巢豆油壶菌与蚕豆相互作用下植物内源激素的动态

 蚕豆火肿病由巢豆油壶菌(Olpidium Viciae Kusano)引起,症状特点是病部产生小瘤状突起。我们对该病的组织病理学研究^［1］表明是一种增生型组织病变,病菌仅定殖在侵入叶、茎的表皮细胞内,病菌的侵染引起邻近侵染点的表皮细胞及其下的叶肉细胞或茎的皮层组织细胞转化为分生细胞,并不断分裂增生和细胞体积增大,导致蚕豆病组织发生畸形增生的病理解剖改变,是病部产生瘤状物的组织学机制。前人对一些增生型组织病变病害的研究结果发现是由于病组织内的内源激素水平改变所致^［2］。蚕豆火肿病这种增生型病变是否与内源激素水平改变有关？其变化特点怎样？至今尚未有研究报导。     

蚕豆油壶菌疱疱病及其防治的初步研究

 蚕豆疱疱病是川西北高原春蚕豆的主要病害之一,发病面积约6万亩。罹病茎、叶起疱,一般因病减产20%左右。病原为巢豆油壶菌(Olpidium viciae)。巢豆油壶菌及其所致病害发生于日本,我国尚属初次发现。该菌寄主范围广,易见的自然寄主为蚕豆、豌豆和歪头菜。在人工接种下,除可侵染大豆、菜豆、箭舌豌豆、兵豆等多种豆科作物外,也能侵染油菜、结球甘蓝、大白菜、萝卜、黄瓜、南瓜、莴苣、菠菜、荞麦等作物。以病株残体内的休眠孢子囊在土内越冬,翌春萌发侵入寄主,在寄主细胞内形成游动孢子囊,通过出管释放单鞭毛游动孢子进行再侵染,潜育期13~18天。游动孢子亦起配子作用,结合形成双鞭毛游动合子,以合子侵入。后期在寄主细胞内形成休眠孢子囊。     

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。     

核盘菌侵入油菜超微结构及侵染机制的研究

 通过电子显微镜观察核盘菌在油菜叶片上侵染过程,发现该菌首先在叶片上形成复合附着器。每个分枝末端一般生出一个侵染钉。侵染钉侵入叶表面腊质、角质层和表皮细胞壁时.不仅靠附着器产生的压力,而且供助于酶对寄主表面的软化、消解作用。该菌通过角质层和表皮细胞壁侵入油菜叶片,尚未发现通过气孔侵入的现象。侵入叶片后,该菌的继续生长,导致了油菜组织的溃烂。然后菌丝在腐烂的叶片上集结形成菌核。     

马铃薯抗癌肿病品种比较试验

马铃薯癌肿病(Potato wart)又名黑瘤病、黑痂病,群众俗称"疙瘩洋芋".病菌(属鞭毛菌亚门真菌)以休眠孢子囊在病组织内或随病残体遗落在土壤中越冬.休眠孢子囊抗逆性很强,可在土壤中存活25 a左右,遇适宜的条件,萌发产生游动孢子和合子,从寄主表皮细胞侵入.     

细胞乳突的形成和小麦白粉菌成功侵染的关系

 用一套己知抗白粉病单基因的小麦材料,研究了小麦白粉菌诱发寄主表皮细胞产生的乳突与成功侵染的关系。抗病小麦叶片上,诱发产生乳突的分生孢子占测定分生孢子数的58.8%,感病品种为63.8%。这表明抗性不同的小麦材料接种白粉菌之后,叶表皮细胞内形成乳突的百分率相近,然从在抗病材料和感病材料中形成的乳突阻止病原物发育和成功侵染的作用不相同,在抗病叶片上,诱发产生乳突的分生孢于中有88%停留在压力孢阶段,不继续发育,仅有12%能突破乳突形成吸器,使得侵染成功。与此对照,在感病叶片上只有32%诱发乳突的分生孢子不能穿透乳突,而68%则能突破乳突。成功地与寄主建立寄生关系。我们的研究表明,乳突能否成功地阻止白粉菌侵入可能与乳突形成的迟早有关。     

栗疫病菌侵染板栗枝条的显微观察

栗疫病是一种严重危害栗属植物的病害。为了明确栗疫病菌侵染板栗枝条的过程及侵染的关键时间点,本研究利用病理组织切片技术、显微镜和扫描电镜技术对栗疫病菌侵染板栗枝条的过程进行了观察。结果表明:接种栗疫病菌后0～5 h,菌丝先降解枝条表皮,进行横向营养生长的同时沿着伤口纵向侵染,为进入皮层做准备;接种后6 h病菌开始在表皮定殖,并侵入皮层;接种后9 h在皮层可观察到侵染性菌丝沿着细胞间隙向相邻细胞延伸;接种后12 h栗疫病菌侵入韧皮部,在皮层的侵染面积扩大。随着侵染程度加深,皮层、韧皮部等处细胞被菌丝降解,最终在形成层附近聚集。接菌后9 h为栗疫病菌侵染板栗枝条的关键时间点。     

蚕豆立枯病和锈病的防治

蚕豆立枯病和锈病的防治王秀芬河北农业大学农学院,保定０７１００１）一、蚕豆立枯病和锈病的症状识别（１）立枯病属真菌性病害。主要侵染蚕豆茎基部或地下部,也侵害种子。茎基部染病后致使茎变黑,有的病班向上扩展达十几厘米。干燥时,病部凹陷,几周后病株枯死。温...     

玉米穗腐病和茎基腐病镰孢菌间相关性的RAPD分析

 利用随机扩增多态性DNA (RAPD)技术对玉米穗腐病和茎基腐病镰孢菌之间的相互关系进行了研究,证实了引起2种病害的串珠镰孢菌同源性很高,在遗传上具有较高的相似性,病菌不易受地域或环境和寄生部位选择作用的影响,同一类型的串珠镰孢菌可以是穗腐病和茎腐病的共同病原菌。禾谷镰孢菌间的遗传变异性很强,易受地域、环境和寄生部位选择作用的影响,出现明显的分化现象,穗腐病和茎基腐病可以由同一禾谷镰孢菌分化类型侵染所致,也可由不同分化类型侵染所致。     

烟草黑胫病田间动态研究

 雨水在黑胫病的重复侵染上起重要作用。烟茎上的孢子囊被雨水冲刷到地面上,释放出大量的游动孢子,随地面活水进行侵染。孢子囊只产生在较幼嫩的组织上,主要着生在烟茎顶端,其次在茎中部,茎基部分则极少发现。山东夏烟产生孢子囊的主要时期为移栽(6月底至7月初)以后到7月下旬。连续大雨有利于产生孢子囊。人工接种后4~5天是产生孢子囊的高峰。     

Visualisation of the uptake of two model xenobiotics into bean leaves by confocal laser scanning microscopy: diffusion pathways and implication in phloem translocation

The diffusion of two fluorescent dyes, Oregon Green 488 (Oregon Green) and Rhodamine B into the leaves of broad bean (Vicia faba L) plants was studied to simulate the foliar uptake process of pesticides. The uptake rate of these model xenobiotics into bean foliage was measured using a standard leaf surface wash-off method. Diffusion into leaf tissues was visualised in vivo by confocal laser scanning microscopy (CLSM). The moderately lipophilic dye (Rhodamine B) showed faster uptake than the hydrophilic one (Oregon Green), despite the former being a larger molecule. While no distinct channels or domains for preferential entry of any of the dyes could be detected in the cuticle layer by CLSM, two different diffusion patterns were identified for the movement of these two dyes after traversing the cuticle. Upon desorption from the cuticle, Rhodamine B diffused extensively into the vacuole of the epidermal cells. Further transport of this dye from the epidermal cells to the mesophyll cells was not observed. In contrast, Oregon Green was found in the epidermal cell walls and cytoplasm, and was also present in the mesophyll cells. Examination of the petioles of the treated leaves revealed that, once absorbed, Oregon Green moved readily out of the treated leaf, whereas Rhodamine B did not show any phloem translocation. It is proposed that these two different diffusion characters may be responsible for the contrasting phloem mobility of the two xenobiotics. The results are discussed in relation to the current knowledge on the uptake, translocation and efficacy of pesticides as influenced by their properties.     

The influence of silicon on cytological interactions between Sphaerotheca fuliginea and Cucumis sativus

The response of epidermal cells of cucumber leaf tissue infected by Sphaerotheca fuliginea was examined by light microscopy to understand how silicon in infected host cells affects host defence mechanisms. Leaf pieces from plants treated with nutrient solutions containing 0·05, 0·50 or 2·3 mm of silicon (Si) were harvested at intervals of 24, 48, 72, 96 and 120 h after inoculation with the pathogen and examined after staining with toluidine blue or aniline blue. Si treatments significantly reduced the time to initiation of production and/or accumulation of phenolic materials in infected host epidermal cells, and increased the number of infected cells that produced and/or accumulated phenolics. The number of haustoria produced per colony of S. fuliginea was significantly reduced over time, and conidiophore development was delayed on the leaves of cucumber treated with 2·3 mm Si nutrient solution.     

Pathogenic characteristics of isolates of Aphanomyces euteiches from pea in France

Aphanomyces root rot ( Aphanomyces euteiches ) has become a very destructive disease in French pea crops since 1993. The host specificity of the French pea-infecting populations of this pathogen was investigated by inoculating pea, common vetch, alfalfa, broad bean and green bean with 91 pea-infecting A. euteiches isolates, originating from the main areas of infestation in France. These isolates were compared to 13 isolates from various countries and hosts (pea, green bean, alfalfa). Virulence phenotypes were defined according to the pathogenicity data on the different hosts: all isolates from France infected two to five legume species, with most infecting pea, vetch, alfalfa and broad bean. Four pathotypes were characterized within the French isolates: one type corresponded to broad host range isolates, the second was composed of isolates preferentially agressive on pea/vetch/alfalfa and weakly aggressive on broad bean, and two others corresponding to more specialized isolates that preferentially infected pea/vetch or pea/vetch/alfalfa. Most isolates from France were preferentially pathogenic on pea, like the pea-infecting isolates from other countries, but were less specialized than the alfalfa- and green bean-infecting isolates from other countries. These results suggest that A. euteiches isolates may be maintained on wild or cultivated legumes other than pea in France.     

Studies of Host—pathogen Interaction Between Maize and Acremonium strictum From Cameroon

Different effects of Acremonium strictum from Cameroon on maize cultivars Ndock 8701, CMS 8704 and CMS 8501 were investigated. Observations of symptoms and re-isolation of the pathogen showed that the disease causes chlorosis, leaf necrosis, stem necrosis, barren plants and wilting symptoms. Reduction in growth and yield is demonstrated. In the cultivar Ndock 8701 the pathogen showed systemic development in the host tissues with inter- and intracellular colonization of the vascular bundle and adjacent tissues including the protoxylem lacuna, xylem vessels, metaxylem, sieve tubes, protophloem and metaphloem. Gels and gums were observed in the maize xylem vessels after fungal invasion and are part of the host defence response. Coloration corresponding to acidic carbohydrates and phenolic compounds was recorded. This is the first demonstration of the pathogenic nature of A. strictum in maize from Cameroon as well as the observation of gels and gums. This pathogen must be regarded as important considering its interaction in maize.     

Localized hemibiotrophy in Colletotrichum: cytological and molecular taxonomic similarities among C. destructivum, C. linicola and C. truncatum

The infection process of hemibiotrophic isolates of Colletotrichum linicola (from flax, Linum usitatissimum ) and C . truncatum (from broad bean, Vicia faba and lentil, Lens culinaris ) was studied by light microscopy. Host surfaces were penetrated directly leading to a symptomless, biotrophic phase characterized by the elaboration of large multilobed, multiseptate, vesicular primary hyphae that were restricted to the initially infected epidermal cells. Biotrophy lasted for the first 48 h of the host-pathogen interaction and was rapidly succeeded by a necrotrophic phase during which narrow, secondary hyphae invaded the surrounding leaf tissues and water-soaked spreading lesions with sporulating, monosetate acervuli were produced on infected host surfaces. Molecular taxonomic analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed very close similarities (97–99%) between these isolates and those of C . destructivum obtained from cowpea ( Vigna unguiculata ) and lucerne ( Medicago sativa ), and also of C . truncatum obtained from pea ( Pisum sativum ). This association was consistent with results from a comparative assessment of some in-planta and in-vitro morphological and growth characteristics of these hemibiotrophic fungi. It was concluded that localized hemibiotrophy is an infection strategy utilized predominantly by a closely-related group of pathogens comprising C . destructivum , C . linicola and C. truncatum , and the formation of multilobed primary hyphae restricted to the first penetrated cell might therefore be a key taxonomic character which correlates consistently with ITS sequence data.     

A transient expression system to assay putative antifungal genes on powdery mildew infected barley leaves

A transient expression system has been established to assay the regulation and impact of putative antifungal proteins at the single cell level in barley (Hordeum vulgareL.) epidermal leaf cells infected with powdery mildew (Erysiphe graminisf.sp.hordei).The system combines biolistic transformation with direct comparison of the fungal attack in transfected and wild type cells. Many epidermal cells showed detectable expression of marker genes including a novel improved version of green fluorescent protein (GFP). The marker was detectable for at least 10 days during which individual host cells supported an unaffected asexual life-cycle of the pathogen.     

Infection of Winter Wheat by a beta-Glucuronidase-Transformed Isolate of Cephalosporium gramineum

ABSTRACT Field-grown winter wheat was inoculated with a beta-glucuronidase-transformed isolate of Cephalosporium gramineum in two field seasons to elucidate the mode of infection in resistant and susceptible cultivars. Colonization of viable root epidermis and cortical cells occurred as soon as 15 days postinoculation and the pathogen was found in the vascular tissues by 20 days postinoculation, well before freezing soil temperatures occurred. Penetration occurred directly through the root epidermis and through wounds adjacent to emerging secondary roots. The pathogen also penetrated through root cap cells and colonized meristematic tissues near root tips to gain access to the vascular system. Lower stem base colonization was observed where the pathogen penetrated directly through the epidermis, wounds, or senescent tissues. Appressorium-like structures, which appeared to aid penetration of cell walls, were often found within cells of both roots and stems after initial colonization. The mechanisms of resistance were not apparent, but less colonization occurred in resistant than in susceptible cultivars.     

Visualization of localized pathogen-Induced pH modulation in almond tissues infected by Colletotrichum acutatum using confocal scanning laser microscopy

Modulation of pH within the host during infection of almond by the anthracnose pathogen Colletotrichum acutatum was studied using confocal scanning laser microscopy and the dual emission fluorescence indicator SNARF-1. This highly sensitive method allowed visualization of the spatial distribution of localized pathogen-induced pH modulation within and in proximity to fungal infection structures in host tissue at the cellular level. Ratiometric measurement of fluorescence at two emission wavelengths and in situ calibration allowed the quantification of pH ranges. After incubation of leaf epidermal tissue with SNARF-1, distinct alkaline (pH 8 to > or =9), red-spectrum (650 nm wave length) fluorescent zones developed as partial or complete halos around many fungal appressoria and in infection vesicles at 24 to 36 h after inoculation. In samples taken after 48 to 72 h, colonizing hyphae in the biotrophic phase and subsequently in the necrotrophic phase were also emitting the red fluorescence that extended into the surrounding host tissue, as also verified by depth analyses. Host epidermal cells were intact and apparently alive during the fungal alkalization process, with no visible disruption of cell structure. Generally, the pH of epidermal cells in noninoculated samples or in areas away from the infection in inoculated samples was lower than pH 7 with green (i.e., 500 to 550 nm wave length) fluorescence detected. Using standard electrodes, a significant increase in pH and ammonia concentration in leaf and fruit tissue was also measured but only at advanced stages of disease. In contrast, hyphae of the pathogen Alternaria alternata were mostly acidic and no change in fluorescence was found inside invaded host cells. The sequence of events in the C. acutatum-almond interaction includes penetration, production of ammonia by C. acutatum, and subsequent pH modulation within almond epidermal tissue to an alkaline environment that leads to further colonization of the host.     

The use of ascospores of the dieback fungus Hymenoscyphus fraxineus for infection assays reveals a significant period of biotrophic interaction in penetrated ash cells

Ascospores, discharged naturally from apothecia growing on rachis debris, were used as inoculum to examine the invasion of ash tissues by Hymenoscyphus fraxineus in order to understand the critical, but poorly understood, early interactions between host and pathogen. Methods were developed to collect ascospores for controlled infection assays on detached leaves, petioles and stem internode tissues. Light microscopy, using plasmolytic techniques, allowed the invasion of living plant cells to be observed. Ascospores were readily available from late May to September. On the plant surface, most spores differentiated directly to form appressoria without germ‐tube growth. Direct penetration was followed by a significant period of biotrophic fungal growth before lesions developed. Following the formation of a vesicle‐like structure after penetration, bulbous and elongated intracellular hyphae were produced in living plant cells. The use of ascospore inoculum, rather than mycelia, will allow natural and rapid screening of ash genotypes for resistance to the devastating dieback disease. The identification of the biotrophic phase of infection suggests that host range is controlled by effector‐triggered immunity.