Ca^2＋信号途径参与稻瘟病菌分生孢子萌发及附着胞形成的调控

为了确定Ca^2 信号途径是否参与、在哪一时期参与稻瘟病菌分生孢子萌发及附着胞形成过程的调控,用四种可从不同位点阻断 Ca^2 信号途径的抑制剂分别处理分生孢子,观察抑制剂对孢子萌发及附着胞形成过程的抑制作用。结果表明：Ca^2 螯合剂 EGTA、Ca^2 通道抑制剂 Verapamil、抑制磷脂酶 C 活性的抑制剂 U-73122、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂 KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂影响孢子萌发和附着胞形成过程在萌发早期(1-4h)最有效;在完全被抑制、不能萌发的孢子内出现了许多颗粒状囊泡;抑制剂可使附着胞形态明显变小甚至不能形成。以上结果表明钙信号途径参与了稻瘟病菌孢子萌发及疏水条件下附着胞形成过程的调控。     

Ca2+信号途径参与稻瘟病菌分生孢子萌发及附着胞形成的调控

为了确定Ca2+信号途径是否参与、在哪一时期参与稻瘟病菌分生孢子萌发及附着胞形成过程的调控,用四种可从不同位点阻断Ca2+信号途径的抑制剂分别处理分生孢子,观察抑制剂对孢子萌发及附着胞形成过程的抑制作用.结果表明Ca2+螯合剂EGTA、Ca2+通道抑制剂Verapamil、抑制磷脂酶C活性的抑制剂U-73122、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂影响孢子萌发和附着胞形成过程在萌发早期(1～4h)最有效;在完全被抑制、不能萌发的孢子内出现了许多颗粒状囊泡;抑制剂可使附着胞形态明显变小甚至不能形成.以上结果表明钙信号途径参与了稻瘟病菌孢子萌发及疏水条件下附着胞形成过程的调控.     

Ca2+信号途径参与稻瘟病菌分生孢子萌发及附着胞形成的调控

为了确定Ca²⁺信号途径是否参与、在哪一时期参与稻瘟病菌分生孢子萌发及附着胞形成过程的调控,用四种可从不同位点阻断Ca²⁺信号途径的抑制剂分别处理分生孢子,观察抑制剂对孢子萌发及附着胞形成过程的抑制作用。结果表明:Ca²⁺螯合剂EGTA、Ca²⁺通道抑制剂Verapamil、抑制磷脂酶C活性的抑制剂U-73122、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂影响孢子萌发和附着胞形成过程在萌发早期(1~4h)最有效;在完全被抑制、不能萌发的孢子内出现了许多颗粒状囊泡;抑制剂可使附着胞形态明显变小甚至不能形成。以上结果表明钙信号途径参与了稻瘟病菌孢子萌发及疏水条件下附着胞形成过程的调控。     

钙信号途径参与小斑病菌致病过程的调控

为确定Ca^2＋信号途径与玉米小斑病菌致病过程的相关性,用可从不同位点阻断Ca^2＋信号转导途径的抑制剂分别处理小斑病菌的分生孢子,结果表明：Ca“螯合剂EGTA、Ca^2＋通道抑制剂Verapamil、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂可使附着胞形态明显变小甚至不能形成。以上结果表明钙信号途径参与了玉米小斑病菌主要致病过程的调控。     

钙信号途径参与小斑病菌致病过程的调控*

为确定Ca²⁺信号途径与玉米小斑病菌致病过程的相关性,用可从不同位点阻断Ca²⁺信号转导途径的抑制剂分别处理小斑病菌的分生孢子,结果表明:Ca²⁺螯合剂EGTA、Ca²⁺通道抑制剂Verapam il、影响钙调素与钙调素依赖蛋白激酶作用位点的抑制剂KN-93,随着浓度的增加,对孢子萌发和附着胞形成过程的抑制作用明显增强;同一浓度下,抑制剂对附着胞形成过程的抑制作用大于孢子萌发过程;抑制剂可使附着胞形态明显变小甚至不能形     

金龟子绿僵菌附着胞分化及其与环腺苷酸cAMP的关联性研究

寄主识别与附着胞分化是虫生真菌启动侵染过程的首要步骤。本文利用先前获得的金龟子绿僵菌基因缺失突变株与其野生型一起进行附着胞分化研究。接种后不同时间下的观察表明,绿僵菌突变株或野生型的附着胞既可以在萌发不久的芽管顶端形成,也可以在伸长菌丝分支的顶端形成。与野生型不同的是,突变株附着胞的分化频率显著下降,附着胞周围也缺乏粘液层的产生。研究表明,绿僵菌的类枯草杆菌类体壁降解酶对于附着胞分化不产生影响,对体壁降解也非完全必需的。与突变株附着胞分化频率显著降低相对应,其胞内环腺苷酸cAMP水平显著下降,而添加外源cAMP能够显著增加其附着胞分化频率,说明绿僵菌cAMP信号途径对于调控附着胞分化起着重要的作用。     

稻瘟病菌微波诱发突变体的分析*

通过诱变获得突变体是研究稻瘟病菌变异机制的基础。本文用微波炉对稻瘟病菌分生孢子进行低强度短时间处理获得了一批形态发育和致病性突变体,并对它们进行了分析。突变体1-40-271菌落呈白色,产孢与萌发均正常,但萌发后即便在人工疏水表面上也不能形成附着胞,且丧失了致病性;突变体2-20-6菌落呈黄色,孢子萌发率为1%,萌发的孢子其附着胞形成率仅为0.01%,致病性减弱;突变体2-30-3菌落呈黄色,形成的附着胞大部分不正常,但致病性正常。Rep-PCR指纹分析发现,突变体2-20-6和2-30-3比其相应野生型少1条带,而突变体1-40-271与其野生型比较没有变化,说明微波可能造成稻瘟病菌基因组DNA缺失或点突变而发生变异。继代分析表明微波处理获得的稻瘟病菌形态和致病性突变体是稳定的。     

稻瘟病菌微波诱发突变体的分析

通过诱变获得突变体是研究稻瘟病菌变异机制的基础。本文用微波炉对稻瘟病菌分生孢子进行低强度短时间处理获得了一批形态发育和致病性突变体,并对它们进行了分析。突变体1-40-271菌落呈白色,产孢与萌发均正常,但萌发后即便在人工疏水表面上也不能形成附着胞,且丧失了致病性;突变体2-20-6菌落呈黄色,孢子萌发率为1％,萌发的孢子其附着胞形成率仅为0．01％,致病性减弱;突变体2-30-3菌落呈黄色,形成的附着胞大部分不正常,但致病性正常。Rep-PCR指纹分析发现,突变体2-20-6和2-30-3比其相应野生型少1条带,而突变体1-40-271与其野生型比较没有变化,说明微波可能造成稻瘟病菌基因组DNA缺失或点突变而发生变异。继代分析表明微波处理获得的稻瘟病菌形态和致病性突变体是稳定的。     

尖角突脐孢菌在稗草和水稻上的萌发和侵染行为比较研究

本试验结合曲利苯兰和荧光素钠两种染色方法的优点,从显微角度研究了尖角突脐孢菌(Exserohilum monoceras)两个菌株X-27和HN-14在稗草和水稻上萌发和侵染行为的差异。结果表明,在寄主稗草上,接种4h后尖角突脐孢菌孢子开始从一端或两端萌发形成初生芽管;10h后芽管顶端膨大形成附着胞,附着在寄主表面,两端萌发的孢子约90%一端败育,仅一端形成成熟附着胞;在接种后24h内成熟附着胞形成率与接种时间成正相关,24h后趋于稳定;16h后在成熟附着胞下方受侵染的细胞内指状吸器开始形成,随后发育为掌状吸器;接种36h后菌丝在组织表面扩展形成网状,同时稗草叶片上显现叶斑病症状,部分菌丝能在细胞间蔓延扩展。在非寄主植物水稻上,同样观察到孢子萌发产生芽管,但是萌发起始时间滞后大约4h,初生菌丝分枝明显减少,且未能观察到附着胞和吸器的产生。     

营养物质对两种炭疽菌潜伏侵染的影响

通过不同浓度的几种糖类对潜伏侵染在青香蕉果实中的colletotrichum musae和芒果果实中的colletotichum gloeosporioides的菌体的影响进行了测定,结果表明,高浓度淀粉可极显著地提高两种炭疽菌的孢子萌发率,并有利于附着胞和分生孢子的形成。单糖和二糖在较低浓度时有利于孢子萌发和产孢,不利于附着胞形成。未成熟果实的坚硬结构和高淀粉含量为病菌提供了以附着胞形式潜伏侵染在寄主中的条件。     

柠檬醛熏蒸对互隔交链孢生长及其产毒的抑制作用

互隔交链孢是一种重要的能产生交链孢酚（AOH）等真菌毒素的植物病原菌。精油是重要的抑制病原菌侵染的挥发性植物提取物,其活性组分包括柠檬醛等。本研究表明柠檬醛可高效地抑制互隔交链孢的生长和AOH毒素的产生。柠檬醛熏蒸能够引起互隔交链孢菌丝断裂影响其延伸,而对其分生孢子结构的影响不明显。柠檬醛能够引起互隔交链孢活性氧生成的紊乱,这可能是导致AOH显著下降的原因之一。由于柠檬醛能高效抑制互隔交链孢生长和产毒,因此其可作为传统熏蒸剂的潜在替代品,以防控互隔交链孢引起的病害以及毒素污染。柠檬醛抑制互隔交链孢生长产毒的研究为其开发与应用奠定了良好的基础。     

链格孢柑橘致病型过氧化物酶体对接复合体蛋白AaPex13和AaPex14的生物学功能

过氧化物酶体是存在于真核细胞中的一类单层膜细胞器,参与多种生理生化代谢过程,而Pex13和Pex14是过氧化物酶体膜上的对接复合体蛋白,参与基质蛋白-受体复合体的跨膜运输。目前,Pex13和Pex14在大多数植物病原真菌中的生物学功能尚不清楚。本研究鉴定了柑橘褐斑病菌链格孢柑橘致病型(the tangerine pathotype of Alternaria alternata)的对接复合体蛋白Pex13和Pex14,并构建基因敲除突变体与回补菌株,探究其生物学功能。结果表明,与野生型和回补菌株相比,ΔAaPex13和ΔAaPex14营养生长、分生孢子形成显著下降,分生孢子的萌发率显著降低,抗氧化能力和抗细胞壁胁迫能力也显著减弱,病菌的ACT毒素产量分别降低30%和33%,在离体叶片上丧失致病力。此外,AaPex13和AaPex14的缺失导致基质蛋白无法定位到过氧化物酶体,过氧化物酶体生物发生存在缺陷。本研究明确了AaPex13和AaPex14在病菌生长发育、过氧化物酶体形成、ACT毒素产生以及维持致病力方面都具有重要的调控作用。     

cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

PEG介导原生质体转化构建链格孢荧光菌株及其侵染苹果花器特性

由链格孢侵染引起的苹果霉心病是一种普遍发生的苹果果实病害。该病菌从侵染到发病的周期较长,对其侵染规律较难开展研究,若借助荧光标记则有望有效解决这些问题。本研究基于PEG介导的菌丝体原生质体转化,将带有GFP基因的外源DNA随机插入链格孢菌基因组中,共获得150个转化子。选取菌落形态、生长速率、致病力均未受影响的转化子NC-1,在田间盛花期接种苹果花组织。48h后花瓣、花柱、花药和花丝发生褐变,出现水渍状病斑,4d后发病严重的花干枯脱落,20d后感病幼果霉变脱落,发病部位菌丝体在荧光显微镜下呈现明显的绿色荧光。试验结果表明,作者成功构建了PEG介导的链格孢菌丝体原生质体遗传转化体系,且获得的链格孢荧光菌株NC-1可用于田间侵染规律的研究。     

青岛胶州湾四种类型湿地AM真菌多样性

对青岛胶州湾4种类型湿地（盐田、滩涂、湖泊和河口）中芦苇Phragmites communis、香蒲Typha orientalis和碱蓬Suaeda glauca 根围土壤中丛枝菌根（arbuscular mycorrhizal,AM）真菌进行孢子分离与鉴定,分析湿地生态系统中植物根围AM真菌群落特征。共分离到AM真菌5属10种,包括斗管囊霉属Funneliformis 2种、无梗囊霉属Acaulospora 3种、近明球囊霉属Claroideoglomus 2种、球囊霉属Glomus 1种、巨孢囊霉属Gigaspora 2种,其中,斗管囊霉属Funneliformis及地斗管囊霉Funneliformis geosporum的分离频度和相对多度最高,分别为湿地中AM真菌优势属和优势种。滩涂和河口湿地中植物AM真菌侵染率显著高于湖泊和盐田湿地植物,AM真菌孢子密度以滩涂湿地最高（572个/20mL）,湖泊湿地最低（220个/20mL）;滩涂湿地的种丰度和Shannon-Wiener指数最高,分别为3.8和1.2。从植物种类来看,AM真菌侵染率总体呈现出香蒲>碱蓬>芦苇,AM真菌孢子密度以香蒲最高,芦苇最低,植物种类对AM真菌种丰度和Shannon-Wiener指数影响不显著（P>0.05）。二因素方差分析和典型RDA相关分析表明,寄主植物种类对AM真菌孢子密度有一定影响,但湿地类型对AM真菌多样性的影响更为显著（P<0.05）,胶州湾湿地土壤因子Ca ²⁺、速效P含量与AM真菌孢子密度、物种丰度和多样性指数显著负相关,而速效K、Na ⁺、pH与其显著正相关。结果表明植物种类主要影响AM真菌孢子密度,AM真菌多样性受植物种类和湿地类型综合影响,滩涂湿地较丰富的AM真菌多样性可能与该湿地较良好的理化性质有关。     

北方蜜环菌菌索生长、发育对环境氧气及土壤湿度的响应

蜜环菌属Armillaria spp.真菌是重要的植物病原菌和兰科Orchidaceae植物天麻Gastrodia elata的专性共生菌,能以菌索形式在土壤中存活和传播,因此,土壤环境变化对蜜环菌菌索生长、发育的影响,是关系天麻栽培和森林蜜环菌病害的重要过程。为了揭示蜜环菌生长发育对土壤水分和氧气的适应特征,本研究以北方蜜环菌Armillaria borealis为研究材料,设计氧气浓度5%、15%、20%和30%的4个水平与土壤湿度20%、40%、60%和80%的双因素实验,在室内24 ℃及黑暗条件下培养,观察菌索发生时间(T)、测定菌索生物量(DW)、菌索长度(L)、菌索数(Nt)、菌索分枝数(Nf)和菌索直径(AD)。结果表明,土壤湿度和氧气浓度对北方蜜环菌菌索发育和生长具有显著影响,在15%的氧气浓度和60%的土壤湿度发育最快。本研究为人工模拟蜜环菌生长发育的环境提供了部分依据,对提高天麻人工栽培的产量,揭示森林蜜环菌病害发病机制具有重要价值。     

Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.     

Trade-off relationship between vein density and vein diameter of Achnatherum splendens in response to habitat changes in Zhangye wetland

Aims The coevolution between vein traits has influences on water use strategies of plant and the formation of leaf economic spectrum, and therefore is important for understanding the trade-off between carbon input in leaf vein construction and the functional feedback from leaf veins. Our aim is to study the allometric relationship between vein density and vein diameter of Achnatherum splendens populations at three natural microhabitats (subcanopy, transitional and open areas) in Zhangye wetland. Methods According to the shade condition of the arbor canopy and the distance to arbor, the A. splendens community were divided into three microenvironments: subcanopy, transitional and open areas. We sampled 10 (4 m × 4 m) A. splendens plots from each microenvironment and investigate the biological characteristics of the plots and leaf traits of the plants within the plots. Then the soil physical and chemical properties, and community photosynthetically active radiation (PAR) were investigated at three gradients. Six individual of A. splendens were selected in each plot and the leaf length, leaf width, vein density and vein diameter of two or three healthy and complete leaves from four directions of each individual were measured in laboratory. The SMA estimation method and correlation analysis were then used to examine the allometric relationship between vein density and vein diameter. Important findings Along the gradient from subcanopy, transitional zone to open areas, soil moisture displayed a pattern of initial decrease of plant community, and soil electric conductivity displayed increase changing trends. Photosynthetically active radiation (PAR), vapor pressure deficit (VPD), vein density (1.28-1.59 mm·mm^-2), leaf width and water use efficiency (WUE) increase gradually, while the leaf length, vein diameter (0.21-0.16 mm) of A. splendens decrease. The average value of plasticity indexes of leaf characteristics was 0.19. Leaf net photosynthetic rate (P_n), transpiration rate (T_r) and bundle density increase first and them decrease. The vein density and vein diameter of A. splendens were negatively correlated with each other in subcanopy environment (p < 0.01), transitional and open areas (p < 0.05). The SMA (0.54-1.50) slope of regression equation in the scaling relationships between vein density and vein diameter decrease gradually from subcanopy to open areas.     

中国稻谷可培养真菌群落组成及多样性

真菌生长导致粮食品质下降及真菌毒素污染,是造成粮食损失的主要原因。我们采用形态学与分子系统发育分析相结合的方法,对中国13个省78份稻谷样品的真菌群落组成及多样性进行研究。结果表明,本次共分离622株真菌,经初步鉴定分属于17属73个物种。南北方因气候条件差异较大,稻谷真菌数量和物种组成都存在很大差异。南方稻谷表面真菌数量（10 ⁴cfu/g）明显高于北方（10 ³cfu/g）。北方收获期的优势种主要属于枝孢属Cladosporium,南方收获期的优势种为黄曲霉Aspergillus flavus、黑曲霉Aspergillus niger和阿姆斯特丹曲霉Aspergillus amstelodami等。进入储藏期后,由于中国仓储管理要求较高,储藏环境相对稳定（aw≤0.75）,耐旱真菌阿姆斯特丹曲霉和多育曲霉Aspergillus proliferans（占比40%以上）成为优势种,这类真菌可作为储藏期粮堆异常的早期预警指示菌。南方收获期感染的黄曲霉和黑曲霉进入储藏期后仍保持优势比例,导致储藏期间毒素污染风险必然增加,因此南方稻谷收储环节均应加强监管。本研究通过分析南北方收获和储藏环节稻谷真菌群落组成差异及产毒真菌的分布规律,以期为中国粮库在稻谷收储环节的真菌毒素防控提供依据。