环境条件对桃褐腐病菌生长和分生孢子萌发的影响

为了探明影响桃褐腐病发生和发展的因素,研究了温度、pH值、光照、营养条件等对桃褐腐病菌的生长和分生孢子萌发的影响。结果显示：桃褐腐病菌营养生长最适温度范围为20～25℃;最适pH值范围为6～7;光暗交替有利于该菌生长;供试的7种培养基中,PDA＋KNO3对生长最有利。分生孢子萌发的最适温度为25℃,最适pH值为8,最适营养液为1％蛋白胨液。     

Berberine as a natural compound inhibits the development of brown rot fungus Monilinia fructicola

Peach brown rot is a severe disease caused by ascomycete fungus Monilinia fructicola and is primarily controlled by chemical fungicides. However, the utilization of fungicides has caused many problems such as microbial resistance, environmental pollution, and fruit contamination. The growing concern for food safety requires alternative management methods that are safe to humans and benign to the environment. Here we report that the extract from Coptis chinensis (a Chinese herb named “Huang Lian”) demonstrates a strong inhibition to M. fructicola. The 50% effective concentration (EC₅₀) of C. chinensis extract to M. fructicola was only 0.91 mg/mL, while the EC₅₀s to other fungi such as Botrytis cinerea and Alternaria solani were 14.09 mg/mL and 27.35 mg/mL, respectively. These results indicate that the extract of C. chinensis has a specific inhibition to M. fructicola. Subsequent partitioning of the ingredients in C. chinensis extract revealed that berberine, the conventional anti-bacterial alkaloid, was the predominant ingredient that exerted robust inhibition against M. fructicola. The EC₅₀ and minimum inhibitory concentration (MIC) against M. fructicola were as low as 4.5 μg/mL and 46.9 μg/mL, respectively. Compared to berberine’s analogs in C. chinensis such as palmatine, berberine tannate and jatrorrhizine, berberine showed the strongest inhibition against M. fructicola. This finding provided insight into the structure–activity relationship between berberine and its analogs. Notably, not only can berberine prevent spore germination and hyphal growth, it also inhibits the activity of cutinase secreted by M. fructicola, implying the potential function of berberine in reducing the pathogenesis of M. fructicola. In addition, the strong in vivo inhibition of berberine against M. fructicola was observed with no visual cytotoxicity noted to peach fruits, even at berberine concentration of 400 μg/mL. Note that this was much higher than its MIC value (46.90 μg/mL). Overall berberine, as a natural compound, may be a promising candidate in control of brown rot.     

鹿蹄草素对桃褐腐病菌的抑制作用及其抑菌机理

 【目的】探讨鹿蹄草素对桃褐腐病菌的抑制活性及作用机理。【方法】采用生长速率法和果实接菌的方法测定鹿蹄草素对桃褐腐病菌菌丝体生长的抑制作用;采用倍比稀释法测定鹿蹄草素最小抑菌浓度（MIC）和最小杀菌浓度（MFC）;观测接上鹿蹄草素和桃褐腐病菌对桃果实多酚氧化酶（PPO）、过氧化物酶（POD）和苯丙氨酸解氨酶（PAL）活性的诱导情况;并用扫描电镜和透射电镜观察鹿蹄草素对桃褐腐病菌超微结构的影响。【结果】鹿蹄草素对桃褐腐病菌有较强的抑制活性,且浓度越高,抑制作用越强,其MIC为0.032 mg?ml-1,MFC为0.156 mg?ml-1;桃果实接上鹿蹄草素＋病原菌后,PPO、POD和PAL活性开始升高,并在整个试验过程中一直保持较高的水平;扫描电镜下可见,菌丝扭曲变形,菌丝体之间相互粘连,细胞壁破裂,表面出现絮状凝集物。透射电镜下可见,细胞壁明显变薄,细胞内部组成紊乱,细胞结构遭到严重的破坏。【结论】鹿蹄草素对桃褐腐病菌具有较强的抗菌作用;可使菌丝体超微结构发生改变。     

桃褐腐病菌对多菌灵抗性的AS-PCR检测技术

由于杀菌剂的长期大量使用, 植物病原菌对杀菌剂抗性问题日趋突出, 严重影响病害防治的效果?褐腐病是一种在全世界范围内广泛发生和流行的重要果树病害?本文以桃褐腐病菌Monilinia fructicola为例, 基于已知的多菌灵抗性机理, 即靶标β微管蛋白基因TUB2的点突变E198A, 建立了一种桃褐腐病菌对多菌灵抗性的等位基因专化性PCR检测技术?结果表明, 碱基错配?内参引物?退火温度?dNTPs?Taq DNA聚合酶?专化性引物浓度及配比等因素均对检测效率有影响?经过对以上因素的优化, 并对优化后的检测技术进行专化性及灵敏度评价, 证实该检测技术能特异性地鉴别桃褐腐病菌M. fructicola对多菌灵抗性基因型E198A, 且检测灵敏度可达到4.342 pg/μL病菌DNA, 有望在实践中推广使用?     

Molecular characterization of the two-component histidine kinase gene from Monilinia fructicola

Brown rot of stone fruit caused by Monilinia fructicola (G. Wint) Honey is one of the most common fungal diseases in California. In this study, two laboratory-induced iprodione-resistant (LIR) mutants of M. fructicola were characterized by osmotic sensitivity, virulence on prune and sequence of the two-component histidine kinase gene (Mfos-1). The LIR mutants showed more sensitivity to osmotic stress and lower virulence on prune than their wild-type parent. Analysis of deduced amino acid of Mfos-1 showed that this protein exhibited all the characteristic features of the two-component histidine kinase genes, including osmotic sensing domain, six 90-amino-acid repeat motifs (coiled coil region) and kinase core and response regulator domains. Comparison of DNA sequences of the Mfos-1 from LIR mutants and the wild-type sensitive (S) isolate showed that LIR mutants had single point mutations in the coiled coil region of Mfos-1.     

瑞香狼毒根的超临界萃取物抑菌活性和成分分析

瑞香狼毒(Stellera chamaejasme)为瑞香科(Thymelaeaceae)植物,广泛分布于我国西北、华北等地(刘欣等,2004)。据《神农百草经》记载,瑞香狼毒与大戟科(Euphorbiaceae)植物狼毒大戟(Euphorbia fischeriana)、月腺大戟(E.ebracteolata)同作狼毒入药,其性味苦平,全草有毒,有逐水祛痰、破积杀虫之功效(张慧文等,2006)。瑞香狼毒也是一种     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

草莓果生炭疽菌LysM效应子CfLysM2的致病功能分析

果生炭疽菌Colletotrichum fructicola侵染引起的草莓炭疽病是草莓生产中的重要病害。前期研究发现一个在果生炭疽菌侵染阶段高表达的含LysM结构域的候选效应子CfLysM2。为进一步研究CfLysM2的致病功能,运用同源重组方法构建CfLysM2缺失突变株ΔCfLysM2和回补菌株CfLysM2c。致病力测定结果表明,接种后15 d,ΔCfLysM2接种的草莓叶片病情指数显著下降。荧光定量PCR分析表明CfLysM2基因在果生炭疽菌接种草莓叶片后24 h相对表达量最高。利用转录组技术对野生型菌株(wild-type strain, WT)和ΔCfLysM2接种后24 h的草莓叶片样品进行比较分析,获得差异表达基因109个。CfLysM2的缺失导致宿主代谢途径尤其是次生代谢物生物合成通路的激活,说明CfLysM2可能在果生炭疽菌侵染草莓过程中通过靶向黄酮醇合成酶等代谢相关基因,抑制植物次生代谢产物合成及其介导的抗真菌免疫,保证其顺利侵染。该研究为揭示CfLysM2机理及对果生炭疽菌和草莓互作研究奠定了理论基础。     

桃褐腐病生防细菌FD6硝吡咯菌素合成基因簇的克隆及prnA功能分析

荧光假单胞菌（Pseudomonas fluorescens）FD6分离自福建闽侯青口青菜根围土壤,采用凹玻片法和离体果实接种法测定菌株FD6对桃褐腐病菌（Monilinia fructicola）的抑制能力。PCR法克隆硝吡咯菌素合成基因簇结构基因（prn）,利用同源重组构建prnA缺失突变体并分析其功能。结果表明,菌株FD6处理桃后可完全抑制桃褐腐病发生;细菌悬浮液对褐腐病菌分生孢子的萌发抑制率达93.18%,细菌培养滤液的抑制率为69.36%;细菌悬浮液对黄瓜根结线虫也具有较强的致死作用。序列分析表明荧光假单胞菌FD6硝吡咯菌素合成基因簇全长为5 868 bp,内含4个开放阅读框prnA、prnB、prnC和prnD,这4个基因组成1个共转录单元。系统发育进化树显示菌株FD6与P. protegens CHA0、Pf-5的prn基因相似性达94%。利用遗传学方法证实prnA基因是硝吡咯菌素合成的必需因子,此外该基因还影响2,4–二乙酰基间苯三酚和藤黄绿脓菌素的合成。     

茶树炭疽菌Colletotrichum fructicola的鉴定及系统发育分析

从福建地区不同品种茶树上分离获得ZHG、WSX、WHG、FTG、ZTG、AHD、WRG、SLH等8株能侵染茶树的病原菌,采用形态学和rDNA-ITS序列分析相结合的方法对其进行鉴定。结果发现,SLH菌株为胶孢炭疽菌Colletotrichum gloeosporioides,其余7个菌株均为Colletotrichum fructicola。C. fructicola为能侵染茶树的新记录种炭疽菌,将其ITS序列与寄主分别为油茶、茉莉和番石榴等木本植物的9株炭疽菌的ITS序列进行聚类分析,结果显示,来自茶树的7个C. fructicola菌株种内存在碱基突变或缺失,且与寄主为茶树、油茶、茉莉等植物的胶孢炭疽菌C. gloeosporioides遗传距离较近。