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(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。     

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。     

稻曲病两个白化菌株的分离与生物学特性

为了筛选带有自然标记的稻曲病菌菌株,2010年从浙江省象山县和陕西省勉县采集和分离到2个稻曲病白化菌株,ZJa0201和SXa0101。它们在PSA培养基上的生长速度约为其他稻曲病菌株的3倍,未见产生厚垣孢子;在PS培养基上只能产生少量分生孢子。rDNA-ITS和rDNA-IGS序列分析表明,两个白化菌株也与稻曲病菌已知所有菌株的ITS序列同源性高于99.6%;rDNA-IGS序列也属于最为常见的类型,含有2个77bp的重复单元序列。由此推断,这两个白化菌株属于稻曲病菌产孢退化的突变体。白化菌株在PSA上     

稻曲病菌厚垣孢子不同破壁方法的比较

为了探究稻曲病菌[Ustiloginoidea virens(Cooke)Takahashi]厚垣孢子的最佳破壁方法,研究采用4种破壁法对该病菌黄色和黑色厚垣孢子进行破壁,血球计数板计算破壁效果,并用考马斯亮蓝法测定不同破壁方法中厚垣孢子壁内可溶性蛋白含量。结果表明,在普通光学显微镜下观察,破壁后厚垣孢子多数为碎片,少数为孢壁内空圆球。4种破壁方法中液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,用该法破壁测得的黄色和黑色厚垣孢子壁内可溶性蛋白质含量也最高。由此可见,液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。     

稻曲病菌厚垣孢子脂肪酸组成的研究

【目的】为探明细胞壁和细胞内脂肪酸成分及含量与细胞抗逆性的关系,【方法】采用酸热法、索氏提取法、有机溶剂法对稻曲病菌的厚垣孢子壁进行脂肪酸提取,并采用气相色谱检测其脂肪酸的组成和含量。【结果】采用酸热法提取脂肪酸效果最好,以该方法提取测定稻曲病菌黄色、黄绿色、黑色厚垣孢子壁饱和脂肪酸相对含量分别为26.92%、17.23%、23.71%,其不饱和脂肪酸相对含量分别为60.46%、61.52%、70.64%;厚垣孢子总(沉淀孢子壁和上清液)饱和脂肪酸相对含量分别为28.87%、21.00%、24.04%,厚垣孢子总不饱和脂肪酸相对含量分别为55.43%、55.87%、63.89%。硬脂酸在厚垣孢子壁中的含量:黄色>黄绿色>黑色;不饱和脂肪酸中顺式-5,8,11,14,17二十碳五烯酸(EPA)在厚垣孢子壁的含量:黑色>黄绿色>黄色。【结论】在3种颜色厚垣孢子中,黑色休眠型厚垣孢子在孢子壁、总不饱和脂肪酸含量均最高,表明不饱和脂肪酸的含量提高,有利于厚垣孢子的休眠越冬。     

稻曲病菌厚垣孢子壁多糖的提取方法优选

探究稻曲病菌Ustiloginoidea virens (Cooke) Takahashi厚垣孢子壁多糖的最佳提取方法,为孢壁多糖含量和组成的研究提供基础.采用5种方法提取该病菌黑色厚垣孢子壁多糖,用苯酚-硫酸法测定多糖含量.经研究比较,最佳提取方法为复合酶-热水浸提-sevag法,最佳提取条件是复合酶量4％,pH 4,浸提温度70℃,浸提时间120 min,物料比1:75(V/V);在优选的方法和条件下,测定稻曲病菌黑色厚垣孢子壁粗多糖相对得率21.2％,多糖含量72 3％;黄色厚垣孢子壁粗多糖相对得率17.5％,多糖含量66.7％,前者明显高于后者.研究表明复合酶-热水浸提-sevag法的工艺简单、可行,适宜稻曲病菌厚垣孢子壁多糖的测定.     

稻曲病菌厚垣孢子壁黑色素提取方法研究

探究稻曲病菌（Ustiloginoidea virens（Cooke） Takahashi）厚垣孢子壁黑色素的最佳提取方法,采用以HCl为提取剂的酸提法和以NaOH为提取剂的碱提法,对该病菌黑色和黄色2种厚垣孢子壁的黑色素进行提取,用3因素3水平进行正交设计试验,结果表明以NaOH作提取剂为佳,其提取黑色素效果最佳的组合条件为3 mol/L NaOH、2 mol/L HC l、水浴温度80℃、水浴时间120 min。     

6株食根结线虫真菌厚垣普奇尼亚菌的形态学比较

采用玻片和平板菌落培养方法,对6株不同来源的食根结线虫真菌——厚垣普奇尼亚菌进行了形态学观察和比较。结果表明:不同菌株在形态和培养性状上存在明显差异。各菌株在CMA培养基上的生长速率不同,QNMP0214和NRRL13094生长最快,气生菌丝发达;QNZFF0601-3菌落背面乳白色;QNAV97-5分生孢子梗细长。6个菌株均能够产生菌丝膨大和厚垣孢子,其中QNAV97-6和QNMP0214厚垣孢子着生方式除间生外还有侧生,NRRL13094厚垣孢子大量间生成串,QNZFF0601-3和QNMP0214厚垣孢子稀少。QNAV97-2、MP0214、NRRL13094和QNAV97-5能产生晶体。     

稻曲病菌厚垣孢子中cAMP提取条件优选

探究稻曲病菌(Ustiloginoidea virens(Cooke.) Takahashi)黑色(休眠)与黄色(非休眠)厚垣孢子中的环磷酸腺苷(cAMP)最佳提取条件,为进一步的研究cAMP功能奠定基础.采用超声-水浴法对cAMP进行浸提,按3因素3水平正交设计,用高效液相色谱法检测cAMP含量;在设定V(甲醇)∶V(0.05 mol/L KH2PO4)=20∶80、流速为0.8 mL/min、检测波长为254 nm、进样量为20 μL的条件下,以黄绿色厚垣孢子为提取样品,其提取cAMP效果最佳组合条件:超声破碎时间10 min(功率400 W、间歇时间2 s),水浴温度80℃,物料比为1∶100,提取的cAMP为6.827 6 μg/mL.在此最佳条件下,测定出黄色厚垣孢子的cAMP为12.805 0±0.533 2μg/mL,黑色厚垣孢子的cAMP为4.171 7±0.097 1μg/mL.此结果表明,由黄色转换为黑色,其厚垣孢子的cAMP含量显著降低.     

Current understanding on Villosiclava virens,a unique flower‐infecting fungus causing rice false smut disease

Villosiclava virens (Vv) is an ascomycete fungal pathogen that causes false smut disease in rice. Recent reports have revealed some interesting aspects of the enigmatic pathogen to address the question of why it specifically infects rice flowers and converts a grain into a false smut ball. Comparative and functional genomics have suggested specific adaptation of Vv in the colonization of rice flowers. Anatomical studies have disclosed that Vv specifically infects rice stamen filaments before heading and intercepts seed formation. In addition, Vv can occupy the whole inner space of a spikelet embracing all floral organs and activate the rice grain‐filling network, presumably for nutrient acquisition to support the development of the false smut ball. This profile provides a general overview of the rice false smut pathogen, and summarizes advances in the Vv life cycle, genomics and genetics, and the molecular Vv–rice interaction. Current understandings of the Vv–rice pathosystem indicate that it is a unique and interesting system which can enrich the study of plant–pathogen interactions. Taxonomy: Ustilaginoidea virens is the anamorph form of the pathogen (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Incertae sedis; Order Incertae sedis; Family Incertae sedis; Genus Ustilaginoidea). The teleomorph form is Villosiclava virens (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Sordariomycetes; Order Hypocreales; Family Clavicipitaceae; Genus Villosiclava). Disease symptoms: The only visible symptom is the replacement of rice grains by ball‐shaped fungal mycelia, namely false smut balls. When maturing, the false smut ball is covered with powdery chlamydospores, and the colour changes to yellowish, yellowish orange, green, olive green and, finally, to greenish black. Sclerotia are often formed on the false smut balls in autumn. Identification and detection: Vv conidia are round to elliptical, measuring 3–5 μm in diameter. Chlamydospores are ornamented with prominent irregularly curved spines, which are 200–500 nm in length. The sclerotia are black, horseshoe‐shaped and irregular oblong or flat, ranging from 2 to 20 mm. Nested polymerase chain reaction (PCR) and quantitative PCR have been developed to specifically detect Vv presence in rice tissues and other biotic and abiotic samples in fields. Host range: Rice is the primary host for Vv. Natural infection by Vv has been found on several paddy field weeds, including Digitaria marginata, Panicum trypheron, Echinochloa crusgalli and Imperata cylindrica. However, the occurrence of infection in these potential alternative hosts is very rare. Life cycle: Vv infects rice spikelets at the late rice booting stage, and produces false smut balls covered with dark‐green chlamydospores. Occasionally, sclerotia form on the surface of false smut balls in late autumn when the temperature fluctuates greatly between day and night. Both chlamydospores and sclerotia may serve as primary infection sources. Rainfall at the rice booting stage is a major environmental factor resulting in epidemics of rice false smut disease. Disease control: The use of fungicides is the major approach for the control of Vv. Several fungicides, such as cuproxat SC, copper oxychloride, tebuconazole, propiconazole, difenoconazole and validamycin, are often applied. However, the employment of resistant rice cultivars and genes has been limited, because of the poor understanding of rice resistance to Vv. Useful websites: Villosiclava virens genome sequence: http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=JHTR01#contigs     

First Report of the Occurrence of a White Smut Infecting Rice in Arkansas

False smut has recently emerged as an important disease of rice in Arkansas. In 2011, 2012 and 2013, spore balls of a white smut similar to the spore balls of false smut were observed in rice fields in eastern Arkansas. As a white false smut was previously reported in China and Japan, we examined the morphology of chlamydospores and spore balls from some of the infected heads and used selected regions of the rDNA to determine the identity of the causal agent of the disease. We also tested the virulence of an isolate of the white smut to two rice cultivars commonly grown in Arkansas. Our results indicate that the morphology of the spore balls, chlamydospores and conidia is similar to those reported for Ustilaginoidea albicans. However, sequences of ribosomal DNA amplicons indicate a high degree of similarity with both U. virens and U. albicans. The isolate of the white smut was virulent to two rice cultivars, producing spore balls similar to those observed in the field and to those previously described for U. albicans.     

Simple transformation of the rice false smut fungus <Emphasis Type="Italic">Villosiclava virens</Emphasis> by electroporation of intact conidia

A simple procedure is reported for transformation of the rice false smut fungus Villosiclava virens (anamorph: Ustilaginoidea virens) using electroporation of intact conidial cells. The transformation vector pCB1004eGFP was constructed with a green fluorescent protein (eGFP) gene under a constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of Cochliobolus heterostrophus. When a linearized vector was applied, eGFP-expressing transformants were successfully acquired. An inoculation test in rice plants showed that the eGFP-expressing transformants were able to form rice false smut balls.     

Endosperm enzyme activity is responsible for texture and eating quality of cooked rice grains in Japanese cultivars

Eating quality of cooked rice grains is an important determinant of its market price and consumer acceptance. To comprehensively assess the variation of eating-quality traits in 152 Japanese rice cultivars, we evaluated activities of eight endosperm enzymes related to degradation of starch and cell-wall polysaccharides. Endosperm enzyme activities showed a wide range of variations and were lower in recently developed cultivars than in landraces and old improved cultivars. Activities of most endosperm enzymes correlated significantly with the eating-quality score and surface texture of cooked rice grains. Principal component analysis revealed that rice cultivars with high eating-quality scores had high stickiness of the grain surface and low levels of endosperm enzyme activities. These results suggest that endosperm enzyme activities control texture and eating quality of cooked rice grains in Japanese rice cultivars.