玉米大斑病菌黑色素缺失突变体的获得及其生物学特性

DHN黑色素是许多植物病原真菌的致病相关因子,为明确黑色素在玉米大斑病菌致病过程中的作用,采用三环唑和紫外线复合诱变的方法获得了黑色素缺失的6个突变菌株,对突变菌株的生长量、产孢量、HT-毒素活性、致病力进行测定。结果显示,突变菌株产毒能力变化不大,但生长量下降,产孢量和致病力显著下降或完全丧失,野生型菌株的产孢量和侵染频率分别是突变菌株的30～50倍和5倍。突变菌株与黑色素共培养后,菌株M01-23a和M01-23b恢复了致病力,但病斑面积和侵染频率较小。玉米大斑病菌黑色素与致病力具有一定的相关性。     

玉米大斑病菌黑色素的一些理化性质和光谱吸收特征

 黑色素是某些植物和动物真菌病害的致病相关因子,不同来源的黑色素其生物合成途径可能不同。对玉米大斑病菌细胞壁结合黑色素和从培养滤液中提取的黑色素进行理化性质、紫外吸收光谱和红外光谱扫描测定,并与标准品黑色素进行比较分析,明确了玉米大斑病菌黑色素具有与标准品黑色素相似的理化性质。DHN黑色素的特异性抑制剂——三环唑,对玉米大斑病菌0号和1号小种黑色素的产生均有抑制作用;以玉米大斑病菌基因组DNA为模板,通过PCR扩增,得到了1,3,8-三羟基萘还原酶基因的同源片段,推测玉米大斑病菌黑色素合成于DHN途径。     

白头翁叶斑病菌黑色素的理化性质及其生物合成途径初步研究

对白头翁叶斑病菌胞壁结合黑色素和胞外黑色素进行了理化性质和红外光谱扫描测定, 结果表明两者具有相似的理化性质, 均易溶于KOH、H2O2和NaClO, 不溶于水、乙醇和丙酮。红外光谱分析表明, 白头翁叶斑病菌YS-24菌株的胞壁结合黑色素与胞外黑色素为同一种类型的黑色素。DHN黑色素的特异性抑制剂—三环唑, 对白头翁叶斑病菌黑色素的产生有明显的抑制作用; 以白头翁叶斑病菌基因组DNA为模板, 通过PCR扩增, 得到了聚酮体合成酶基因的同源片段 AaPKS , 初步推断白头翁叶斑病菌黑色素合成属于DHN途径。     

桑椹菌核病菌(Scleromitrula shiraiana)黑色素生物合成相关基因的克隆与功能分析

小柱孢酮脱水酶(scytalone dehydratase,SCD)及羟基萘还原酶(hydroxynaphthalene reductase,HNR)是真菌多聚二羟奈类(DHN)黑色素生物合成途径中的关键酶。根据已知真菌的小柱孢酮脱水酶及羟基萘还原酶的保守结构域设计兼并引物并利用RACE技术,获得桑椹菌核病菌(核地仗菌,Scleromitrula shiraiana)SsSCD1和Ss4HNR1的DNA和c DNA序列。SsSCD1和Ss4HNR1均含2个内含子和3个外显子,分别编码169和263个氨基酸残基。进化分析表明SsSCD1和Ss4HNR1与灰葡萄孢和核盘菌中小柱孢酮脱水酶和四羟基萘还原酶基因的亲缘关系最近。DHN黑色素合成特异性抑制剂三环唑处理核地仗菌,结果显示三环唑可抑制核地仗菌菌丝生长和黑色素合成,对SsSCD1的表达无显著影响,但Ss4HNR1的表达水平显著提高。这些结果表明三环唑能够特异性的抑制四羟基萘还原酶,且DHN黑色素是核地仗菌生长发育的重要产物。     

玉米大斑病菌CaM基因的克隆及三氟拉嗪的抑菌作用

 以玉米大斑病菌为材料,根据同源序列法分别进行了3'端和5'端的RACE扩增,获得了玉米大斑病菌钙调素基因cDNA全长。利用genomic walking技术获得了CaM基因启动子序列,该序列含有多个与转录调控有关的保守序列(如TATA-box、Spl、AP-2和TFⅡD)。Southem杂交结果表明,玉米大斑病菌CaM基因在基因组中以单拷贝形式存在。用CaM特异性抑制剂三氟拉嗪(trifluoperazine)处理玉米大斑病菌,发现其对孢子萌发和附着胞形成的抑制作用与浓度呈正相关,同一浓度下,抑制剂对附着胞形成的抑制作用大于对孢子萌发的抑制。推测CaM基因在玉米大斑病菌的致病过程中起着至关重要的作用。     

多菌灵、三环唑对大丽轮枝菌微菌核、黑色素形成及致病力的影响

 在离体和活体条件下测定了多菌灵、三环唑对大丽轮枝菌的微菌核黑色素形成的影响,以及经2种杀菌剂处理产生的形态变异菌株对棉苗的致病力。结果表明:多菌灵在培养基内含量超过0.1μg/ml时,即可抑制大丽轮枝菌微菌核的形成,随着培养基内多菌灵浓度的增加,微菌核形成时间逐渐延长,形成量逐渐减少;三环唑浓度为0.5μg/ml时,可抑制微菌核的黑色素形成,微菌核从黑色变为浅红至红褐色。三环唑对微菌核黑色素形成呈可逆抑制,变色的微菌核菌落移入不含药的培养基后,大多可恢复形成黑色素。培养基内三环唑浓度的提高,也可抑制大丽轮枝菌微菌核的形成;因2种杀菌剂的抑制而丧失形成微菌核的白色菌丝体移入不含药的培养基,微菌核形成能力也不能恢复。多菌灵和三环唑经棉株吸收后均能抑制植株内大丽轮枝菌微菌核的形成,但对微菌核色素的形成未见有明显影响。2种杀菌剂处理产生的形态变异菌株的致病力与野生型菌株致病力差异不显著。     

基底硬度对玉米大斑病菌主要侵染结构和黑色素沉积的影响

 基底硬度是调控植物病原真菌侵染结构形成的重要物理信号。为探讨基底硬度对玉米大斑病菌(Setosphaeria turcica)侵染能力的影响,本研究利用不同硬度的PDA培养基进行模拟试验,并在不同叶龄的玉米叶片上进一步验证,结果表明,基底硬度越大,气生菌丝越少,菌丝分枝越多;硬基底上,病菌的分生孢子产量高,是软基底和适中硬度基底上的29.93和6.82倍,且硬基底上病菌附着胞形成率显著高于软基底。研究发现,在硬基底上生长的大斑病菌菌落颜色较深,菌丝中黑色素含量高。利用实时荧光定量PCR技术分析病菌黑色素合成途径中相关基因的表达情况,发现在硬基底上StMR1、StPKS、St4HNR、StSCD、St3HNR、StLAC1、StLAC2和StLAC4基因的相对表达量均高于软基底。上述结果说明,基底硬度影响了病菌形态结构的发育,导致了与侵染相关的黑色素含量的变化和侵染结构的形成,研究结果为探究植物病原真菌的侵染机制和病害防控提供了理论依据。     

玉米茎腐病菌毒素致病力初报

通过提取玉米茎腐病菌毒素粗提液,证实玉米茎腐病原菌(镰刀菌和腐霉菌为主)在镰刀菌毒素培养液、查氏培养液和普通培养液中,能产生和玉米小斑病菌毒素(简称HM毒素,下同)类似的致病毒素。该物质能抑制种子根的生长。接种在玉米叶片上能产生典型的萎蔫枯死斑;用上述3种培养液培养病原菌,产生的毒素致病力差异不显著;不同类型病原菌产生的毒素致病力和同一类型病原菌不同菌株产生的毒素致病力差异都极显著。玉米品系对茎腐病原菌产生的毒素抗病性差异也极显著。     

希金斯炭疽菌自噬相关基因ChAtg26的功能分析

希金斯炭疽菌(Colletotrichum higginsianum)是一种重要的植物病原真菌,可引起严重的十字花科蔬菜炭疽病,对蔬菜生产造成了很大的影响。为了探究希金斯炭疽菌自噬相关基因ChAtg26在致病过程中的作用,本研究以希金斯炭疽菌侵染拟南芥Col-0后的cDNA为模板,通过qRT-PCR技术测定了ChAtg26基因在侵染过程中的表达模式,并利用同源重组技术构建了ChAtg26基因的敲除与回补突变体,分析了敲除ChAtg26基因对希金斯炭疽菌生长发育与致病能力的影响。结果表明,ChAtg26基因在病菌侵染寄主后0～40 h中有较高的表达量,而敲除ChAtg26基因后,病菌在生长速率、孢子萌发、附着胞形成以及对氧化胁迫敏感性方面没有明显变化,但是会在菌落黑色素累积与对细胞壁胁迫的敏感性方面有所下降,同时其产孢量与致病力会明显降低,说明ChAtg26基因参与了希金斯炭疽菌的黑色素合成、细胞壁胁迫应答反应、产孢与致病过程,并在这些过程中起着重要作用。     

玉米灰斑病菌生理分化的鉴别寄主体系

玉米灰斑病现已成为北方玉米产区的主要病害之一，但国内外关于玉米灰斑病菌Cercospora zeaemaydis Tehon ＆ Daniels致病力分化等方面的研究较少，尚未建立该病菌生理分化的鉴别寄主系统，无法明确我国是否存在玉米灰斑病菌致病性分化类群或生理小种及优势菌群和分布，限制了有针对性地进行品种合理布局和抗病育种工作，因此研究玉米灰斑病菌致病性分化鉴别寄主体系是一项重要的基础性工作。作者采用田间成株期鉴定技术，对不同的玉米灰斑病菌菌株进行致病力划分，研究其鉴别寄主体系。     

Melanin biosynthesis in Pyricularia oryzae: Site of tricyclazole inhibition and pathogenicity of melanin-deficient mutants

Tricyclazole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) inhibits melanin synthesis in Pyricularia oryzae at concentrations less than 0.01 μg/ml. The primary site of inhibition in the biosynthetic pathway occurs between scytalone and vermelone. Accumulation of several metabolites derived from melanin precursors along branch pathways is associated with inhibition of melanin biosynthesis. At low tricyclazole concentrations (0.01–1 μg/ml), predominant accumulation of 2-hydroxyjuglone and 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone (3,4,8-DTN) occurs as a result of the primary block between 1,3,8-trihydroxynaphthalene and vermelone. As the concentration of tricyclazole is increased from 1 to 10 μg/ml, flaviolin accumulation is markedly enhanced, whereas that of 3,4,8-DTN and 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone is depressed, indicating possible secondary sites of inhibition in the main and branch pathways. Five melanin-deficient mutants of P. oryzae that phenotypically resemble the tricyclazole-treated wild-type strain were nonpathogenic or rarely infected two rice varieties. Three of the mutants studied were genetically defective in the melanin biosynthetic pathway at the site blocked by tricyclazole in the wild type. The wild-type strain converted both scytalone and vermelone to melanin; whereas the three mutants and the tricyclazole-treated wild type converted only vermelone to melanin. The data suggest a relationship between melanin biosynthesis and pathogenicity in P. oryzae.     

草莓多主棒孢霉小柱孢酮脱水酶基因CcSCD1的功能研究

 多主棒孢霉(Corynespora cassiicola)是世界范围内重要的植物病原真菌,其引起的草莓棒孢霉叶斑病对草莓产业的健康发展具有潜在威胁。小柱孢酮脱水酶(scytalone dehydratase,SCD)是真菌多聚二羟萘类(DHN)黑色素生物合成途径中的关键酶,在植物病原菌致病过程中发挥重要作用。本研究通过同源重组的方法获得了草莓多主棒孢霉小柱孢酮脱水酶基因(CcSCD1)的敲除突变体,并进行了回补和RT-PCR验证。与野生型相比,敲除突变体△CcSCD1-2表现为菌落无色素沉积、菌丝稀疏、产孢量显著下降、分生孢子无色较小以及致病力明显减弱。结果表明,CcSCD1参与调控多主棒孢霉的黑色素生物合成、营养生长、分生孢子产量及致病力。     

三环唑对稻瘟病菌再侵染的抑制作用初探

在实验室条件下模拟稻瘟病菌Magnaporthe grisea再侵染发生的全过程,研究了三环唑对稻瘟病菌再侵染的抑制作用及其机制。在接种稻瘟病菌48 h后喷雾处理,10 μg/mL三环唑的防病效果只有28.58%,表明三环唑对稻瘟病的治疗效果较差;10 μg/mL三环唑处理过的发病植株作为再侵染源引起周围未经药剂处理的健康水稻的发病程度要比未经药剂处理的病株作为再侵染源引起的低63.28%。进一步的研究表明,三环唑抑制稻瘟病菌再侵染的主要机制是抑制再侵染源的孢子产生和降低分生孢子的释放及致病能力。     

Effect of tricyclazole on growth and secondary metabolism in Pyricularia oryzae

Tricyclazole [5-methyl-1,2,4-triazolo (3,4-b)-benzothiazole] controls rice blast disease caused by Pyricularia oryzae at concentrations (5–10 μg/ml) which do not inhibit growth of the pathogen in vitro. However, concentrations of 1 μg/ml or less inhibit melanin formation in the fungus. Production of pyriculol by the pathogen is usually enhanced by 10 μg/ml of tricyclazole, whereas production of 3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone is strongly inhibited or markedly reduced and delayed. Evidence suggests that tricyclazole blocks aspects of polyketide metabolism in P. oryzae which may have a role in pathogenicity.     

Melanin biosynthesis as a prerequisite for penetration by appressoria of Colletotrichum lagenarium: Site of inhibition by melanin-inhibiting fungicides and their action on appressoria

Melanin biosynthesis by appressoria was studied in relation to their penetrating ability using tricyclazole [5-methyl-1,2,4-triazolo(3,4-b)benzothiazole], pp 389 [4,5-dihydro-4-methyltetra-zolo(1,5-a)quinazolin-5-one], and pyroquilon [1,2,5,6-tetrahydropyrrolo(3,2,1-i,j)quinolin-4-one], and color mutants of Colletotrichum lagenarium. Tricyclazole at 100 μM inhibited melanin biosynthesis by appressoria of C. lagenairum 104-T, and caused accumulation of 3,4-dihydro-4,8-dihydroxy-1(2H)naphthalenone (DDN) in the culture medium. By contrast, DDN was not detected in culture media of tricyclazole-treated mutant 8015, which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene (1,3,8-THN). Vermelone restored melanization of appressoria of albino mutant 79215 and of the parent strain 104-T treated with tricyclazole, pp 389, and pyroquilon; however, scytalone restored melanization only in appressoria of albino mutant 79215. These results indicate that tricyclazole, pp 389, and pyroquilon inhibit the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway of appressoria of C. lagenarium. Colorless appressoria formed in the presence of the three melanin-inhibiting chemicals germinated laterally on nitrocellulose membranes and rarely penetrated the membranes. On the other hand, when pigmented appressoria were restored by application of vermelone in the presence of the three chemicals, lateral germination of the appressoria was largely suppressed, and the membranes were effectively penetrated. From these results, it is concluded that the major effect of tricyclazole, pp 389, and pyroquilon on appressoria of C. lagenarium, causing failure of penetration, is the inhibition of melanization. Effects of the chemicals on other metabolic functions can be precluded as significant factors affecting the penetration process.     

Localized Melanization of Appressoria Is Required for Pathogenicity of Venturia inaequalis

ABSTRACT During formation of appressoria produced from conidia and ascospores of Venturia inaequalis, a dark brown ring structure was detected at the base of appressoria. This melanized appressorial ring structure (MARS) was attached to the leaf surface like a sealing ring and formed the fungus-plant interface; it is believed to be required for pathogen penetration of the cuticle. Neither germ tubes nor infection structures beneath the cuticle were found to be visibly melanized. MARS were formed not only on apple leaves but also on leaves of nonhost plants and artificial surfaces differing in hydrophobicity; the formation of appressoria and MARS was confined to hard surfaces. The melanin nature of the ring was confirmed by using melanin biosynthesis inhibitors. Applications prior to inoculation largely inhibited the melanization and reduced infection rate by 45 to 80%; curative applications were not effective. Transmission electron microscopy verified a localized melanization of the cell wall around the penetration pore, and melanin was incorporated into all layers of the fungal cell wall. Appressoria without MARS were not able to infect the plant, suggesting that this structure can be considered to be a pathogenicity factor in V. inaequalis.     

硫与三环唑混配对抑制稻瘟病菌的协同增效作用

试验证明, 硫与三环唑(7∶1) 混配成悬浮剂, 对抑制稻瘟菌的菌丝生长、分生孢子萌发、附着胞的形成以及产孢能力等都具有较明显的协同增效作用, 协同作用比率(S R ) 分别为2. 1、2. 5、1. 8 及8. 6。混配剂集合了硫及三环唑两者的优点, 并显示出耐雨水冲刷、持效期长的特点。田间试验亦表明其防病增产效果比较显著。