稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻绿核菌无性孢子形成过程及厚垣孢子萌发率测定

对马铃薯蔗糖人工培养基(PSA)上绿核菌(稻曲病菌)不同培养时期的产孢情况进行了系统的扫描电镜观察。研究结果表明,在培养的前期(前20d),菌落表面往往形成集结状菌丝结构,其上开始产生大量分生孢子;一些分散的菌丝上也可产生少量的分生孢子。而在培养的后期,菌落表面往往形成黄色子实体,内部集生大量厚垣孢子。说明绿核菌在人工培养基上前期以形成分生孢子为主,后期则以厚垣孢子为主,且厚垣孢子的量远远大于分生孢子。萌发试验表明,成熟的厚垣孢子会随着保存时间的延长萌发率急剧下降。因此,新鲜的成熟厚垣孢子是最为理想的接种体。     

进境美国加州脐橙中丁香疫霉Phytophthora syringae的截获

从产自美国加利福尼亚州的新鲜脐橙样品中发现多个腐烂病果,通过分离培养得到3个疑似丁香疫霉Phytophthora syringae菌株,对3个菌株进行形态学研究、致病性测定和分子序列比对分析。结果表明病菌在V8A培养基上菌落稀疏、平铺,呈星状,菌丝紧贴培养基生长或埋于基质内生长;在PDA培养基上菌落呈菊花花瓣状,菌丝致密,乳白色;游动孢子囊和菌丝膨大体在无菌水和土壤浸出液中黑暗条件下48h后产生;菌株为同宗配合,卵孢子在带有新鲜脐橙果实组织或杜鹃叶片的V8A培养基中大量产生;创伤接种脐橙果实,7d后接种脐橙出现典型的褐腐症状;通用引物ITS1/ITS4扩增测序,Blastn分析表明序列与GenBank中P. syringae序列相似性为99%。依据上述研究结果,将分离获得的3株菌鉴定为丁香疫霉Phytophthora syringae,系国内首次截获的一种植物检疫性真菌病害。     

稻曲病菌的Nested PCR检测

为设计稻曲病菌(Ustilaginoidea virens)专化性PCR引物,测定了1991-2001年采集的多个水稻品种、不同水稻产区的菌株的ITS和5.8S rDNA区序列。U. virens的ITS1、ITS2和5.8S rDNA区域的长度为 624-625bp, 序列高度保守。在与麦角菌科其它种比较的基础上,设计了U. virens专化性嵌合引物。采用PCR方法可以灵敏地检测目标真菌,并且与传统的组织观察结果很好地吻合。这一结果为深入研究稻曲病的侵染规律和建立田间早期诊断技术提供了可能。     

一株产赭曲霉毒素A黑曲霉的筛选与鉴定

【目的】筛选出可产生赭曲霉毒素A (OTA)的霉菌菌株。【方法】利用CYA和YES培养基从实验室32个霉菌样品中筛选目的菌株。利用高效液相色谱-荧光检测法对OTA产生菌进行初筛,利用高效液相色谱-质谱联用对OTA初筛菌株进行复筛。通过菌落形态、菌丝及分生孢子形态、ITS DNA序列、β-Tubulin基因序列及Calmodulin基因序列分析等鉴定目的菌株。【结果】得到一株OTA产生菌株1062,该菌株能在25 °C条件下,在CYA、YES和CA培养基中很好生长。结合形态学、对培养基的要求以及上述3个基因序列的进化树分析,该菌株属于黑曲霉(Aspergillus niger)。【结论】菌株1062具有OTA产生能力,是一株黑曲霉。     

金沙江干热河谷地区芒果畸形病的病原菌

从金沙江干热河谷地区采集芒果畸形病组织,运用柯赫氏法则证实分离物MG6为该病的致病菌。菌株MG6在马铃薯蔗糖琼脂培养基（PSA）上菌丝白色,无色素产生,米饭培养基浅粉红色;在康乃馨叶片培养基（CLA）上以单瓶梗或复瓶梗假头状产孢,不产生链状孢子;小型分生孢子卵形或长椭圆形,具0－1个分隔,3.1－10.2×1.5－2.2μm;大型分生孢子呈镰刀形,通常3个分隔,18－38×1.8－2.4μm。EF-1α测序结果在Fusarium数据库中进行同源性分析显示,菌株MG6与F. mangiferae的同源性最高,达99.68%。综合培养性状、形态学特征和EF-1α序列分析,将菌株MG6鉴定为Fusarium mangiferae。     

稻曲病菌毒素的活性测定、抗体制备与细胞定位

稻曲病菌在PD 液体培养基中生长良好,并能产生对植物细胞具有高度生物抑制活性的毒素。生物学活性测定袁明,用100%的甲醇能提取稻曲病菌液体培养物中的粗毒素。粗毒素对小麦胚根胚芽的生长有强烈的抑制作用。把毒素主要成分Ustiloxin A 和BSA 偶联后,制备了抗血清,ELISA 检测表明用两种偶联剂偶联所制备的抗体效价分别为1∶20000和1∶6000。进一步的免疫胶体金标记分析表明,所制备的抗体能与茼丝中分泌的毒素特异性结合,说明所获得的抗体是特异性的。     

武汉梅花炭疽病病菌的多样性研究

炭疽病是梅花（Prunus mume）栽培中的重要病害,对梅花的栽培构成严重威胁。本研究从武汉发病的梅花叶片样品上分离、获得了170个炭疽病菌菌株,它们在形态特征、致病性、分子遗传水平等方面都表现出较大的差异。按菌落形态、色素分泌、拟菌核产生、分生孢子及孢子梗形态和大小等形态特征将梅树炭疽病菌分为7种类型,其中Ⅵ型和Ⅶ型菌株在PDA培养基上可以连续产生大量的有性后代。7种类型的菌株只能侵染梅花、樱树、梨树、苹果、桃树、杏树等蔷薇科园艺植物,并且存在着明显的致病力分化,但不侵染吉祥草、柑桔、大叶黄杨、豇豆、紫荆、高粱等供试的其它科植物。依据致病力可将梅树炭疽病菌分为强、中、弱3类。ITS序列表明它们均属于胶孢炭疽（Colletotrichum gloeosporioides）。对其中7种类型36个梅树炭疽病菌菌株的进行了RAPD聚类分析,在55%相似水平上,供试菌株可以分为3组,所聚类群与形态学类型和致病力分化所形成的强、中、弱3类没有明显的相关性。表明梅花炭疽病菌菌株间存在丰富的遗传多样性。     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

Histopathological studies of sclerotia of Rhizoctonia solani parasitized by the EGFP transformant of Trichoderma virens

Aims:  The aim of the study was to investigate the antagonistic interactions of Trichoderma species against Rhizoctonia solani sclerotia by enhanced green fluorescence protein (EGFP)-tagged transformant of Trichoderma virens TY009.
Methods and Results:  An EGFP was used as a report gene for transforming T. virens strain, and a stable EGFP transformant GF5 was obtained with the mycoparasitic activity against sclerotia of R. solani . Observation of parasitized sclerotia by fluorescence microscopy showed hyphae of transformant GF5 was able to invade into sclerotia and its colonization was mainly intercellar with uniformly distributed mycelium in sclerotia. The host cells were colonized, penetrated, and then the whole cells were replaced by transformant GF5 hyphae. Chlamydospores were seen after 10 days but mature ones after 20 days. Sclerotia became soft and decayed after 40 days but a few cells seemed not to be colonized completely.
Conclusions:  Trichoderma virens was able to parasitize sclerotia to make sclerotia soft and decayed, and its colonization was mainly intercellar in sclerotial tissues.
Significance and Impact of the Study:  This is first report of parasitism of sclerotia of R. solani by EGFP-tagged transformant, providing useful information for using T. virens as effective biocontrol agent.     

稻绿核菌(稻曲病菌)分离方法的比较研究

作者对不同情况下稻曲病菌的分离方法进行了比较研究。结果表明成熟早期稻曲球上的绝大多数新鲜的厚垣孢子具有萌发能力,及时进行分离培养是病原菌成功分离的关键。随保存时间的延长,厚垣孢子萌发能力急剧下降;消毒处理可杀死大部分的厚垣孢子。菌核可长期保存并保持极高的萌发生长能力,是稻曲病菌分离最为理想的材料。稻曲球中部的致密菌丝组织分离难度较大,只能作为稻曲病菌分离的一种补救方法。     

Role of two G-protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens

G-protein alpha subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein alpha subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Galphai class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of Rhizoctonia solani and Sclerotium rolfsii in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The tgaA mutants germinated by straight unbranched germ tubes, while tgaB mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both tgaA and tgaB mutants and the wild type overgrew, coiled, and lysed the mycelia of R. solani, but tgaA mutants had reduced ability to colonize S. rolfsii colonies. In the soil plate assay, both mutants parasitized the sclerotia of R. solani, but tgaA mutants were unable to parasitize the sclerotia of S. rolfsii. Thus, tgaA is involved in antagonism against S. rolfsii, but neither G protein subunit is involved in antagonism against R. solani. T. virens, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.     

稻曲病菌毒素的活性测定、抗体制备与细胞定位

稻曲病菌在PD液体培养基中生长良好,并能产生对植物细胞具有高度生物抑制活性的毒素。生物学活性测定表明,用100％的甲醇能提取稻曲病菌液体培养物中的粗毒素。粗毒素对小麦胚根胚芽的生长有强烈的抑制作用。把毒素主要成分Ustiloxin A和BSA偶联后,制备了抗血清,ELISA检测表明用两种偶联剂偶联所制备的抗体效价分别为1：20000和1：6000。进一步的免疫胶体金标记分析表明,所制备的抗体能与茵丝中分泌的毒素特异性结合,说明所获得的抗体是特异性的。     

Genetic Diversity of Rice False Smut Fungus, Ustilaginoidea virens and its Pronounced Differentiation of Populations in North China

Rice false smut caused by Ustilaginoidea virens is an important constraint affecting rice yield and quality in Asia. In China, rice false smut is especially severe in the japonica rice-growing areas in the North China. Nothing is known neither about the diversity of the pathogen in this region nor about the characteristic of its local population. In this study, 110 U. virens isolates sampled from Liaoning and Beijing of North China were analysed using amplified fragment length polymorphism (AFLP) markers to primarily understand the genetic diversity of this pathogen and its population characteristics. At the genetic distance of 0.32, all isolates were divided into two groups. Group A consists of two subgroups differentiated at the genetic distance of 0.55, subgroup 1 included all isolates from Liaoning with an average genetic similarity over 0.82 and subgroup 2 included 27 isolates from Beijing with similarity of 0.74. Group B consists of 28 isolates from Beijing having a diversity of 0.054. The isolates from the Liaoning province, where rice false smut has generated different take-all epidemic for 20 years, showed a genetic diversity of 0.305, which was approximately equally distributed within and among populations. Whereas genetic diversity was 0.458 among isolates from Beijing, an extremely high level of genetic differentiation among 55 isolates was observed in this disease hotspot. Our results suggested that the populations among different locations where sexual stage of the pathogen was rare to be found within ecological region were similar, and the variation of this pathogen has mainly arisen via asexual mechanisms. The migration through human activities in breeding perhaps provides a means of transporting the pathogen from one region to another.