白粉寄生孢寄生黄瓜白粉菌的特性研究

采用白粉寄生孢（Ampelomyces quisqualis Ces.AQ）接种离体条件下赛璐酚上黄瓜白粉菌［Sphaerotheca fuliginea (Schlecht)Poll.］和活体条件下黄瓜白粉菌后,通过棉兰染色和显微观察分析,初步明确了白粉寄生孢的侵染寄生过程。白粉寄生孢分生孢子产生芽管可入侵黄瓜白粉菌的分生孢子、菌丝、分生孢子梗;有时黄瓜白粉菌串生的分生孢子、分生孢子梗可被2~3条白粉寄生孢的菌丝寄生,随着寄生过程的进一步发展,黄瓜白粉菌的分生孢子梗基部膨大成无色的椭圆形或球形,其上逐渐产生由黄色至褐色的白粉寄生孢的分生孢子器,器内含有大量的分生孢子。     

茎基腐亚洲镰孢菌侵染抗感小麦品种的组织病理学观察

小麦茎基腐是由多种镰孢菌侵染的世界性土传病害,亚洲镰孢菌(Fusarium asiaticum)是我国冬小麦主产区茎基腐镰孢菌的优势种群,对小麦生产造成巨大损失。本研究利用绿色荧光蛋白报告基因标记亚洲镰孢菌,研究其侵染抗感小麦的病理组织学过程,建立了茎基腐病菌与寄主互作的直观性的研究体系,对病害防治及抗病育种具有重要意义。基于PEG-CaCl_2介导原生质体转化法将gfp导入亚洲镰孢菌株CF0915,对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性及致病力分析,选取与野生型表现相近的转化子进行侵染分析。结果表明,绿色荧光蛋白基因(gfp)与潮霉素基因(hyg)PCR扩增表明gfp已整合入真菌基因组中,转化子菌丝与分生孢子表现强烈绿色荧光信号,gfp能够在转化子中稳定遗传,菌落形态、生长速度及致病力与野生型菌株无显著差异;将gfp标记病菌分生孢子接种感病品种1 d后,大量孢子附着于根毛及根表皮细胞开始萌发,接种2 d后观察到抗性品种分生孢子萌发;感病品种接种3 d后,菌丝直接侵入表皮细胞或沿表皮细胞间层定殖生长,扩展至皮层组织,8 d后菌丝从根部迅速扩展至茎基部,至第10 d大量菌丝充塞根皮层细胞,叶鞘维管束也被菌丝侵染,并产生大量大型分生孢子,植株表现褐色病斑,14 d后根部及茎维管束被大量菌丝体填充,而后产生大量厚垣孢子,至25 d大部分感病品种幼苗萎蔫死亡;与感病品种相比,抗性品种在整个侵染过程中表现时间滞后。本研究对引起茎基腐病的亚洲镰孢菌侵染小麦的组织学过程观察,为病菌致病机理的阐释及抗病资源的利用提供了重要理论依据。     

玉米大斑病菌越冬生态研究

1.陕西关中夏玉米栽培区玉米大斑病菌主要越冬菌态是病斑内菌丝体,其次是病残体上的分生孢子。干枯病叶中的菌丝体和病斑表面的分生孢子可存活两年以上。2.越冬病斑内菌丝体可从气孔抽生出新分生孢子梗或由营养菌丝分化出分生孢子梗产生孢子。产孢温度范围为10-35℃,适温15-28℃,并要求保持90%以上的相对湿度或叶面呈湿润状态。3.地面以上的病残体堆积物或秸垛是主要越冬基地,因病残体绝对数量多,越冬率高而稳定,其次是散布地面的病残体。越冬病源出现时期相当长,可以作为玉米大斑病初侵染及后续侵染菌源。     

荔枝炭疽病菌生物学特性的研究

 本文主要研究了温度、湿度、pH值、光照、营养对荔枝炭疽病菌(Colletotrichum gloeosporioides Penz.)生长、产孢和孢子萌发的影响。该菌菌丝生长的温度范围为8~38℃,最适28℃;产生分生孢子的温度范围为12~36℃,最适28~32℃;分生孢子萌发的温度范围为8~38℃,最适28~32℃。在pH 3~10的范围内该菌均能生长和产孢,菌丝生长最适pH 5~6;产生分生孢子最适pH3~4,分生孢子萌发最适pH 6~7。分生孢子在饱和湿度或水滴中萌发快,相对湿度低于85%时不能萌发。光照处理对该菌生长发育无显著性影响。葡萄糖、蔗糖、麦芽糖、果糖对分生孢子萌发有促进作用。分生孢子的致死温度为50℃ 10 min,菌丝体的致死温度为60℃ 30 min。     

促进镰刀菌产孢的培养基

 镰刀菌是真菌中一个重要的属,其中多种是重要病原,如麦类赤霉病菌,棉枯萎病菌等,但也有一些是有益的,如寄生于昆虫和锈菌及产生植物激素的种。镰刀菌的研究和鉴定主要依据大型分生孢子、小型分生孢子、产孢细胞、厚壁孢子及菌落性状等。镰刀菌在常用的PSA、PDA等基物上,往往气生菌丝茂盛,色泽明显,但不易产生典型的孢子以供鉴定,产孢细胞亦难观察,这是研究中经常遇到的难题,因此作者开展了筛选培养基试验,初步结果简报如下。     

环境因素对芸苔链格孢生长发育的影响

研究结果表明,芸苔链格孢(Alternaria brassicae)在0—35℃下均可萌发,最适温度15—20℃。菌丝在0—30℃下均能生长,最适温度20—25℃;孢子及菌丝的致死温度为50℃,10分钟。该菌对湿度要求较严格,相对湿度在90％以上时孢子才能萌发,最适相对湿度为98％;病菌在相对湿度达93％时可以侵染,98％以上最适。脱落后孢子曝露在室内(相对湿度约63％)经1小时萌发率下降32.4％,4小时下降80％。耐旱能力较差。一般情况下紫外光能促进该菌产孢,但是对某些菌系的促进作用不明显。过长的紫外光照射对该菌产孢不利,照射160分钟孢子萌发率下降近80％。麦芽糖、蔗糖为该菌的最佳碳源;硝态氮、有机氮是该菌的良好氮源。pH值为4—6时有利于分生孢子萌发,强酸、强碱对分生孢子萌发有抑制作用。     

稗草生防菌新月弯孢菌株J15（2）的生物学特性

研究了分离自稗草上的一个致病菌新月弯孢Curvularia lunata菌株J15（2）的基本生物学特性。结果表明,该菌菌丝生长、产孢的最适温度为28～32℃,pH为6～8。菌丝生长对光照无要求,黑暗利于增加产孢量,培养至15d可达产孢高峰。碳、氮、磷和硫等元素是该菌菌丝体生长、产孢的必需元素,钾、镁和铁对菌丝体的生长、产孢有极大的促进作用。分生孢子萌发的适宜温度范围为20～35℃,最适温度28℃;适宜培养基初始pH值在4～10之间。在58℃下,分生孢子10min失活。     

拟康宁木霉T-51菌株生物学特性及其生物防治潜力

为进一步扩宽拟康宁木霉Trichoderma koningiopsis T-51菌株的生防应用范围,该研究测定T-51菌株菌丝生长、产孢和分生孢子萌发的最适温度、光照和碳氮源条件及其对灰葡萄孢Botrytis cinerea菌核的重寄生能力和对12种植物病原真菌的生防潜力。结果表明,拟康宁木霉T-51菌株在26℃下菌丝生长最快,生长速度达到2.5 cm/d,在20℃光照条件下产孢量最大,达到1.64×109个/皿,26～28℃光照条件下适宜T-51菌株分生孢子萌发;供试氮源中硫酸铵和硝酸铵适宜T-51菌株菌丝生长、产孢和分生孢子萌发,供试碳源中葡萄糖和可溶性淀粉适宜T-51菌株菌丝生长和分生孢子萌发,果糖和乳糖适宜T-51菌株产孢;T-51菌株能够重寄生灰葡萄孢菌核,当分生孢子悬浮液浓度为1×108个/mL时,重寄生效果最好;T-51菌株对核盘菌属Sclerotinia和丝核菌属Rhizoctonia植物病原真菌重寄生能力较强,对镰刀菌Fusarium spp.、暹罗刺盘孢菌Colletotrichum siamense、桃褐腐病菌Monilinia fructicola和稻瘟菌Mag...     

柿树炭疽菌侵染柿树叶柄的超微结构观察

 利用透射电镜技术研究了柿树炭疽菌侵染柿树叶柄的超微结构。结果表明:病原菌侵入寄主细胞后,产生细胞内的初生菌丝,其表面沉积凹凸不平的电子不透明物质。一层界面基质(interfacial matrix)把表初生菌丝细胞壁和凹陷的寄主原生质膜分开。随着初生菌丝定殖下一个细胞,原先细胞中的细胞膜消失,形成许多泡囊,随后叶绿体消失,内质网和高尔基体也逐渐降解,最后细胞内物质全部被降解成电子不透明的颗粒,降解的物质沿着初生菌丝和细胞壁表面沉积。初生菌丝穿透细胞壁的过程中,菌丝顶端接触细胞壁后膨大,并在中部产生一个隔膜,然后顶端细胞产生一个较细的穿透菌丝,穿透寄主细胞壁。穿透菌丝在寄主细胞壁中的狭窄处产生一个隔膜,一旦穿透寄主细胞壁后,迅速膨大。次生菌丝在细胞间和细胞内扩展,通过菌丝体对细胞壁施加的机械压力引起寄主细胞壁破裂,或同初生菌丝一起使细胞壁解体。侵染90 h后,形成垫形分生孢子盘。在分生孢子盘周围的表皮细胞中,次生菌丝不断形成子座组织,使原来的子座扩大,子座不断分化形成产梗细胞,产梗细胞产生分生孢子梗,分生孢子梗生长和发育对角质层和表皮细胞壁组织折叠处施加机械压力,使角质层和表皮细胞壁组织进一步折叠,分生孢子盘也相应扩大。     

青杨叶锈病菌(Melampsora larici-populina Kleb.)侵染过程的超微结构研究

 采用电子显微镜技术对青杨叶锈病菌(Melampsora larici-populina Kleb.)的侵染过程进行了研究。发现该菌夏孢子萌发产生1~3个芽管,且具较多的树杈状分枝。芽管由气孔侵入,侵入前不形成明显的附着胞或仅个别芽管形成附着胞。芽管侵入气孔后在气孔腔内形成气孔下囊,再分化出圆形的膨大体而产生1~2支初生菌丝。初生菌丝在寄主细胞间扩展,与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞中的细胞器与胞间菌丝相同,双核。吸器母细胞产生侵入钉侵入叶肉细胞内部形成吸器,成熟吸器由细长具颈环的管状颈部和膨大的吸器体组成,此时胞间菌丝在吸器母细胞处分化出次生菌丝,在叶肉细胞间扩展形成次生菌落,产生孢子堆。病菌在寄主细胞间隙或沿寄主细胞壁延伸时,寄主细胞仍保持正常状态。     

Infection and disease development of Quambalaria spp. on Corymbia and Eucalyptus species

Quambalaria spp. are eucalypt leaf and shoot pathogens of growing global importance, yet virtually nothing is known regarding the manner in which they infect and colonize their hosts. A study of the infection process of Q. pitereka and Q. eucalypti on Corymbia and Eucalyptus species was thus undertaken using light, scanning and transmission electron microscopy after artificial inoculation. Conidial germination was triggered when relative humidity levels exceeded 90% and commenced within 2 h in the presence of free water. Light reduced germination but did not prevent germination from occurring. Conidial germination and hyphal growth occurred on the upper and lower leaf surfaces with penetration occurring via the stomata or wounds on the leaf surface or juvenile stems. There was no evidence of direct penetration of the host. Following penetration through the stomata, Q. pitereka and Q. eucalypti hyphae grew only intercellularly without the formation of haustoria or interaction apparatus, which is characteristic of the order Microstromatales. Instead, the presence of an interaction zone is demonstrated in this paper. Conidiophores arose through stomatal openings producing conidia 7 days after infection.     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

Fungal development and plant response in detached onion, onion bulb scales and leaves inoculated with Botrytis allii, B. cinerea, B. fabae and B. squamosa

Fungal development and plant responses were examined in detached leaves and mid-bulb scales of Allum cepa. Following inoculation with suspensions of 105 conidia/ml distilled water Botrytis squamosa consistently produced spreading lesions in leaves and bulb scales. B. allii produced spreading lesions at most sites in bulbs but was very inconsistent in its infection of leaves; lesions were often confined to inoculation sites. Limited lesions were usually produced by B. cinerea but R. fabae failed to produce symptoms at most sites. Extensive colonization by B. allii and B. tauamosa required rapid penetration and totally necrotrophic fungal growth. During development of a spreading lesion, plant cell walls became very swollen around intramural hyphae and wall swelling appeared to precede epidermal cell death. Resistance to colonization was due to poor germination, failure to produce distinct infection hyphae (associated with accumulation of deposits of granular reaction material [RM] in underlying live cells) or restriction of infection ryphae amongst small groups of dead cells (limited lesion formation). Only B. fabae germinated poorly, and germ-tubes produced often failed to attempt penetration but grew over the leaf or bulb scale surface. Reducing numbers of conidia increased the frequency of sites associated with RM accumulation; granular deposits being particularly common at sites inoculated with low numbers of B. allii conidia. Electron microscopy revealed that RM granules were osmiophilic aggregates formed between the plasma membrane and epidermal cell wall. In the absence of RM, growth of avirulent species was restricted within the swollen walls of dead epidermal cells. Results ae compared with those from studies on tulip and broad bean leaves.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Scanning electron microscopy study of infection structure formation of Puccinia graminis f.sp. tritici in host and non-host cereal species

The development of uredospore-derived infection structures of Puccinia graminis f.sp. tritici in wheat, barley, sorghum and maize was examined by scanning electron microscopy (SEM). Germ tubes grew over the leaf surface until a stoma was located. An appressorium formed over the stoma and the leaf was penetrated by an infection peg. Within the substomatal chamber of all species the infection peg developed a substomatal vesicle by 6 h post-inoculation (hpi). from which a primary infection hypha developed parallel to the long axis of the leaf. In wheat, barley and maize, when a primary infection hypha abutted onto a host cell, a septum was laid down between the tip of the hypha and the substomatal vesicle, delimiting a haustorial mother cell by 12 hpi; haustorial mother cells did not form in sorghum. Secondary infection hyphae arose on the substomatal vesicle side of the septum; infection did not progress further in maize, but in wheat and barley secondary infection hyphae branched, and proliferated intercellularly forming the fungal thallus. A haustorial mother cell was delimited when an intercellular hypha abutted onto a host cell. Infection sites with haustorial mother cells were observed at 12 hpi in barley and 24 hpi in wheat. In all four plant species, some atypical substomatal vesicle initials, substomatal vesicles and primary infection hyphae were observed.     

立枯丝核菌侵染玉米的研究

 用获自水稻及玉米的立枯丝核菌(Rhizoctonia solani Khn) AG-11A接种玉米发生典型纹枯症状,其致病力显著强于AG-4。玉米拔节期,上位叶鞘抗性较强,抽雄及抽丝期抗性减弱,下位叶鞘无论在拔节期或抽雄、抽丝期,均较上位叶鞘感病。接种玉米后8小时,形成侵染垫及附着胞,从这些结构上形成侵入钉侵入,AG-4侵染上位叶鞘时,常以菌丝直接穿透表皮或从气孔侵入。在去掉菌体的叶鞘表面,发现有周边光滑或稍破损的侵入孔。接种后12小时,在叶鞘细胞中发现菌丝,它们在穿过细胞壁进入邻近细胞时,明显变细。接种后16小时,新生出的菌丝从气孔成丛出现。     

Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.     

香蕉枯萎病菌侵染香蕉根系的组织学过程

 为探明香蕉枯萎病菌侵染香蕉根系的过程,利用绿色荧光蛋白标记的香蕉枯萎病菌4号生理小种（Fusarium oxysporum f. sp. cubense race 4 tagged with green fluorescent protein,GFP-FOC4）,接种香蕉根系以观察病原菌侵染香蕉根系的组织学过程。结果表明,接种1 d后病原菌以菌丝体、大型分生孢子和小型分生孢子的形式附着于根系表皮细胞,优先沿细胞胞间层生长。接种7 d后,观察到病原菌以菌丝体、大型分生孢子和小型分生孢子的形式直接侵染维管束,在维管束内以两种方式扩展繁殖,一种在维管束内横向扩展,菌丝体随机分支,逐步形成网状分布;另一种是菌丝体在维管束内纵向生长,倾向于呈束状沿维管束单侧生长繁殖,形成大量菌丝体。本研究首次从组织病理学的角度观察并分析了GFP-FOC4侵染香蕉根系的过程,为研究香蕉枯萎病菌的致病过程机理提供参考。     

小麦叶锈菌在感病寄主上发育的组织病理学和超微结构研究

 应用荧光显微技术、微分干涉技术和生物电镜技术,系统地研究了小麦叶锈菌在感病寄主上的发育过程及其超微结构特征。小麦叶锈菌在感病品种上的发育过程可分为几个明显的阶段,即孢子的萌发、附着胞的形成、气孔下囊的分化、初生菌丝和次生菌丝的形成和生长、吸器母细胞和吸器的形成、夏孢子床和夏孢子堆的产生以及夏孢子的形成。小麦叶锈菌的胞间菌丝呈丝状,生长和分枝通常沿寄主细胞壁进行。胞间菌丝与寄主细胞的接触诱导了吸器母细胞的分化,吸器母细胞在与寄主细胞壁的接触部位发育形成入侵栓,穿透寄主细胞壁后于细胞内形成吸器。胞间菌丝和吸器母细胞均含有双核,而成熟吸器则含有单核。经常规染色后,胞间菌丝和吸器母细胞的壁与隔膜均可分辨出由多层构成。