稻曲病菌休眠与非休眠厚垣孢子的MSAP分析

本研究以稻曲病菌Ustilaginoidea virens黄色(非休眠)厚垣孢子、黑色(休眠)厚垣孢子、厚垣孢子萌发后产生的分生孢子(非休眠)3种孢子为材料,采用DNA甲基化敏感扩增多态性技术对供试材料基因组DNA进行甲基化分析,探究稻曲病菌休眠与非休眠厚垣孢子DNA甲基化的水平和模式。结果显示,3种形态孢子的基因组DNA甲基化水平差异明显,分生孢子基因组DNA中CCGG序列的总甲基化率为20.56%,黑色休眠厚垣孢子为25.63%,黄色非休眠厚垣孢子为33.52%。黄色和黑色的厚垣孢子主要是全甲基化模式,其全甲基化率高于半甲基化率,其中黄色厚垣孢子全甲基化率高达17.68%;分生孢子则是以半甲基化模式为主。结果对揭示稻曲病菌厚垣孢子休眠的分子机制,探究真核生物孢子的分子休眠机制有普遍意义。     

稻曲病菌厚垣孢子侵染时期和侵染途径的研究

用田间自然发病病粒上新形成的稻曲病菌厚垣孢子,喷雾或注射接种不同生育期的稻穗获得成功。水稻孕穗末期至破口期是稻曲病菌厚垣孢子侵染的主要时期。在孕穗末期喷雾接种2次萌发孢子的发病率高达50.8%。解剖736个病粒内部都含有花药,镜检119条花药和35个柱头发现,病粒内大多数花粉粒的内容物已基本充实,柱头大多处于分枝状突起阶段。解剖刚发病的病粒柱头上有多根菌丝,并向花药等处扩展。萌发的厚垣孢子可以侵染处于乳头状突起期的柱头,并形成菌丝及产生小孢子。稻曲病菌厚垣孢子主要在水稻破口前1—4天至破口时从水稻的柱头侵入引起发病。     

稻曲病菌无性孢子萌发及在不同培养基上生长特性

采用不同的培养基研究稻曲病菌孢子萌发及病菌生长特性.结果表明,稻曲病菌厚垣孢子和分生孢子在蒸馏水和2%蔗糖溶液中3h不能萌发;6h时分生孢子、厚垣孢子分别萌发产生次生分生孢子和分生孢子;新采集的黄色和黑色稻曲球上的厚垣孢子在2%蔗糖溶液中的萌发率均高于其在蒸馏水中的萌发率.供试的9种固体培养基中,以马铃薯蔗糖琼脂培养基(PSA)最适于病原菌生长.生长速率为2.54 mm/天;病菌在7种培养液中振荡培养7天后,在马铃薯葡萄糖培养液(PDB)中菌丝干重最大,达24.5mg/mL,在大米汁和马铃薯蔗糖培养液(PSB)中产生的分生孢子量最多,孢子浓度分别为2.63×107和2.28×107个/mL;而固、液态的Czapek、稻秆汁和胁本哲氏培养基均不适宜于稻曲病菌的培养.     

稻曲病菌越冬厚垣孢子萌发力及其染力的研究

通过３年系统测定试验，稻曲病菌越冬厚垣孢子在室内和水泥池的土中可以存活５－６月。在种植油菜、苕子、蚕豆、小麦的露天水泥池中，越冬的厚垣孢子均有萌发力。在油菜、苕子、小麦３种越冬作物大田土中越冬的厚垣孢子的萌发率以油菜田最高，为２．０４‰。越冬后种子上的厚垣孢子数量大，萌发率可达０．４％－２．９７％。稻茬中越冬的厚垣孢子和在３ｃｍ浅土中的厚垣孢子，其萌发率分别较冬闲田或科作田土中和１５ｃｍ深土中高。     

稻曲病菌越冬厚垣孢子萌发力及其侵染力的研究

通过3年系统测定试验,稻曲病菌越冬厚垣孢子在室内和水泥池的土中可以存活5—6月。在种植油菜、苕子、蚕豆、小麦的露天水泥池中,越冬的厚垣孢子均有萌发力。在油菜、苕子、小麦3种越冬作物大田土中越冬的厚垣孢子的萌发率以油菜田最高,为2.04‰。越冬后种子上的厚垣孢子数量大,萌发率可达0.4％—2.97％。稻茬中越冬的厚垣孢子和在3cm浅土中的厚垣孢子,其萌发率分别较冬闲田或冬作田土中和15cm深土中高。越冬厚垣孢子可以侵染早稻的种子、芽鞘、苗叶和苗根,并引起穗期发病。用水泥池内和种子上越冬的厚垣孢子液注射稻穗和涂抹穗苞均可致病。在上年发病的水泥池栽种水稻亦能引起发病。     

影响稻曲病菌厚垣孢子萌发因素的研究

成熟过程中的稻曲病菌孢子球表面桔黄色的厚垣孢子萌发率很高,后随孢子球老熟其萌发率急剧下降而进入休眠期。在26℃和高湿条件下处理老熟孢子球20天以上就能打破厚垣孢子的休眠,促其顺利地萌发并产生小孢子,萌发率可达30％。这两类厚垣孢子萌发方式差异较大。适温高湿处理10天后的厚垣孢子其呼吸强度明显增强。打破休眠后的厚垣孢子萌发时仍需足够的水分和氧气,其萌发盛期长达35天以上,处理约60天后孢子球开始解体,萌发率急剧下降。日光、荧光灯、紫外线灯等光照对厚垣孢子萌发无明显的抑制或促进作用,但抑制小孢子的形成。偏酸至中性有利于萌发和产孢,过酸或过碱(pH3.0和10.0)则明显地抑制孢子的萌发和产孢。1％的葡萄糖、果糖、蔗糖和甘露糖有利于萌发和产孢。     

稻曲病菌的形态学观察研究

 利用光学和电子显微镜对稻曲病菌的有性子座和不同来源的繁殖体进行了形态学观察。子囊壳单层环状埋生于子座外层,顶端突出子座表面而形成许多乳突结构。子囊发育成熟后,子囊壳壁的顶端可自行消解,露出里面的子囊。子囊孢子无色单胞,线状。厚垣孢子圆形或稍椭圆,黄褐色至黑褐色,胞壁厚密,外表有许多疣状突起。子囊孢子和厚垣孢子萌发都产生次生分生孢子。这些次生分生孢子与液体培养获得的分生孢子在大小和形态特征上是一样的,它们都是薄壁分生孢子。薄壁分生孢子卵圆形或长圆形,大小为2.6~8μm×2~5μm,无色透明,外表光滑。薄壁分生孢子萌发直接形成新一代薄壁分生孢子,此繁殖过程可不断重复进行。     

谷子腥黑穗病的侵染途径和生物学特性研究

 以谷子腥黑穗菌(Tilletia setariae Ling)冬孢子接种正开花的谷子小花后引起发病,为花器局部侵染,当年侵染当年发病的非系统侵染病害。以二甲基亚砜处理,紫外光线照射和土壤越冬处理的冬抱子均能打破其休限期。在15-30℃里均能萌发,最适萌发温度为25℃,在pH4-7里均可萌发,以pH6为最适。碳、氮源对冬孢子萌发无明显的刺激作用。病菌在人工培养基上生长最适温度是20℃,最适pH值为7.0。在PDA上生长最佳。棉子糖、葡萄糖和可溶性淀粉为最佳碳源,DL-丝氨酸为最佳氮源。冬孢子经扫描电镜观察为T型(瘤突型)。萌发产生的担孢子数多达百个,虽"毛刷状"。在人工培养基上能产生厚垣孢子。以0.5% KOH-safranin O快速核染色剂染色,看到菌丝细胞里多含有双核,及锁状联合。     

稻区杂草上的梨孢菌与稻瘟病发生的关系

 新发现的稻曲病菌白化菌株其分生孢子座白色,厚垣孢子表面光滑,大小为3.5-6.0×3.5-6.8μm。在麦芽糖-稻芽浸渍液中可产生较多的分生孢子。pH值为6时生长最好。普通培养基中加入几种供试碳源和氮源均有利于菌落生长,以蔗糖为碳源,浓度为2%或以L-天门冬酰胺为氮源浓度为0.2%~0.3%时时其菌落生长有促进作用。培养过程中可产生对稻种萌发有抑制作用的毒素,而固体培养时产毒能力高于液体培养。白化菌株回接到水稻上可重新产生白色稻曲球。该菌和稻曲病菌的酯酶同功酶和RAPD扩增谱带明显不同。可见白化菌株具有独立于稻曲病菌的新的分类地位。     

绿粘帚霉(Gliocladium virens)厚垣孢子的诱导

 通过对8种培养基的筛选,获得了绿粘帚霉产厚垣孢子的最佳培养基(PD培养基),进一步研究得到了厚垣孢子产生的最佳培养条件为14 w、24 h/d光照,120 r/m in振荡,pH 3,30℃。在此条件下可完全抑制其分生孢子的产生,并在1周内产生8.00×10⁶个/mL厚垣孢子。     

制约稻粒黑粉病菌冬孢子萌发的关键因素

在相同条件下，从稻田直接收集的病粒中的稻粒黑粉病菌厚垣孢子不能萌发，而取之于仓库中的孢子能萌发。为探明其制约因素，对该病菌进行了光照和浸水时间试验。研究结果表明：稻粒黑粉病菌冬孢子形成后，必须经光照射后，方可进入休眠期；度过5～6个月休眠期的冬孢子，必须浸水48h以上才开始复苏；复苏后的冬孢子必须在光照条件下才能萌发。未完成后熟作用的冬孢子和完成后熟但尚未复苏的冬孢子即使在光照条件下也不能萌发。即稻粒黑粉病菌冬孢子必须经历后熟、休眠、复苏三个阶段后，在适宜的光照与温湿条件下，方可萌发。光照对其冬孢子具有双重作用，促使后熟进入休眠和刺激萌发。     

Effect of calcined lime (CaO) on chlamydospores of the rice false smut pathogen,Villosiclava virens,and their root attachment and infection

Rice false smut caused by Villosiclava virens is a devastating disease. The smut balls contain chlamydospores, which fall onto paddy soils to become the primary inoculum. After transplanting, the chlamydospores subsequently germinate and infect rice roots. The application of a CaO-containing fertilizer to paddy soils can inhibit the development of rice false smut disease; however, the underlying mechanism is unknown. In this study, we evaluated the suppression of chlamydospore behaviour due to CaO. Specifically, we clarified the effects of the following on chlamydospore morphology and germination: (a) pH, (b) calcium (Ca) concentration, (c) Ca concentration (pH adjusted to 6.5), and (d) CaO-added soil solution. Germination was suppressed at pH 4 and 10, in contrast with the normal germination at pH 6, 7, and 8. Treatments with more than 10 mg/L Ca melted the outer layer of chlamydospores and suppressed germination regardless of whether the pH was adjusted to 6.5. A saturated CaO solution induced bursting of chlamydospores. Suppressed germination and a melted outer chlamydospore layer were also observed, even though the soil buffering effect initially prevented the CaO-mediated pH increase. Furthermore, the chlamydospores within 15 mm from the CaO small lump exhibited suppressed germination in soil. In addition, there was no effect of CaO treatment on chlamydospore attachment to rice roots and hyphal invasion of rice roots in in vitro inoculation tests. These results suggest that Ca concentration is an important factor for inhibiting the occurrence of rice false smut disease.     

稻曲病菌白化菌株生物学特性研究

 从水稻白叶枯病菌培养滤液中提取到毒素。菌体培养滤液和毒素对抗、感水稻品种的作用不一致,培养滤液中可能含有激发子。毒素稀释液抑制水稻出苗,引起水稻幼苗萎蔫和烟草坏死反应。该毒素薄层层析显示含有七种组分,野生菌与毒性基因突变体毒素组分大体相同,但含量有明显差异。不同小种间的毒素产量有区别,组分区别不大。     

高粱花黑穗病生物学特性及侵染途径研究

 高粱花黑穗病(Ustilago Kenjiana Ito)是我国高粱上发生的一种病害,对产量影响轻微。冬孢子萌发适温是25-30℃,在水滴里萌发率最高,最适pH是6.0-6.5。在不同营养液里以酵母汁及花粉汁里萌发率最高,在人工培养基上病菌生长最适温度是25℃,最适pH是5.9.碳源以葡萄糖、半乳糖、甘露糖和果糖里生长最佳。氮源以蛋白胨和天冬素生长最好.菌瘿黑冬孢子变褐后不经休眠即可萌发。冬孢子在室外可存活一年半,在土壤里经一冬便丧失生活力,室内能存活三年.以冬孢子接种土壤和种子均未发病,属于花器局部侵染,当年侵染当年发病的非系统侵染病害.冬孢子在高粱孕穗或尚未开花的小花护颖与外颖缝隙中侵入子房,而不是在开花期或开花后侵入,也不是从柱头、花柱或其它部位侵入,当年尚能重复侵染.     

稻粒黑穗病菌孢子萌发中的感光作用

 自从高桥良直在1896年发现稻粒黑穗病菌[Neovossia horrida(Tak.) P.et K.]以来,长时期内学者们只能偶然地观察到该菌厚垣孢子的萌发。邓叔群教授(1931)首先对于这个问题进行了多方面的研究,并且提出了与该菌孢子萌发可能有关的各种因素。     

稻曲病菌不同颜色厚垣孢子超微结构比较

 The surface and internal structure difference of yellow and black dormant chlamydospores of Ustilaginoidea virens on mid-and late-season rice were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM revealed that the spherical yellow and black chlamydospores had prominent spines, with the diameters from 3 to 6 μm and 4 to 6 μm, respectively. The spines were 200-700 nm and 400-1 100 nm long in yellow and black chlamydospores, respectively. Variance analysis showed that there was no difference in diameter of the yellow chlamydospores but significant difference among the black ones collected from both rice cropping types. Similarly, there was no difference in length of the spines on the same color chlamydospores but extremely different between the yellow and black chlamydospores collected from the two cropping types. TEM revealed that the cell wall of both color chlamydospores were composed of two layers, the thick endosporium and thin exosporium, and the thickness of the former layer of the black chlamydospores was nearly two times as that of the yellow ones. The organelles in yellow chlamydospores such as the nucleus were obviously visible, but invisible in black ones. The cytoplasm of black chlamydospores was occupied by a big lipid globule. This study suggests that the formation of dormant chlamydospores of U. virens is related with the structural changes in the cell.     

Epiphytic colonization of Ustilaginoidea virens on biotic and abiotic surfaces implies the widespread presence of primary inoculum for rice false smut disease

Ustilaginoidea virens (Uv), the causative agent of rice false smut disease, infects developing rice spikelets at the booting stage, and transforms individual grains of the panicle into smut balls. Epidemics of the disease occur when the rice booting and heading stages coincide with rainy days. Using a green fluorescent protein (GFP)‐labelled Uv isolate that can form false smut balls on rice panicles, it was found that under high humidity and free water conditions the Uv isolate could colonize leaves of plants belonging to various families including the Poaceae (Oryza sativa, Echinochloa crusgalli, Digitaria sanguinalis and Leptochloa chinensis), the Brassicaceae (Arabidopsis thaliana) and the Solanaceae (Nicotiana benthamiana) without symptoms. Over several days, some conidia could germinate on the leaves of these plants and in water on the surface of Parafilm and cellophane, form hyphae and differentiate conidiophores to generate a large number of secondary conidia, while other conidia were able to directly produce secondary conidia. Conversely, in the absence of water some conidia could either bud to form new conidia or were converted into chlamydospores. These data indicate that Uv is one of a few fungal pathogens reported to have epiphytic characteristics. The rapid generation of a large number of spores on biotic and abiotic surfaces greatly increases the inoculum that can infect rice spikelets, resulting in the occurrence of rice false smut disease epidemics. These findings are important in the development of disease control strategies.     

向日葵锈菌夏孢子萌发条件的研究

以向日葵锈菌330生理小种为材料,研究了夏孢子萌发的适宜条件和培养载体。向日葵锈菌夏孢子萌发适宜温度范围10~25℃,最适温度15℃。15℃下在琼脂薄膜上1h即可萌发,12h达到萌发高峰,萌发率最高可达97%。孢子在无菌水中萌发最适浓度为20mg/L,萌发率随浓度增大而降低。光照在前5h均抑制夏孢子萌发,但7h后光照对夏孢子总萌发率基本无影响。新鲜夏孢子萌发率较高,在室温放置270d后基本丧失萌发能力。最适条件下,在供试的6种不同萌发载体上,夏孢子萌发率、芽管长度及萌发形态显著不同,琼脂薄膜载玻片、尼龙纱网、玻璃纸较适宜夏孢子萌发,萌发率超过90%;在PVDF膜和亲水滤膜上可观测到芽管内部黄色物质转移和特殊结构的形成。     

水稻粒黑穗病侵染的研究

 稻粒黑穗病在川东焉蔓延已久的病害。解放前万孙熊家乡长民曾企图用温汤浸梗法以防治此病,结果无效。