采后番木瓜果实炭疽病病原菌分离鉴定及其拮抗菌的筛选

从采后发病番木瓜果实上分离炭疽病的病原菌,同时采用平板对峙法和滤纸片法从番木瓜果园土壤中筛选得到炭疽菌的拮抗菌W-2。通过形态学和分子生物学分别鉴定了病原菌和拮抗菌相关种属,并测试了拮抗菌W-2的拮抗活性。结果表明:番木瓜炭疽病病原菌鉴定为番木瓜炭疽菌Glomerella magna,拮抗菌W-2为萎缩芽孢杆菌(Bacillus atrophaeus)。拮抗菌W-2能够引起Glomerella magna菌丝体膨大和孢子畸形,且W-2浓度为1×108 CFU/mL时,对菌丝生长抑制作用最强,浓度为1×108和1×109 CFU/mL时,对孢子萌发的抑制作用最为显著(P<0.05)。活体生防实验表明,浓度为1×108 CFU/mL的W-2菌悬液处理能够有效控制番木瓜果实炭疽病病斑的扩展,并显著降低果实的发病率(P<0.05)。     

刺梨白粉病拮抗菌的筛选及其抑菌机制初探

为筛选出对刺梨白粉病具有良好抑制作用的拮抗菌,从健康刺梨及其根系土壤进行微生物分离,以刺梨白粉病病原菌(Sphaerotheca pannosa)为靶标菌株,采用叶盘法对拮抗菌进行筛选,通过形态学、生理生化特征以及16S rRNA对筛选的拮抗菌进行鉴定,测定其OD值绘制生长曲线,并探究拮抗菌对病原菌孢子萌发和菌丝的影响及其产酶情况。结果表明:共分离得到48株细菌,其中JK-6和JK-32具有显著抑制作用,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和空气芽孢杆菌(Bacillus aerius),对刺梨白粉病的防治效果达到了60%和57%;拮抗菌作用下致使病原菌的孢子在萌发过程中发生异常膨大现象,菌丝表面变得粗糙、膨大、缠绕和融断;产酶情况表明两株拮抗菌均可产生蛋白酶、纤维素酶以及果胶酶。为刺梨白粉病的生物防治提供了理论基础,具有进一步研究价值。     

芒果露水斑病病原菌分离鉴定及其拮抗菌筛选

目的:明确芒果"露水斑病"的病原菌,并对病原菌拮抗菌进行筛选,为病害的进一步防治提供理论基础。方法:采用扫描电镜观察病果表皮状况,通过组织分离法对发病样品进行病原菌分离,采用平板对峙法对病原菌拮抗菌进行筛选,最后对病原菌及拮抗菌进行形态学以及分子生物学鉴定。结果:明确"露水斑病"是由真菌引起,并鉴定为枝状芽枝霉(Cladosporium cladosporioides)。针对病原菌从芒果果园土壤中分离筛选出8株对其具有拮抗效果的菌株,其中细菌JS-8、酵母菌Y-1拮抗效果最为显著(p0.05),其抑菌圈直径分别达15.17 mm、14.93 mm。经形态特征、生理生化特征以及分子生物学将菌株JS-8、Y-1分别鉴定为唐菖蒲伯克霍尔德菌(Burkholderia gladioli)、汉逊德巴利酵母(Debaryomyces hansenii)。结论:"露水斑病"主要由枝状芽枝霉引起,唐菖蒲伯克霍尔德菌、汉逊德巴利酵母可作为该病害潜在生防菌株。     

小白杏软腐病病原菌的分离鉴定

小白杏软腐病腐烂组织中分离纯化出1株病原菌,并应用rDNA-ITS分子标记分析和传统真菌形态学鉴定相结合的方法,对病原菌进行了鉴定。结果表明:引起小白杏软腐病的病原菌为半知菌亚门(Deuteromycotina)、丝孢纲(Hyphomycetes)、丝孢目(Hyphomycetales)、丛梗孢科(Moniliaceae)、曲霉属(Aspergillus)、白曲霉(Aspergillus niger)。     

具有拮抗胶孢炭疽菌活性酵母菌的分离和筛选

从芒果果皮、叶片和芒果园土壤中分离筛选得到具有拮抗胶孢炭疽菌(Colletotrichum gloeosporioides)活性的酵母菌,研究其对芒果中胶孢炭疽菌的抑制作用。采用平板对峙法和活体实验进行筛选,采用形态学及r DNA-ITS序列分析等方法进行鉴定。通过平板对峙和活体实验筛选发现,菌株T18对芒果炭疽病病原菌胶孢炭疽菌的拮抗作用明显。抑菌实验表明,当T18菌悬液浓度为1×108cfu/m L时,对胶孢炭疽菌的抑菌半径为14.33mm。活体实验中,经T18处理的芒果果实炭疽病病斑直径仅为1.58mm,具有较强抑菌效果。通过菌落和菌体形态、生理生化、ITS序列分析对菌株T18进行鉴定,最终确定为尼泊尔德巴利酵母(Debaryomyces nepalensis)。     

常温条件下拮抗酵母菌对葡萄果实的抗性诱导研究

以从草莓果实上筛选的、对由灰葡萄孢(Botrytis cinerea)引起的灰霉病具有较好抑制作用的葡萄有孢汉逊酵母(Hanseniaspora uvarum)菌株为供试拮抗酵母菌,研究其对"巨峰"葡萄果实抗性的诱导。将葡萄果实分组,并分别进行喷洒和人工接入浓度为1×108cfu/mL的拮抗酵母菌悬液,常温条件下贮藏,观察果实感官品质变化,并测定抗性相关酶酶活变化。结果表明,拮抗酵母菌处理不影响果实感官品质,甚至可以较好地维持果实的感官品质;人工接入的拮抗酵母菌可以较好的诱导果实的POD、PAL、CAT、PPO及APX的酶活,而对喷洒拮抗酵母菌的整个果实可以较好的诱导POD、SOD、PAL及APX的酶活。整体而言,拮抗酵母菌可引起果实的应激反应,诱导寄主产生抗性。     

拮抗酵母控制樱桃番茄采后病害及其机制研究

通过体外和体内试验,从实验室保存的酵母菌中筛选对樱桃番茄采后黑斑病具有控制作用的拮抗酵母,考察所筛选的拮抗酵母对樱桃番茄自然腐烂的防治效果及对果实品质的影响。通过拮抗酵母对黑斑病病原菌链格孢孢子萌发、芽管伸长和菌丝结构的影响,在樱桃番茄伤口处和表面的生长动态,探讨其对病原菌的直接和间接抑制作用。采用RT-qPCR技术测定拮抗酵母对樱桃番茄抗性相关基因表达水平的影响,分析其对樱桃番茄果实抗病性的影响。结果表明,试验菌株中,卡利比克毕赤酵母对樱桃番茄采后黑斑病的控制效果最好,该处理组果实发病率仅为16.25%,显著低于对照组（68.76%）,并且该拮抗酵母还可显著降低樱桃番茄采后自然腐烂,缓减果实品质劣变。卡利比克毕赤酵母通过抑制链格孢孢子萌发、菌丝伸长以及寄生于菌丝上直接抑制病原菌生长,还在樱桃番茄伤口处和表面与链格孢竞争营养和空间而间接抑制病原菌生长。卡利比克毕赤酵母还能诱导樱桃番茄抗氧化基因（SOD,CAT和APX）以及病程相关基因（PR基因）（GLU和CHI）的表达。卡利比克毕赤酵母可以显著控制樱桃番茄采后黑斑病和自然腐烂,对果实品质无显著不良影响。该拮抗酵母可以通过抑制病原菌生长...     

烟草镰刀菌根腐病（Fusarium spp.）拮抗菌L210的鉴定及生防潜力评价

为筛选烟草镰刀菌根腐病（Fusarium spp.）的高效拮抗菌株,通过平板对峙、盆栽和大田复筛试验,从平顶山烟区烟株根际土壤中分离筛选拮抗菌;利用形态观察、生理生化测试和16S rDNA序列分析对拮抗菌进行鉴定,并对拮抗菌抗镰刀菌根腐病的机理进行研究。结果表明：从10份土壤样品中共分离土壤微生物菌株512株,其中以L210菌株对镰刀菌根腐病抑制效果最好,平板对峙抑菌直径71.4 mm,抑菌率为68.2%;盆栽和大田防病试验的发病率分别为0、6%,病情指数分别为0、1.5,防治效果分别为100%和77.9%。初步鉴定L210菌株为液化沙雷氏菌（Serratia liquefaciens）,其抑菌机理为溶解尖孢镰刀菌的菌丝,使其失去侵染能力,进而抑制病原菌的生长。菌株发酵液中含有几丁质酶和纤维素酶,可溶解病原菌的细胞壁,还可显著诱导提高烟苗中多酚氧化酶（PPO）、过氧化物酶（POD）和苯丙氨酸解氨酶（PAL）等防御酶的活性。因此,菌株L210在烟草镰刀菌根腐病的生物防治中具有较大的应用潜力。     

褪黑素采前喷施对采后番茄果实抗病性和贮藏品质的影响

为研究褪黑素采前喷施番茄植株对采后果实抗灰葡萄孢菌能力和品质的影响,本实验以'Ailsa Craig'番茄为材料,采用1、50、100、150 μmol/L褪黑素采前喷施番茄植株,测定采后番茄果实品质、相关酶活力及基因表达量。结果表明:与蒸馏水喷施对照组相比,100 μmol/L褪黑素采前喷施处理可以显著降低番茄果实接种灰葡萄孢菌后的发病率、病斑面积(P0.05),诱导提高果实中几丁质酶、β-1,3-葡聚糖酶、多酚氧化酶和苯丙氨酸解氨酶活力,增加抗病相关基因PR1、NPR1、PI Ⅱ和LoxD的相对表达量,提高VC、可滴定酸、可溶性糖和可溶性蛋白含量。综上,100 μmol/L褪黑素采前处理对采后番茄果实抗灰葡萄孢菌的效果最佳,褪黑素可提高番茄果实的抗病相关酶活性和贮藏品质。     

桃褐腐病菌拮抗酵母的筛选、鉴定及其抑菌活性初探

以北京平谷桃果实表皮上分离得到的45株酵母菌为出发菌株,采用平板对峙法,筛选对桃褐腐病菌（Monilinia fructicola）具有较高拮抗作用的生防酵母菌。结果表明,共筛选出具有较好抑制效果的酵母菌6株,其中菌株MLL-9-1拮抗活性最强,对桃褐腐病菌的抑菌率达77.91%;活体筛选试验结果表明,该菌株对桃褐腐病的抑制率达50.30%。生防相关性状检测结果显示,该菌株能够产生蛋白酶,透明圈直径达30.5 mm,说明该菌主要通过分泌蛋白酶达到抑制病原菌的效果。经形态学、生理生化试验及26S rDNA序列分析,鉴定该菌为葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）。     

海水酵母菌的分离鉴定及其在果实贮藏保鲜中的应用

本文研究了海水酵母菌对果实腐烂病的防治效果以及其在梨和苹果果实采后贮藏保鲜中的作用。通过显微观察结合分子生物学方法对分离的菌株进行鉴定,结果表明该菌与酵母菌(Galactomyces geotrichum)同源性相近,属于酵母菌类。通过平板抑菌试验及活体试验研究该菌对腐烂病菌的拮抗作用,发现该菌对腐烂病菌具有明显的拮抗作用,活体试验结果表明,5×107 CFU/m L该菌对接种了5×104 spores/m L病原菌的梨和苹果果实腐烂病具有良好的防治效果。经该菌悬浮液浸泡处理的采收后的梨果实,贮藏20 d时的腐烂率比对照少20%,贮藏60 d苹果的腐烂率比对照减少了30%。另外,该菌处理能够延缓梨和苹果果实的软化速率,保持果实较高的营养品质。本研究表明,从海水中分离的酵母菌在果实腐烂病的生物防治和果实保鲜方面具有极大的应用潜力。     

Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis

The consumption of fresh-cut fruit has substantially risen over the last few years, leading to an increase in the number of outbreaks associated with fruit. Moreover, consumers are currently demanding wholesome, fresh-like, safe foods without added chemicals. As a response, the aim of this study was to determine if the naturally occurring microorganisms on fruit are “competitive with” or “antagonistic to” potentially encountered pathogens. Of the 97 and 107 isolates tested by co-inoculation with Escherichia coli O157:H7, Salmonella and Listeria innocua on fresh-cut apple and peach, respectively, and stored at 20 °C, seven showed a strong antagonistic capacity (more than 1-log unit reduction). One of the isolates, CPA-7, achieved the best reduction values (from 2.8 to 5.9-log units) and was the only isolate able to inhibit E. coli O157:H7 at refrigeration temperatures on both fruits. Therefore, CPA-7 was selected for further assays. Dose-response assays showed that CPA-7 should be present in at least the same amount as the pathogen to adequately reduce the numbers of the pathogen. From the results obtained in in vitro assays, competition seemed to be CPA-7's mode of action against E. coli O157:H7. The CPA-7 strain was identified as Pseudomonas graminis. Thus, the results support the potential use of CPA-7 as a bioprotective agent against foodborne pathogens in minimally processed fruit.     

采前喷施壳聚糖复合膜对猕猴桃软腐病的防控及其保鲜作用

以‘贵长’猕猴桃为试材,通过病原菌分离、致病性测定和DNA测序鉴定了修文县猕猴桃软腐病病原菌,并选用壳聚糖、钙盐和糊精分别与茶多酚、甘氨酸、柠檬酸、抗菌肽混合制备和筛选了复合膜剂,研究了采前幼果期和壮果末期果面喷施壳聚糖复合膜对猕猴桃软腐病的防控及其保鲜作用。结果表明,修文县猕猴桃软腐病病原菌为葡萄座腔菌（Botryosphaeria dothidea）和拟茎点霉菌（Phomopsis sp.）;采前喷施不同壳聚糖复合膜对其软腐病的防效均达60%以上（添加茶多酚防效86.54%、甘氨酸防效61.54%、柠檬酸防效71.15%、抗菌肽防效69.23%）,显著降低丙二醛（MDA）积累,提高果实的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）活性,诱导增强果实的抗病性。同时,该复合膜能有效地增加果实单果质量和体积,显著提高果实VC、可溶性固形物、可溶性总糖、可滴定酸、叶绿素和蛋白质的含量,促进猕猴桃产量的增加和品质的改善。此外,施用该复合膜还能有效提高和维持果实硬度、降低果实呼吸强度以及果实质量损失率和营养物质的损失,明显抑制果实可溶性固形物和可溶性总糖含量的上升速率和延缓组织的衰老软化,从而提高了猕猴桃耐贮性。研究结果为猕猴桃优质栽培、病害有机防控和果实绿色保鲜提供了科学依据和新途径。     

灰霉病菌拮抗放线菌的筛选鉴定及对草莓的防腐保鲜效果

为丰富草莓采后病害拮抗菌资源,以草莓灰霉病菌（Botrytis cinerea）为指示菌,采用琼脂块法对草莓根际土壤分离的78 株放线菌进行筛选,并测定拮抗效果最强的菌株T3-5的拮抗谱。根据形态学特征、生理生化特性及16S rDNA序列进行鉴定,并初步研究其无菌发酵液对贮藏草莓腐烂发生及果实品质的影响。结果表明：草莓根际土壤分离的菌株T3-5对草莓灰霉病菌具有较强拮抗作用,拮抗圈直径为22.5 mm;对3 株常见细菌和6 株果蔬采后病原真菌均具有不同程度的抑制效果。该菌株经鉴定为黄暗色链霉菌（Streptomyces xanthophaeus）。菌株无菌发酵液能够显著降低贮藏草莓腐烂率（P＜0.05）,延缓果实硬度、可溶性固形物、可滴定酸及VC的降低。研究表明拮抗链霉菌T3-5对贮藏草莓具有良好的防腐保鲜作用,在草莓采后贮藏和生物保鲜中具有潜在的应用价值。     

晚熟桃采后褐腐病致病菌的分离鉴定及拮抗菌对其防治效果的研究

为了明确晚熟桃采后褐腐病的主要致病菌以及拮抗菌对其防治效果,以晚熟桃品种"映霜红"为试材,采用传统的形态学方法和18S-rDNA分子生物学方法对褐腐病病原菌进行分离鉴定,同时,进一步选用四种拮抗菌对晚熟桃褐腐病进行预防处理,研究其对晚熟桃褐腐病的生物防治效果。结果表明,从晚熟桃上分离的褐腐病病原菌属于真菌界子囊菌门(Ascomycota),柔膜菌目(Helotiales),核盘菌科(Sclerotiniaceae),核盘菌属(Sclerotinia),该病菌有伤接种发病率为100%。四种芽孢杆菌B001、B1、HB-2、B579对褐腐病病斑扩展均具有较好抑制效果,其中,B001、B579提前1 d和2 d预防效果显著好于0 d预防效果(p<0.05);B1、HB-2提前1 d预防处理显著好于其他两组(p<0.05)。四种拮抗菌还能够抑制晚熟桃贮藏过程中的V_C、可溶性固形物、可滴定酸含量降低和硬度下降。芽孢杆菌在晚熟桃采后褐腐病的生物防治中具有较好的应用潜力。     

Biological control of Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative mechanisms of antagonism

Treatment of stone fruits (apricot, peach and nectarine) with Pantoea agglomerans strain EPS125 decreased the incidence and diameter of lesions of brown rot caused by Monilinia laxa and soft rot caused by Rhizopus stolonifer. Root control was achieved on fruits either wounded and subsequently inoculated with the pathogens or non-wounded and naturally infected from orchards. The efficacy of biocontrol was dependent on the concentration of the biocontrol agent and pathogen. At medium to low pathogen dose, optimal EPS125 concentrations were above 10(7) CFU ml(-1). The median effective dose (ED(50)) of EPS125 was 4.5x10(4) in M. laxa and 2.2x10(5) CFU ml(-1) in R. stolonifer. However, EPS125 was more effective in M. laxa than in R. stolonifer as indicated by the ratio between ED(50) of the biocontrol agent and pathogen (K(z)/K(x)) which was 166 and 1263, respectively. Interactions between the strain EPS125 and the fruit surface, and M. laxa and R. stolonifer, were studied to determine the mechanisms of protection from postharvest rots. The strain EPS125 colonizes, grows and survives on stone fruit wounds. Significant inhibition of conidial germination and hyphal growth of R. stolonifer and M. laxa was achieved when the fungal and EPS125 cells were cocultivated on peel leachate or nectarine juice. However, no effect was observed when the antagonist and the pathogen cells were physically separated by a membrane filter which permits nutrient and metabolite interchange. Therefore, a direct interaction between the strain and the pathogen cells is necessary for antagonism, without a significant contribution of the production of antibiotic substances or nutrient competition. Preemptive exclusion by wound colonization and direct interaction with the pathogen is proposed as the mechanism of biocontrol.     

芋头软腐病拮抗菌的筛选及生防效果研究

分别从蔬菜田土壤和采后芋头表面黏附土壤中分离纯化到267和244 株菌株,通过平板拮抗实验,发现其中6 个菌株对胡萝卜软腐欧文氏菌（Erwinia carotovora subsp. carotovora,Ecc）产生了清晰且明显的拮抗圈（直径＞10 mm）。利用芋头对它们的生物防控效果进行复筛,结果发现BGP14菌株发酵液处理芋头的发病率仅为6.7%,显著低于其他5 个菌株（P＜0.05）。根据BGP14的形态学特征、芽孢产生及16S rRNA和gyrB基因的部分核酸序列,该菌株被鉴定为解淀粉芽孢杆菌BGP14（Bacillus amyloliquefaciens）。BGP14在芋头伤口上具有较强的定殖能力,并将病原菌Ecc的群体数量压制在一个较低水平。BGP14与病原菌Ecc联合处理芋头的苯丙氨酸解氨酶（phenylalanine ammonia-lyase,PAL）活性显著高于其他处理。以上结果表明,解淀粉芽孢杆菌BGP14对病原菌E. carotovora具有强大的拮抗活性,能够有效防治贮藏期芋头软腐病,具有潜在的应用开发前景。     

Control of <Emphasis Type="Italic">Penicillium Expansum</Emphasis> and <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> on Apple Fruit by Mixtures of Bacteria and Yeast

Antagonistic activity of the mixtures Rahnella aquatilis–Rhodotorula glutinis and R. aquatilis–Cryptococcus laurentii was assessed against Penicillium expansum (cause of blue rot) and Botrytis cinerea (cause of grey rot) on apple fruit at 4 °C and 95% relative humidity (RH). Under these cold-storage conditions, the mixture R. aquatilis–R. glutinis inhibited the development of B. cinerea and P. expansum in apples stored for 40 days and reduced the incidence of disease produced by these moulds to nearly zero. The other mixture, R. aquatilis–C. laurentii, was less effective; the incidences of the grey rot and the blue rot were about 25% and 15%, respectively. Population dynamics of the mixtures showed that the growth of R. aquatilis was strongly stimulated by the presence of the yeast R. glutinis, but in the case of the mixture R. aquatilis–C. laurentii, the same effect could not be observed. In this study, it was demonstrated that the combination of two microorganisms with different requirements and antagonistic abilities resulted in a successful mixture against the two pathogen molds, B. cinerea and P. expansum. In addition, it was also proved that it is possible to improve the biocontrol of these pathogens without increasing the inoculum size of the antagonist (alone or in mixtures), which was always 10⁶cell/ml, despite the high concentration of the pathogen (10⁶ conidia/ml) utilized.     

桃杏果实内生细菌多样性分析及软腐病原菌的分离与验证

为了解桃和杏采后果实内生细菌群落组成,并对潜在病原细菌进行筛选与验证,为其贮藏保鲜、软腐病害防治等相关研究奠定基础,利用高通量测序技术,对新疆地产油桃和库车小白杏采后果实内生细菌群落组成进行分析;同时,利用微生物传统分离培养,采用16S rDNA序列分析对潜在病原细菌进行分子鉴定,并利用离体接种法和再分离法,对相关菌株的致病能力进行验证。研究结果表明,油桃和库车小白杏内生细菌共包括128个操作分类单元,涉及9个门117个属,其中变形杆菌门(Proteobacteria)为绝对优势菌门,其次为厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在属水平上,油桃以泛菌属(Pantoea)、芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、拟杆菌属(Bacteroides)等菌属为优势菌群;库车小白杏以葡糖杆菌属(Gluconobacter)、泛菌属(Pantoea)、克里斯滕森菌(Christensenella)、普氏菌属(Prevotella)等菌属为优势菌群。通过分离筛选获得了1株潜在采后致腐病原细菌XAAS-P1,经分子鉴定初步确定其归属于泛菌属(Pantoea)。XAAS-P1对采后油桃和库车小白杏均具有较强的致腐能力,并呈现出与自然软腐一致的病状。接种4 d后,XAAS-P1致腐率可达100%,与阴性对照组相比,接种组样品的软腐现象提前了1 d。对接种组病灶中的微生物进行再分离、鉴定,结果显示,菌株XAAS-P1是病灶组织中的绝对优势菌群,证明了菌株XAAS-P1是导致采后油桃和库车小白杏软腐变质的主要病原细菌。研究表明,桃和杏果实中存在丰富多样的内生细菌,其内生泛菌XAAS-P1可导致采后油桃和库车小白杏的软腐变质。