桃果实褐腐病拮抗菌的筛选、鉴定及其拮抗活性

以桃褐腐菌(Monilinia fructicola)为靶菌,采用稀释分离法和平板对峙法从桃园土壤中筛选出对病原菌有较强拮抗作用的菌株12a和14b。通过离体和活体实验对拮抗菌抑菌活性进行研究,经菌株形态、生理生化特性及16S rRNA基因序列分析进行菌株鉴定。结果表明:菌株12a和14b分别鉴定为死谷芽孢杆菌(Bacillus vallismortis)和高地芽孢杆菌(Bacillus altitudinis),其无菌发酵滤液和挥发性代谢产物对桃褐腐菌的生长有显著的抑制作用,其中12a挥发性代谢产物对其菌丝生长的抑菌效果更为显著(p0.05)。两株拮抗菌的菌液浸泡和熏蒸桃果实,延缓了桃褐腐病发病时间,有效地控制了病斑的扩展。     

不同芽孢杆菌对极早熟桃采后链格孢菌的防治效果及其发酵液挥发性物质成分分析

为了明确芽孢杆菌及其发酵液挥发性物质对极早熟桃采后主要致病菌链格孢菌的防治效果,以"津柳早红"桃为试材,通过有伤接种法、采后浸泡法和平板对峙法对三种芽孢杆菌(Bacillus strains) HB-2、B1和X的抑菌活性以及对极早熟桃采后品质保鲜效果的影响进行探究,同时利用平板倒扣法研究三种芽孢杆菌发酵液挥发性物质的抑菌活性,并采用顶空固相微萃取结合气相色谱-质谱联用法(SPME-GC-MS)对发酵液挥发性物质进行成分分析。结果表明:与对照组相比,三种芽孢杆菌发酵液均能显著抑制链格孢菌菌落的生长(p 0.05),同时可有效降低极早熟桃采后腐烂率,延缓果实VC含量、可滴定酸含量和硬度的降低(p 0.05),较好地维持果实品质,且效果HB-2 X B1;此外,三种芽孢杆菌发酵液产生的挥发性气体对抑制链格孢菌菌落的扩展有明显的抑制作用(p 0.05),抑菌率均在50%以上,并通过对其主要挥发性物质成分进行鉴定分析,从芽孢杆菌HB-2、B1和X发酵液中分别得到28种、31种和30种化合物,主要包括醇类、酯类、酮类、酸类、酚类、烃类和其他类化合物。芽孢杆菌HB-2、B1和X发酵液对极早熟桃采后病害的生物防治具有良好应用潜力。     

“美早”甜樱桃褐腐病病原菌的分离及内生拮抗细菌的筛选鉴定

为明确山东泰安地区"美早"甜樱桃褐腐病的主要病原菌,并进行内生拮抗细菌的筛选鉴定,采集当地具有褐腐病特征的"美早"甜樱桃进行病原菌分离,利用形态学和分子生物学技术鉴定,并将分离的病原菌菌株回接甜樱桃进行致病性验证;针对褐腐病原菌,从健康甜樱桃本身筛选优质拮抗菌株,开展生防研究。结果显示,导致"美早"甜樱桃褐腐病的病原菌为美澳型核果褐腐病菌(Monilinia fructicola),回接试验表明,与腐败"美早"甜樱桃自然发病特征一致;从健康甜樱桃本身筛选获得内生拮抗细菌X-16,经形态学、生理生化特征以及16S r DNA序列鉴定为特基拉芽孢杆菌(Bacillus tequilensis),抑制率达到(72. 27±0. 55)%,对拮抗菌X-16的菌悬液、发酵液和发酵上清液进行拮抗效果验证,发酵液和发酵上清液对褐腐病菌具有较好抑制效果,抑制率分别为(71. 93±0. 18)%和(67. 14±0. 43)%。结果表明,美澳型核果褐腐病菌为山东泰安地区"美早"甜樱桃褐腐病主要病原菌;该研究筛选出的内生拮抗细菌X-16抑菌效果显著,具有优质生防菌株的潜力。     

人参根腐病拮抗菌的筛选、鉴定及其抑菌活性

目的：从人参根际土壤中筛选出拮抗人参根腐病的菌株,初步研究其抑菌作用。方法：采用平板稀释法从人参根际土壤中分离获得菌株,以人参根腐病菌为靶标菌采用平板对峙法筛选出拮抗菌;通过形态学特征、生理生化特征及16S rDNA序列分析对筛选出的拮抗菌进行鉴定;检测拮抗菌无菌发酵液对根腐病菌菌丝生长、孢子萌发的影响,同时测定拮抗菌的抑菌谱。结果：分离获得一株对人参根腐病原菌拮抗作用较强的菌株HB-3,经鉴定菌株HB-3为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。菌株无菌发酵液对人参根腐病菌菌丝生长和孢子萌发均有显著抑制作用;菌株HB-3的抑菌谱较广,对根腐病菌、疫霉病菌、锈腐病菌、菌核病菌、灰霉病菌均有一定的抑菌活性。结论：筛选得到一株解淀粉芽孢杆菌HB-3,能显著抑制人参根腐病菌的生长,对人参根腐病有较强的生物防治效果。     

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）。     

枯草芽孢杆菌QDH-1-1对采后苹果青霉病的抑制效果

目的研究一种从番茄叶面分离获得的拮抗菌--枯草芽孢杆菌(Bacillus subtilis)QDH-1-1对苹果采后青霉病的防治效果.方法通过拮抗菌的不同处理、使用浓度及与钙配合等方法研究了枯草芽孢杆菌对青霉病的抑制作用.结果表明拮抗菌使用浓度明显地影响苹果中病斑发生和病斑扩展,且使用浓度越大,其抑病效果越好.当拮抗菌的使用浓度达到109CFU/mL时,可完全抑制接种在苹果上的青霉菌(Penicillium expansum,104CFU/mL)的侵染.用108～109CFU/mL的拮抗菌与2%CaCl2配合对苹果采后青霉病的抑制效果明显地好于单独使用相同剂量的拮抗菌或2%CaCl2.拮抗菌的不同处理对病原菌孢子萌发的抑制效果与体内试验结果一致.结论枯草芽孢杆菌QDH-1-1主要通过营养和空间竞争作用抑制病原菌生长.     

葡萄灰霉病生防菌株的筛选及其拮抗机理初探

以新疆哈密瓜、蟠桃及红枣中分离出的59株内生细菌为出发菌株,采用离体实验和活体实验,从中筛选出11株对葡萄灰霉菌具有较好抑制作用的菌株;经低温作用、农药耐受性及致病菌广谱性等复筛实验,从红枣中获得1株性能优良的目标菌株T2,该菌在0 ℃条件下对葡萄灰霉菌的抑菌率可达58.43%,农药耐受性达68.2%～98.4%,对致病菌具有广谱抗性,其中桃褐腐病菌和草莓灰霉菌的病原菌菌落直径均小于30 mm,葡萄炭疽菌病原菌菌落直径小于50 mm。经过形态、生理生化和16S rDNA同源性分析,确定菌株T2为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,该菌主要依靠菌体本身和重寄生作用对葡萄灰霉菌发挥拮抗作用。     

采后莲雾果实软腐病病原菌的分离鉴定和拮抗菌的筛选及防治机理

目的:以采后"大叶"莲雾为对象,对其病原菌进行分离及鉴定,筛选病原菌拮抗菌,并对其拮抗机理进行初步探究。方法:通过组织分离法得到引起莲雾果实软腐病的病原菌,采用平板对峙法筛选出拮抗菌,对病原菌、拮抗菌进行形态学和分子生物学鉴定。通过空间竞争和抗病相关酶活性测定探究其机理。结果:莲雾软腐病病原菌鉴定为小孢拟盘多毛孢(Pestalotiopsis microspora)。针对病原菌分离出拮抗菌143株,效果最明显的菌株命名为D-1,其抑菌圈直径达11.10 mm,鉴定为沙福芽孢杆菌(Bacillus safensis)。体内实验结果表明,拮抗菌D-1可有效抑制莲雾软腐病病斑的扩展,明显降低果实发病率,并且其能在果实伤口处快速定殖形成空间竞争。而且,D-1可明显提高果实抗病相关酶苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶的活性,从而提高果实的抗病能力。     

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。     

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)。     

Effect of Pichia membranaefaciens combined with salicylic acid on controlling brown rot in peach fruit and the mechanisms involved

BACKGROUND: Fruits are usually stored at low temperature to slow physiological metabolism, maintain quality and extend storage life. However, brown rot caused by Monilinia fructicola often occurs in peach fruit at low temperature, resulting in serious losses. As a promising alternative to the use of synthetic fungicides, the antagonistic yeast Pichia membranaefaciens showed a potential effect on controlling post‐harvest brown rot. To improve biocontrol efficacy of the yeast against fungal pathogen, the biocontrol efficacy of P. membranaefaciens combined with salicylic acid (SA) in inhibiting brown rot in peach fruit caused by M. fructicola was investigated. RESULTS: As compared to P. membranaefaciens used alone, the yeast combined with SA significantly reduced lesion diameter caused by M. fructicola, and increased the activities of chitinase, β‐1,3‐glucanase and peroxidase (POD), as well as enhanced the expressions of chitinase and β‐1,3‐glucanase genes in peach fruit. Moreover, SA at 2 mmol concentration did not affect yeast growth, but inhibited pathogen growth. CONCLUSION: The mechanism by which SA enhances the biocontrol efficacy of the antagonistic yeast against brown rot may be related to inducing biochemical defence responses in peach fruit, and a moderate inhibitory effect of SA on growth of M. fructicola. Copyright © 2008 Society of Chemical Industry     

桃采后病原菌种类及侵染规律研究

本试验以大久保、京玉等北方地区桃主栽品种为试材，研究采后病原菌种类、侵染和发病规律。结果表明：引起桃采后贮藏过程中腐烂的病原菌有8种。不同菌种在不同温度条件下的回接试验表明8种病原真菌的致病性有较大差异，其中灰霉菌（Botrytis cinerea）致病力最强；芽枝霉（Cladosporium sp.）最弱；根霉和黑曲霉多在常温中致病，且病程一般很短：灰霉、交链孢霉在桃低温和高温中均致腐。     

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。     

Difference between chitosan and oligochitosan in growth of Monilinia fructicola and control of brown rot in peach fruit

Chitosan (CS) and oligochitosan (OCS), as natural antifungal agents, have been primarily used as alternatives to synthetic chemical fungicides to control postharvest diseases of fruits. The effectiveness of these two agents on the growth of Monilinia fructicola to control brown rot has not yet been reported. Both spore germination and mycelial growth of M. fructicola were strongly inhibited by CS and OCS treatments in this study. Moreover, the inhibitory effect of both agents was more obvious on mycelial growth than on spore germination. By comparison of the half maximal inhibitory concentration (IC₅₀) of each, OCS treatment conferred a little better inhibitory effect on fungal growth in vitro, but the effectiveness of both treatments on brown rot control in peach fruit stored at 25 °C was very similar. The integrity of the plasma membrane of CS- or OCS-treated spores was obviously lower than that of the control. Cytoplasm leakage was significantly higher in chitosan-treated mycelia than that of the control. These results suggest that both chitosan and oligochitosan used in this study are promising natural fungicides for control of brown rot of peach.     

L-半胱氨酸处理对采后李果实褐腐菌的抑制作用

为探究L-半胱氨酸对采后李果实褐腐菌的抑制作用,本实验通过离体实验探究不同质量浓度（1、10、100、1000 mg/L）L-半胱氨酸对李果实褐腐菌Monilinia fructicola的抑制效果及可能的抑菌机制;采用损伤接种的方法评价L-半胱氨酸处理对李果实采后褐腐病的防治效果。结果表明：在所选的4 个L-半胱氨酸处理质量浓度中,只有1 000 mg/L L-半胱氨酸处理与对照相比可以显著延缓褐腐菌孢子的萌发,抑制菌丝体的生长,增加褐腐菌菌丝体胞外电导率和核酸物质的释放量,破坏褐腐菌孢子细胞膜的完整性（P＜0.05）;且在果实损伤接种实验中,1000 mg/L L-半胱氨酸处理可显著降低李果实的损伤接种发病率和病斑直径（P＜0.05）,而1、10、100 mg/L L-半胱氨酸处理对采后李果实褐腐病无明显控制效果。     

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。     

一株防治花生采后黄曲霉污染的拮抗菌的筛选及其防控效果

为预防花生储藏过程中的黄曲霉污染,从农田土中分离出2株拮抗芽孢杆菌,通过平板对峙法优选出1株作为研究对象,研究其对黄曲霉的抑制效果及对花生采后黄曲霉污染的防控效果。采用平板对峙法检测拮抗菌的抑菌广谱性并绘制生长曲线,并通过生理生化实验、形态特征观察及16S rDNA序列分析对该菌株进行鉴定。结果表明：优选出拮抗菌B419作为研究对象,其可有效抑制黄曲霉的生长,在拮抗菌浓度为1.0×10⁶ CFU/mL,黄曲霉孢子浓度为1.0×10³ CFU/mL时,黄曲霉孢子萌发抑制率达到98%;相比拮抗菌无菌发酵滤液和拮抗菌细胞悬浮液,拮抗菌发酵液防治花生黄曲霉污染效果最好;该菌株对互隔交链孢霉、枝孢霉、青霉、拟盘多毛孢等多种霉菌都有抑制作用,对大肠杆菌、酵母菌也有抑制作用;该菌株在培养4 h后进入对数期,14 h达到最大,26 h后进入衰亡期;该菌株被鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。综上,分离的拮抗菌B419对多种微生物都有抑制作用,不仅可以用来预防花生储藏过程中的真菌污染,在防治植物微生物病害方面也具有良好的应用潜力。     

Development of biocontrol agents from food microbial isolates for controlling post-harvest peach brown rot caused by Monilinia fructicola

An unconventional strategy of screening food microbes for biocontrol activity was used to develop biocontrol agents for controlling post-harvest peach brown rot caused by Monilinia fructicola. Forty-four microbial isolates were first screened for their biocontrol activity on apple fruit. Compared with the pathogen-only check, seven of the 44 isolates reduced brown rot incidence by >50%, including four bacteria: Bacillus sp. C06, Lactobacillus sp. C03-b and Bacillus sp. T03-c, Lactobacillus sp. P02 and three yeasts: Saccharomyces delbrueckii A50, S. cerevisiae YE-5 and S. cerevisiae A41. Eight microbial isolates were selected for testing on peaches by wound co-inoculation with mixtures of individual microbial cultures and conidial suspension of M. fructicola. Only two of them showed significant biocontrol activity after five days of incubation at 22 degrees C. Bacillus sp. C06 suppressed brown rot incidence by 92% and reduced lesion diameter by 88% compared to the pathogen-only check. Bacillus sp.T03-c reduced incidence and lesion diameter by 40% and 62%, respectively. The two isolates were compared with Pseudomonas syringae MA-4, a biocontrol agent for post-harvest peach diseases, by immersing peaches in an aliquot containing individual microbial isolates and the pathogen conidia. Treatments with isolates MA-4, C06 and T03-c significantly controlled brown rot by 91, 100, and 100% respectively. However, only isolates MA-4 and C06 significantly reduced brown rot by 80% and 15%, respectively when bacterial cells alone were applied. On naturally infected peaches, both the bacterial culture and its cell-free filtrate of the isolate C06 significantly controlled peach decay resulting in 77 and 90% reduction, respectively, whereas the treatment using only the bacterial cells generally had no effect. Isolate C06 is a single colony isolate obtained from a mesophilic cheese starter, and has been identified belonging to Bacillus amyloliquefaciens. The results have clearly demonstrated that isolate C06 has a great potential for being developed into a biocontrol agent.     

中草药提取液对桃果实采后致腐真菌抑制效应

为探讨甘草、高良姜、黄柏、北豆根、艾叶、连翘等6种中草药提取液对桃果实采后致腐真菌毛霉和青霉的抑制效果,采用生长速率法与活体果实接种孢子悬液法,对不同中草药提取液的抑菌效果进行对比,筛选出抑菌效果良好的中草药种类,并进一步通过体积浓度梯度稀释法,对其最小抑菌浓度(MIC)和最小杀菌浓度(MBC)进行了探讨。结果表明:不同中草药提取液对桃果实采后致腐真菌毛霉和青霉均有不同程度的抑制作用,其中甘草和连翘提取液的抑菌效果较强,黄柏和高良姜提取液的抑菌效果一般,艾叶和北豆根提取液的抑菌效果较弱。甘草和连翘提取液对毛霉的MIC均为培养液体积分数的25%、MBC均为培养液体积分数的50%;甘草和连翘提取液对青霉的MIC均为培养液体积分数的12.5%、MBC均为占培养液体积分数的25%。