,几丁质对海洋酵母R.paludigenum生防效力的提高作用依然显著。
(3)几丁质与氯化钠单独诱导培养均能在一定程度上增强海洋酵母R.paludigenum的拮抗效力,但是这两者之间不存在协同增效作用,且在结合诱导培养中起主导作用的可能是几丁质。
2株浅白隐球酵母对葡萄灰霉病和柑橘青霉病采后防治效果的研究
2株浅白隐球酵母对葡萄灰霉病和柑橘青霉病采后防治效果的研究张岳;杨俊颖;王旭东;宋海慧;张莉莉;石玉莹;陈秀玲;王傲雪【期刊名称】《江苏农业科学》【年(卷),期】2017(045)002【摘要】以2株浅白酵母Ca63(Cryptococcus albidus 63)、Ca64(Cryptococcus albidus 64)为研究对象,通过平板对峙、显微观察致病菌和浅白酵母的共培养物.结果表明,2株浅白酵母均能显著抑制葡萄灰霉病菌和柑橘青霉病菌的生长.Ca63和Ca64对葡萄灰霉病菌菌丝生长量的抑制率分别为83.1％、80.7％;Ca63和Ca64对柑橘青霉病菌菌丝生长量的抑制率分别为82.8％、77.1％.采后果实防治试验发现2株酵母菌对葡萄灰霉病菌和柑橘青霉病菌都有良好的抑制效果,菌株Ca63对2种病菌的防治效果均好于菌株Ca64,Ca63防治灰霉病效果为62.6％,防治柑橘青霉病效果为72.6％.该研究为进一步将这2种酵母菌应用于果实采后保鲜提供了基础.【总页数】5页(P96-100)【作者】张岳;杨俊颖;王旭东;宋海慧;张莉莉;石玉莹;陈秀玲;王傲雪【作者单位】东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学生命科学学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学园艺学院,黑龙江哈尔滨150036;东北农业大学生命科学学院,黑龙江哈尔滨150036【正文语种】中文【中图分类】S436.631.1+9;S436.661.1+9【相关文献】1.浅白隐球酵母产油工程菌液体深层发酵技术研究2.罗伦隐球酵母对柑橘采后酸腐病的抑制效果3.罗伦隐球酵母对草梅采后灰霉病害的生物防治4.浅白隐球酵母产油工程菌液体深层发酵技术研究5.罗伦隐球酵母对蒜薹采后病害防治效果的研究因版权原因，仅展示原文概要，查看原文内容请购买。
第七章酵母拮抗菌防治水果采后病害及其作用机理ppt课件
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研究发现将季也蒙假丝酵母(Candida guilliermondii)接种到桃 果实的伤口处，在有病原菌存在的时候该拮抗菌的数量在 24h时可以增长到200多倍。这种快速的繁殖活动反应出拮抗 菌与病原菌之间的营养竞争。
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酵母拮抗菌与病原菌争夺的营养物质主要是碳水化合物、 氮源等。研究中发现，相同浓度的拮抗菌细胞悬浮液比 培养原液有更好的拮抗效果，原因很可能是培养液中的 丰富营养削弱了拮抗菌与病原菌进行营养竞争的力度。 Filonow（1998）用C14标记的果糖、葡萄糖、蔗糖检验 在苹果果实伤口处拮抗菌Cryptococcus laurentii与病原菌 Botrytis cinerea在营养利用上的差别，发现拮抗菌比病原 菌孢子消耗了更多的营养物质。
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此外，一些食品添加剂如碳酸氢钠以及钼酸铵与拮抗菌混 合使用提高拮抗菌的抑菌效果，降低了拮抗菌有效防治病 害的浓度，从而降低了生产成本。
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二、 几种拮抗菌的混合使用 在使用拮抗菌生物防治水果采后病害的时候，必须考虑到 一种拮抗菌不可能有效地抑制控制一种水果上所有的病菌， 这一点甚至连杀菌剂通常也未必能做到。正如Baker和 Cook所说：“几种拮抗菌的混合物比单一使用一种拮抗菌 进行生物防治得到成功的机会更多，同时这样的组合防治 效果也比较稳定。” 其中，混合使用具有不同拮抗机理的拮抗菌进行生物防治 将是一种最佳的组合方式，这种方法的使用可以减少病原 菌因选择压力的存在而对一种拮抗菌产生抗性。
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Fan等发现，无论在体外还是体内，季也蒙假丝酵母 (Candida guilliermondii)和膜醭毕赤酵母(Pichia membranefaciens)均可诱导产生外切、内切几丁质酶和β-l， 3-葡聚糖酶，这些水解酶可以明显地抑制病原真菌 Rhizopous stolonfier孢子的萌发以及芽管伸长。
果蔬采后病害生物防治的研究进展_韦莹莹
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。现代意义上的生物防治不仅利用微生物之间的拮抗作用 ， 还包括病原菌、 生防
［8 ］ 菌和它们所处的微环境之间的相互作用 ， 改变微环境的平衡， 以达到防治病害的目的 。 果蔬采后病害 的生物防治， 是指在贮藏过程中， 通过引入生防菌， 使果实上的微生物达到平衡， 从而抑制病原菌在此微生
果蔬采后病害生物防治的研究进展
韦莹莹， 毛淑波， 屠康
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Hale Waihona Puke （ 南京农业大学食品科技学院 ， 江苏 南京 210095 ） 摘要：采用生防菌进行果蔬采后病害的生物防治是一种安全有效的方法 。过去 20 年内， 国内外研究者在生防菌的筛选 、 作 提高生防效果的途径等方面做了大量的研究 ， 而且目前国际市场上已有几种商业化生防产品 。 尽管如此， 未来仍 用机制、 需要筛选效力高的生防菌 ， 深入研究其生防机制以及生防菌 、 病原菌、 寄主之间的互作模式， 使生物防治成为能真正替代化 学杀菌剂的方法。本文综述了近年来果蔬采后病害生物防治的研究成果 ， 探讨了未来提高生防效果的研究方向 。 关键词：果蔬； 采后病害； 生物防治； 生防菌 中图分类号：TS255． 3 文献标志码：A 文章编号：1000－2030 （ 2012 ） 05－0183－07
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生物防治的定义
传统意义上的生物防治主要是指利用微生物之间的拮抗作用 ， 选择无害的微生物抑制病原菌的生长 ，
收稿日期：2012－07－05 基金项目：国家自然科学基金项目 （ 31101282 ） ； 江苏省高校优势学科建设工程项目； 江苏省现代农业技术创新项目（ CX（ 11 ） 2063 ）
果品采后病害及生物防治研究进展
23(增刊)97-102中国生物防治Chinese Journal of Biological Control2007年9月果品采后病害及生物防治研究进展李静1,于建娜2,张利莉1*(1新疆生产建设兵团塔里木盆地生物资源保护利用重点实验室,阿拉尔843300;2塔里木大学植物科技学院)Advances of Research on Postharvest Diseases and Biocontrol of FruitsLI Jing1,YU Jian-na2,ZHANG L-i li1(11Key Laboratory of Protection and Utilizati on of Biological Resources in Tari m Basi n of Xinjiang Production&Construction Corps,Alar Xinjiang843300;21College of Plant Science and Technology,Tari m University,China)提要:综述了果品采后病害病症、生防微生物种类、生防机理以及生防方法,并展望了果品采后病害生物防治的应用前景。
关键词:采后病害;拮抗机理;生物防治中图分类号:S432198;S476文献标识码:A文章编号:1005-9261(2007)增刊-0097-06新鲜水果采后腐烂是一个全球性的问题。
发达国家有10%~30%的新鲜果蔬损失于采后腐烂,我国果品出口运输过程中的腐烂率高达30%~50%[1]。
腐烂主要归因于病原微生物的致病作用,而控制采后病害的主要方法是使用化学杀菌剂。
随着社会的发展,人们逐渐认识到:长期使用化学杀菌剂不但会导致病菌产生抗药性,而且果品上残留农药也会对人类健康造成威胁和环境污染。
自20世纪80年代中期,在大田农作物病害生物防治蓬勃发展的引领下,水果采后生物防治方法作为一种控制采后病害的新途径,渐渐为人们所重视,并逐步成为研究热点。
酵母菌对果蔬采后病害的生物防治
酵母菌对果蔬采后病害的生物防治摘要：采用生防菌进行果蔬采后病害的生物防治是一种安全有效的方法，酵母菌因其拮抗效果好、不产生毒素、可以和化学杀菌剂共同使用等优点，备受人们关注。
该文对酵母菌对果蔬采后病害防治的研究现状进行了综述, 其中包括防治效果、防治机理、防治的优势。
许多酵母菌对果蔬采后病害具有明显的抑制作用, 酵母菌抑制霉菌的机理主要在于营养与空间的竞争、对病原菌的直接寄生作用及诱导寄主产生抗病性。
该文还分析了酵母菌在果蔬采后病害生物防治中存在的问题，拮抗酵母菌商业化应用的局限性，并对其发展前景进行了展望。
关键词：生物防治；酵母菌；采后病害前言果蔬采后病害导致的巨大损耗己成为全球性的问题，造成采后果蔬腐烂变质的原因有自身的生理失调和衰老，病原菌侵染以及采收贮运过程中的机械损伤等，其中以病原微生物侵染为主。
在发达国家约有10%～30%的新鲜果品由于采后病害导致腐烂损失，而在发展中国家，由于缺乏贮运冷链设备，腐烂损失率则更高，达40%～50%[1]。
据报道，中国每年因采后腐烂导致的果蔬损失约8000万t，造成经济损失约750亿元，占全国果蔬产业总值的30%以上[2]。
长期以来，防治果蔬采后病害的方法主要是采用化学杀菌剂，然而，化学杀菌剂毒性较大，易污染环境。
目前越来越多原先用于果蔬采后处理的化学杀菌剂己被禁止使用，如苯来特、多菌灵、双胍盐等[1]。
此外，频繁使用化学杀菌剂会使病原菌产生抗药性而逐渐降低其防治效果，为达到灭菌效果不得不增加剂量，从而造成果蔬农药残留超标，危害人类的健康。
因此，迫切需要寻求一些新的、无毒高效的防腐技术，以逐步取代化学杀菌剂在采后果蔬上的使用。
用微生物进行采后病害的生物防治是近年来发展起来的一个新的研究领域。
自从20世纪50年代Guter等首次报道枯草杆菌（Bacillus Subtilis）对水果病原菌有拮抗作用以来，国外学者对用微生物拮抗菌对果蔬采后病害的防治进行了广泛的研究。
生防酵母菌防治果品采后病害机理的研究进展
明显 防治效 果 的细菌 、 酵母 菌和 小型 丝状 真菌 ＿ ２ ］ 。 Ｓ ｔ ｙ ｒ ｉ ａ ｋ 等 Ｊ 发现 Ｓ ａ ｃ ｃ ｈ ａ ｒ ｏ ｍｙ ｃ ｅ ｓ ｓ ｐ ｐ ． 、 Ｋｌ ｕ ｙ ｖ ｅ ｒ ｏ ｍｙ 关 于 果 品采 后 病 害 的生 防 机理 ， 早 期 的研究 主 ｃ ｅ ｓ ｓ ｐ ｐ ． 和 Ｒｈ ｏ ｄ ｏ ｔ ｏ ｒ ｕ ｌ ａ ｓ ｐ ｐ ． 等 都 可 以 分 解 赭 曲 霉 要 集 中于生 防细 菌 。研 究 发现 生防 细菌 的主要 抗菌 素 Ａ、 雪 腐镰 刀菌烯醇 （ ｎ ｉ ｖ ａ ｌ ｅ ｎ ｏ １ ） 、 脱 氧 萎 镰 菌 醇 机 理是 分泌 抗生 素 ， 因大 多 消 费者 对 细 菌 缺 乏足 够 （ ｄ ｅ ｏ ｘ ｙ ｎ ｉ ｖ ａ ｌ ｅ ｎ ｏ １ ） 和伏 马毒 素 Ｂ １ （ ｆ ｕ ｍｏ ｎ ｉ ｓ ｉ ｎ Ｂ １ ） 。
［ ６ ３ ； 酿酒 酵母 （ Ｓ ａ ｃ ｃ ｈ ａ ｒ ｏ ｍｙ ｃ ｅ ｓ ｃ ｅ ｒ ｅ ｖ ｉ ｓ ｉ ａ ｅ ） 和 粘红 替 代化 学杀 菌剂 已迫 在眉 睫 。生 物 防治作 为相对 安 Ａ）
全 和无 毒 的防腐 手段 已引 起广 泛关 注 ， 在过去的 ３ Ｏ 酵母 （ Ｒ ｈ ｏ ｄ ｏ ｔ ｏ ｒ ｕ ｌ ａ ｇ ｌ ｕ ｔ ｉ ｎ ｉ ｓ ） 可 以 分 解 展 青 霉 素 年里 ， 专 家们 已经 筛 选 出多种 对 果 品采 后 病 害具 有 （ ｐ ａ ｔ ｕ ｌ ｉ ｎ ） ¨ ７ Ｊ ， 降 低 展 青 霉 素 在 果 实 上 的 积 累ｌ ＿ ８ ］ ；
机理研究进展迅速 ， 笔者从 营养或空间竞争、 直接病原菌寄 生 、 诱导 寄主产生抗 性 、 分 泌抗 菌素等 ４个方面对 生 防机理研究 的历史和最新状况进行综述 ， 分析存 在的问题 并展 望了研究 前景。 关键词 果实 ；采后病害 ；防治 ；生防酵母 菌 ； 作用机理
间型假丝酵母菌株对多种水果蔬菜腐败霉菌的拮抗效果和拮抗机制的研究
YPD 液态培养基 (质量浓度为 20 %的蔗糖) 直径[8 ] 。重复 3 次 。
产
30 ℃摇床培养 24 h ,稀释到 10 - 3 ,取 100μL
取洋葱 ,选择大小基本一致者 ,以 10 g/ 与
涂 YPD 平板 ,30 ℃温箱培养 24 h ,取大的菌 落镜检后接种到 YPD 斜面上 。
霉菌均分离自正在腐败的果蔬中 。 114 高抑制活力酵母菌的筛选
2 结果与分析
25 ℃培养 ,观察霉菌生长情况[4 ] 。
211 酵母菌的分离 、筛选和抑制霉菌效果
115 酵母菌的分类鉴定
从霉腐玉米分离到了几十株酵母菌 ,经
鉴定方法见参考文献[ 6 ] 。
过初步筛选得到拮抗效果较好的 10 株酵母
116 间型假丝酵母抑制霉菌的机制研究
菌 (C1～C10) ,同时利用本实验室现有的酵
0
0
0
4
6
8
10
10
0
0
5
10
10
10
12
13
0
2
7
12
12
12
13
15
4
6
9
14
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6
9
10
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15
16
18
8
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18
++
++
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表 4 不同浓度间型假丝酵母在 25 ℃下对橙子青霉的抑菌效果
1d
2d
3d
4d
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6d
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第19卷第4期2003年7月农业工程学报T ransacti ons of the CSA E V o l .19 N o.4July 2003酵母菌对果蔬采后病害防治的研究进展张红印,蒋益虹,郑晓冬,席王与芳,孙 萍(浙江大学食品科学与营养系,杭州310029)摘 要:该文对酵母菌对果蔬采后病害防治的研究现状进行了综述,其中包括防治效果、防治机理、防治的优势及最新研究成果。
许多酵母菌对果蔬采后病害具有明显的抑制作用,酵母菌抑制霉菌的机理主要在于营养与空间的竞争、对病原菌的直接寄生作用及诱导寄主产生抗病性。
酵母菌与其它生防微生物相比,具有拮抗效果好、不产生毒素、可以和化学杀菌剂共同使用等优点。
使用生物工程技术改造拮抗酵母菌从而提高其生防效果,是酵母菌对果蔬采后生物防治的最新研究领域。
该文还指出了酵母菌对果蔬采后生物防治存在的问题,并对未来的研究提出了新的思路。
关键词:果蔬采后病害;生物防治;酵母菌;生物防治效果与机理中图分类号:T S 201.3 文献标识码:A 文章编号:100226819(2003)0420023205收稿日期:2002209225基金项目:国家自然科学基金资助项目(30170659)作者简介:张红印,男,讲师,博士研究生,杭州市凯旋路268号 浙江大学食品科学与营养系,3100291 引 言果蔬采后病害造成的损失是巨大的。
据报道,发达国家有10%～30%的新鲜果蔬损失于采后的腐烂,而在发展中国家,由于缺乏储运冷藏设备,果蔬腐烂损失率更高[1]。
目前我国的果蔬采后损耗约占总产量的30%～40%[2]。
果蔬采后腐烂大多由真菌引起。
水果在采收、包装、贮存和运输过程中若受到机械损伤[3],霉菌很容易通过伤口侵蚀水果,并在水果上大量生长,从而造成水果的腐烂。
长期以来防治真菌病害的方法主要是采用化学杀菌剂。
然而,连续使用化学杀菌剂会使病原菌产生抗药性,由于农药对环境的污染和对公众健康的危害[4],迫使人们寻求安全、无毒和有效的新方法。
生物杀菌剂可分为植物杀菌剂、动物杀菌剂和微生物杀菌剂三种,用微生物进行采后病害的生物防治是国内外近年来发展起来的一个新的研究领域。
自从Gu ter 首次报道枯草杆菌(B acillus S ubtilis )对水果病原菌有拮抗作用以来,国外学者对用微生物拮抗菌对果蔬采后病害的防治进行了广泛的研究[5,6]。
目前,已研究可作为果蔬采后病害拮抗菌的微生物有细菌、霉菌和酵母菌等,其中酵母菌由于具有拮抗效果好、不产生毒素、可以和化学杀菌剂共同使用等优点而成为果蔬采后生物防治研究的热点[7,8]。
2 酵母菌对果蔬采后病害防治的效果目前已证明对果蔬采后病害具有防治作用的酵母菌品种很多。
在国外Chalu tz 等从柠檬果实表面分离得到的一株汉逊德巴利酵母(D eba ry o m y ces hansen ii ),对柑桔青霉病、绿霉病和酸腐病有显著的防治效果,其对青霉病的抑制效果在11℃或22℃条件下可以维持21d [9];W isn iew sk i 等研究发现,假丝酵母(C and id aoleop h ila )对苹果灰霉病有良好的防治效果,Ca 2+可促进这种效果,而M g 2+没有这样的作用[10];B enbow 等研究了隐球酵母属的C ry p tococcus .inf ir m o 2m in ia tus 、C .lau ren tii 和红酵母属的R hod otoru la .g lu tin is 对梨病害的生物控制效果,结果表明,隐球酵母属的C .I nf ir m o 2m in ia tus 和红酵母属的R hod otoru la .g lu tin is 在采收前一天对梨进行处理,能有效地控制梨的采后腐烂[11];Zahavi 等的研究发现,假丝酵母(C and id a g u illier m ond ii )对葡萄由灰葡萄孢霉及黑曲霉引起的腐败具有明显的抑制效果,与对照相比,分别可以减少腐败损失16.81%和60%[12]。
在国内,范青等研究了季也蒙假丝酵母(Cand id a g u illier m ond ii )对桃采后果实软腐病的抑制效果,用5×108CFU mL 悬浮液处理的果实贮藏在25℃下经4d 、15℃经7d 和3℃经30d 后软腐病的发病率都为零,和1000m g L 扑海因处理的效果一样[13]。
范青等研究了丝孢酵母(T richosp oron sp )的不同处理和接种时间对“富士”苹果灰霉病和青霉病的抑制效果,结果表明,当接种灰霉菌和青霉菌孢子浓度分别为1×105个 mL和5×104个 mL 时,在25℃,1×108CFU mL 的酵母悬浮液完全抑制这两种病害的发生;在1℃冷藏30d 后,灰霉病和青霉病的发病率分别为13%和0[14]。
范青等对果实病害生物防治拮抗菌进行了筛选和分离,结果表明:季也蒙假丝酵母(C .g u ilier m ond ii )、柠檬形克勒克酵母(K loeckera ap icu la ta )、汉逊德巴利酵母(D eba ry o m y ces hansen ii )等3种酵母菌对甜樱桃褐腐病都表现出显著的抑制效果,季也蒙假丝酵母和柠檬形克勒克酵母能有效地防治核果类果实采摘后软腐病;从桃果实表面分离得到的浅白隐球酵母(C ry p tococcus a lbid us (sa ito )sk inner )和丝孢酵母(T richosp oron sp )能防治苹果灰霉病和青霉病,而从桃果伤口处分离到的膜醭毕赤酵母(P ich ia m e m branef aciens H ansen )则对核果类果实软腐病有较好的抑制作用[15]。
刘绍军等研究了啤酒酵母对草莓的保鲜作用,结果表明,以活细胞数为1.75×107个 mL 的酵母液处理草莓,常温下可延长保鲜期2～3d [16]。
梁泉峰等从腐败玉米中分离到一株间型假丝酵母(C .in ter m ed ia ),对苹果青霉病及洋葱黑曲霉病都具有显著的防治效果,接种致病菌后3225℃贮藏7d的苹果,对照组(未接酵母菌)的病斑直径达12mm,而接种酵母菌的果实未见病斑;同样接种酵母菌的洋葱25℃贮藏20d未见病斑,相同条件下对照组(未接酵母菌)则发生严重的黑曲霉病[17]。
近年来,我们课题组承担了国家自然科学基金项目“酵母菌对柑橘采后病害的生物防治及其防治机制的研究”,对应用拮抗酵母菌来保鲜果蔬的效果及机理进行了深入的研究,并取得了许多有意义的成果。
研究涉及到的拮抗酵母菌有隐球酵母、红酵母、假丝酵母等,涉及到的果蔬有柑橘、苹果、梨、葡萄、杨梅等,证实了隐球酵母、红酵母、假丝酵母对多种果蔬由霉菌导致的腐烂具有明显的抑制作用。
目前,我们正在利用基因工程技术来进行拮抗酵母菌的改造,期待能提高拮抗酵母菌的抑菌能力,扩大果蔬采后病害生物防治的研究领域,为微生物在果蔬采后生物防治中的应用打下良好的基础。
3 酵母菌对果蔬采后病害防治的机理产生抗菌物质是大部分生防细菌和生防霉菌的作用机制。
例如枯草芽孢菌(B23)能产生拮抗物质伊枯草菌素(itu rin)7,对多种重要果蔬的病原菌具有拮抗性;木霉能分泌吡喃酮,对由灰葡萄孢引起的草莓、苹果灰霉病有很强的抗生作用等[18]。
但酵母菌不产生抗菌物质。
酵母菌对果蔬采后病害防治的机理主要包括以下4个方面:1)营养或空间的竞争;2)酵母菌与病原菌的直接作用;3)酵母菌诱导寄主产生抗病性;4)其它物质对拮抗效果的影响。
3.1 营养或空间的竞争营养或空间的竞争是酵母菌产生生防作用的主要机制。
酵母菌由于对环境的适应性较强,在温度、湿度、pH值或渗透压不利于病原菌生长的情况下,这些拮抗菌能有效地利用果蔬表面或侵入点附近低浓度的营养物质而生长存活,利用果蔬表面或伤口的营养大量繁殖,尽可能快地消耗掉伤口营养,并占领全部空间,使得病原菌得不到合适的营养与空间条件,不能生息繁衍,从而抑制病害的发生[19]。
范青等研究了季也蒙假丝酵母(C.Gu illier m ond ii)接种到桃果实伤口上的生长动态,结果发现,在有病原菌存在的情况下,15℃培养72 h,酵母菌数可以增长34.4倍,而25℃下培养72h,酵母菌数可以增长45.6倍,这种高速的繁殖活动反映出拮抗菌与病原菌之间的营养竞争[13]。
D roby等在用拮抗酵母U S27来防治柑桔采后绿霉病病源菌指状青霉(P en icillum.d ig ita tum)时发现,向共存有拮抗菌和病原菌的柑桔伤口处添加营养物质,酵母菌U S27抑制柑桔绿霉病的效果降低[20];范青和田世平在研究中发现,相同浓度的拮抗菌细胞悬浮液比培养原液有更好的拮抗效果,原因很可能是培养液中的丰富营养削弱了拮抗菌与病原菌之间营养竞争的力度[13]。
3.2 酵母菌对病原菌的直接寄生作用许多酵母菌可以分泌胞外水解酶(几丁酶,Β21,32葡聚糖酶等),从而分解病原菌的细胞壁或菌丝体;某些酵母菌还可以附着在病原菌上,形成对病原菌的直接寄生作用。
Casto ria等在研究红酵母及隐球酵母对苹果采后病害的防治机制时发现,展青霉(P.ex p ansum)及灰葡萄孢(B otry tis.cinerea)细胞壁都可以诱导红酵母及隐球酵母产生胞外Β21,32葡聚糖酶;在利用展青霉作为唯一碳源时,隐球酵母的Β21,32葡聚糖酶活性高于红酵母,而隐球酵母对苹果采后青霉病的防治效果也好于红酵母[21]。
W isn iew sk i等的研究也发现,毕赤氏酵母(P.g u illier m ond ii)对苹果灰霉病的防治效果强于汉逊德巴利酵母(D eba ry o m y ces hansen ii),而前者的Β21, 32葡聚糖酶活性高于后者;同时W isn iew sk i还发现,毕赤氏酵母可以附着在灰葡萄霉菌丝体上从而对其进行破坏[22]。
3.3 酵母菌诱导寄主产生抗病性某些酵母菌在果蔬上大量生长,可以诱导宿主产生具有抑菌作用的物质;而有些酵母细胞可以诱导果蔬产生几丁酶、葡聚糖酶及其它酶类,这些酶类可以分解果蔬病原菌的细胞壁,从而抑制病原菌的生长[23];还有一些酵母菌可以使宿主细胞组织结构发生变化,增加宿主的抗病能力。
D roby等在研究假丝酵母C.oleop h ila对葡萄采后青霉菌的拮抗作用时发现,向葡萄果皮组织上添加假丝酵母细胞悬浮液可以增加乙烯的生物合成,诱导苯丙氨酸氨基裂解酶及植保素的积累,并能增加几丁质酶、Β21,32内切葡聚糖酶的活性,从而诱导宿主产生抗性[24];I ppo lito等发现拮抗酵母菌A u reobasid ium p u llu lans在苹果上可以显著提高几丁酶、Β21,32葡聚糖酶和过氧化物酶的活性[25];范青等的研究也发现拮抗酵母菌P.m e m branef aciens和C.g u illier m ond ii可以诱导苹果产生较高水平的几丁酶和Β21,32葡聚糖酶活性,增强对软腐病的抑制效果[26];E I2Ghaou th发现假丝酵母C.sa itoana在苹果伤口上可以诱导宿主细胞变形,产生乳突结构,抑制病原菌的入侵[27]。