稻瘟病菌（Magnaporthe oryzae）的凋亡诱导和检测

 使用低浓度H2O2和高浓度NaCl诱导稻瘟病菌的凋亡,检测了产生的凋亡特征。结果显示：诱导后稻瘟病菌具有典型的凋亡特征,如胞内活性氧累积、磷脂酰丝氨酸从细胞膜的内表面转移到外表面、染色体浓缩和DNA断裂。进一步研究发现：凋亡关键性酶类caspase的抑制剂Z-VAD-FMK能够显著影响稻瘟病菌的孢子萌发和附着胞形成过程。典型的稻瘟病菌孢子由3个细胞组成;在Z-VAD-FMK的作用下,2个以上细胞同时萌发产生芽管的比例由34.1％增加到66.0％,同时形成附着胞的源细胞也以顶端细胞为主转变为以基部细胞为主。这些结果说明凋亡参与了稻瘟病菌的孢子萌发和附着胞形成过程。     

Effect of carpropamid on secondary infection by rice blast fungus

Carpropamid (WIN™, KTU 3616) provides good control of leaf and panicle blast by ‘one-shot’ nursery-box treatment. It inhibits melanin biosynthesis in appressorial cells of Pyricularia oryzae, making them hyaline. Penetration by infection hyphae from the hyaline appressoria into rice epidermal cells is substantially hindered. In addition, the spread of rice blast spores from primary lesions to the other parts of the plant leading to secondary infection is largely prevented when the plants are treated with carpropamid by spray or water surface application. Secondary infection was simulated in a glass chamber fitted with an ultrasonic humidifier. On treated plants, many blast spores formed in the lesions, but the number of air spora that were dispersed from the lesions decreased significantly. A similar suppression of the spore liberation was observed in vitro when lesions on rice leaf segments, or discs from Pyricularia cultures on oatmeal agar were treated with the chemical. Spores from treated lesions or from the cultures on oatmeal agar amended with the chemical germinated normally and produced well-melanized appressoria on cellophane membranes. In addition, the spores proved to be fully pathogenic towards rice seedlings, producing normal disease symptoms. These results strongly suggest that carpropamid reduces the secondary infection of rice by Pyricularia by specifically hindering spore liberation. © 1999 Society of Chemical Industry     

Assessment of blast disease resistance in transgenic PRms rice using a gfp-expressing Magnaporthe oryzae strain

Rice blast caused by the fungus Magnaporthe oryzae (anamorph Pyricularia grisea ) is one of the most devastating diseases of cultivated rice worldwide. In this study, a green fluorescent protein ( gfp )-expressing M. oryzae strain was generated and used to investigate the infection process in a commercial rice cultivar. Expression of the gfp gene did not affect the pathogenicity of the M. oryzae transformants. Confocal microscopy allowed in vivo imaging of this pathogen during infection of rice tissues. Magnaporthe oryzae pathogenicity was examined on both leaf and root tissues. In roots of wild-type plants, the fungus penetrated into epidermal and cortical cells, and colonized the central cylinder and xylem vessels. However, the dimorphic growth pattern typically observed during the biotrophic and necrotrophic stages of leaf colonization was not observed during colonization of root tissues. Furthermore, events occurring during infection of rice plants constitutively expressing the maize pathogenesis-related PRms gene were characterized and compared with those occurring during the interaction of this pathogen with untransformed rice plants. Fungal penetration was drastically reduced and delayed in tissues of PRms plants compared to untransformed plants. These results indicated that the gfp -expressing M. oryzae represents a strategic tool for the assessment of blast disease resistance in transgenic rice which can be also applied to the analysis of the M. oryzae interaction with other cultivars or mutants of important crop species.     

Selection and mutation of the avirulence gene AVR-Pii of the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae avirulence (AVR) genes are predicted to be involved in pathogen invasion and their virulence functions are restricted by the presence of the cognate resistance (R) genes. In this study, the distribution and variation of the avirulence (AVR-Pii) gene of M. oryzae in Yunnan province, China were analysed to understand haplotype diversity of AVR-Pii under field conditions. The presence of AVR-Pii in 454 field isolates of M. oryzae collected in Yunnan province was examined using gene-specific PCR markers. The results showed that 82 M. oryzae isolates carried AVR-Pii. Among them, 39 (35.5%), 5 (15.2%), 4 (14.3%), 2 (13.3%), 25 (12.8%) and 7 (9.7%) of the M. oryzae isolates carried AVR-Pii from central, southeastern, southwestern, northwestern, western and northeastern Yunnan province, respectively. Of these isolates, 55 were sequenced, while the remaining 27 isolates were not suitable for PCR-based sequencing and were not used for further analysis. Moreover, three AVR-Pii haplotypes were identified among the 55 isolates, in which H1 was identical with a previous sequence in GenBank (accession no. AB498874 ) and H2 and H3 were novel variants. All DNA sequence variations were found to occur in the protein-coding region resulting in amino acid substitutions. One virulent haplotype of AVR-Pii to Pii was identified among 55 field isolates, suggesting that the AVR-Pii gene has lost avirulence function through base substitution. These findings suggest that AVR-Pii is under positive selection and AVR-Pii mutations are responsible for overcoming race-specific resistance in nature.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

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.     

Investigation of rice blast development in susceptible and resistant rice cultivars using a gfp‐expressing Magnaporthe oryzae isolate

In this study, an isolate of Magnaporthe oryzae expressing the green fluorescent protein gene (gfp) was used to monitor early events in the interaction of M. oryzae with resistant rice cultivars harbouring a blast resistance (R) gene. In the resistant cultivars Saber and TeQing (Pib gene), M. oryzae spores germinated normally on the leaf surface but produced morphologically abnormal germ tubes. Germling growth and development were markedly and adversely affected in leaves of these resistant cultivars. Penetration of host cells was never seen, supporting the idea that disruption of germling development on the leaf surface might be one of the resistance mechanisms associated with Pib function. Thus, this particular R gene appeared to function in the absence of host penetration by the fungal pathogen. Confocal laser scanning microscopy of M. oryzae‐infected susceptible rice cultivars showed the dimorphic growth pattern that is typically observed during the biotrophic and necrotrophic stages of leaf colonization in susceptible cultivars. The suitability of the gfp‐expressing M. oryzae isolate for further research on R‐gene function and identification of resistant genotypes in rice germplasm collections is discussed.     

GTP酶激活蛋白MoGcs1参与调控稻瘟病菌的无性繁殖,附着胞分化及对外界胁迫的应答

稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是水稻上的一种毁灭性真菌病害,每年导致的稻谷产量损失可养活6千万人口。从分子水平了解该病菌的致病机理,对新型药剂靶标的挖掘和病害防控策略的制定具有重要的理论和实践指导意义。生物体存在两类GTP结合蛋白,一类是异三聚体G蛋白,另一类是小G蛋白。小G蛋白分子量为20~30 KD,具有GTP酶活性,在多种细胞反应中具有分子开关作用。小G蛋白结合GTP时被激活,可作用于下游分子使之活化。当GTP水解成为GDP时,则恢复为非活化状态。细胞中存在一些专门控制小G蛋白活性的调节因子,如鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factor,GEF)和GTP酶激活蛋白(GTPase activating protein,GAP)。GEF能够催化GTP的转换,而GAP的作用是加速GTP的水解,使小G蛋白向失活的状态转变。本研究在稻瘟病菌中鉴定到一个GAP蛋白MoGcsl,并对该蛋白编码基因的生物学功能进行了研究。对MoGCS1基因进行敲除突变发现,△Mogcsl突变体产孢量显著下降,分生孢子形态异常且附着胞形成加快,但突变体的营养生长和致病能力与野生型比较无明显变化。进一步研究发现,突变体对外界盐胁迫敏感性升高,对细胞壁胁迫敏感性降低。上述结果表明,MoGcsl是稻瘟病菌生长发育过程中一个重要的蛋白,参与调控病菌的无性繁殖、附着胞分化及对外界胁迫的应答。     

三环唑和多菌灵对水稻抗病品种与水稻稻瘟病菌互作的影响

分析了三环唑和多菌灵对水稻稻瘟病毒力及对品种抗病性的影响,结果表明三环唑和多菌灵对水稻品种与稻瘟病菌互作有重要影响。当喷雾0.2μg/mL三环唑于13个日本已知抗性基因品种时,原对ZE₃和ZG₁混合菌株表现抗性的爱知旭表现感病,当喷雾2μg/mL三环唑时,爱知旭则表现抗病,而在0、0.2μg/mL三环唑时均表现抗病的露明则在2μg/mL三环唑时表现感病;喷雾0.2μg/mL多菌灵于13个日本已知抗性基因品种时,ZB₁、ZE₃、ZG₁混合菌株在0μg/mL多菌灵时表现抗性的爱知旭则表现感病,当多菌灵浓度为2μg/mL时,露明又表现为抗病,而在0、0.2μg/mL多菌灵时,对ZG₁表现为抗病的草笛则表现感病;13个已知抗性基因品种中其他品种在使用了三环唑和多菌灵后抗性也发生了变化,这说明稻瘟病菌的分布和组成与这类杀菌剂的长期使用可能有一定关系。     

湖北省随州地区稻瘟病菌群体结构分析及生态模式菌群的建立

采用人工接种方法,用7个中国鉴别品种和95个自选资源材料对2014年至2015年间采自湖北省随州地区的76个稻瘟病菌单孢菌株进行致病型鉴定。分别利用传统生理小种种群划分方法和近邻传播聚类法(affinity propagation clustering,AP聚类法)进行菌群结构分析,比较两种方法的差异。结果表明,传统生理小种种群划分方法将76个菌株分为6个菌群,14个中国小种, ZB菌群为优势种群,占比65.79%,ZB15为优势生理小种,占比达到60.53%;近邻传播聚类法将76个菌株分为14个菌群,Cluster 2、Cluster 6、Cluster 7、Cluster 8为随州地区优势菌群,占比分别为18.42%、19.74%、22.37%、17.11%,Cluster 6、Cluster 7和Cluster 8具有相对较高的致病力;两种方法划分结果虽不完全一一对应,但划分趋势相同,近邻传播聚类法在稻瘟病菌种群结构研究中具有一定应用前景;根据试验结果筛选出1个代表随州地区稻瘟病总致病谱的菌群,菌群由12个菌株组成,将其命名为“随州生态模式菌群”。     

Impact of weedy rice populations on the growth and yield of direct-seeded and transplanted rice

To examine the impact of weedy rice ( Oryza sativa L. f. spontanea ) populations on the growth and yield of direct-seeded and transplanted rice ( Oryza sativa ), a field experiment with a random two-factor design that included cultivation methods (direct-seeding and transplanting) and the density of weedy rice (0, 5, 25, and 125 plants per m²) was conducted. The data from the experiment showed that weedy rice had a significantly poorer performance in the direct-seeded fields than in the transplanted fields in terms of its vegetative (plant height) and reproductive traits (panicle and seed production). In contrast, with the interference of weedy rice, cultivated rice showed an improved performance in the direct-seeded fields than in the transplanted fields, with a significantly higher tiller production and grain yield. The results suggest that cultivated rice can tolerate more successfully the infestation of weedy rice in direct-seeded fields because of its enhanced competitive ability compared to that in transplanted fields.     

湖南桃江病圃稻瘟病菌对24个水稻抗稻瘟基因的毒性分析

稻瘟病是水稻生产上的重要病害,了解稻瘟病菌群体毒性组成是水稻抗病品种合理布局的重要基础。2012-2015年从湖南桃江病圃中不含已知抗瘟基因的水稻品种‘丽江新团黑谷’上成功分离出351个稻瘟病菌单孢菌株,在温室于水稻5叶期采用离体接种法测定了其对24个水稻抗稻瘟病单基因系的毒性,结果表明,病圃中稻瘟病菌以广谱强致病性的菌株为主,病菌对不同抗瘟基因的毒力频率在50.56%~96.67%之间,而不同年度间对24个抗性基因均具毒性的菌株出现频率在0~15%之间。对2015年的每2个抗瘟基因的联合毒力分析表明,基因两两搭配后的联合抗病系数最高、联合致病系数最低的组合是Pi-3*Pi-k(RAC=0.19,PAC=0.43)。     

水稻和稻瘟病菌互作中的信号传导及防御反应基因诱导表达的研究

 水稻和稻瘟病菌互作是研究植物与病原菌互作的模式体系。本文利用3个水稻抗稻瘟病近等基因品系(CO39、C101LAC和C101A51)和2个特异性菌株(M209和M210)构成不同亲和程度的互作关系,研究了稻瘟病菌对3个水稻信号传导途径关键酶合成基因、5个防御反应病程相关蛋白基因和1个防御反应转录因子调控基因诱导表达的作用。结果表明:稻瘟病菌诱导了各种互作关系中水稻OsLOX、OsAOS和OsPAL酶合成基因的表达,水稻启动了茉莉酸和水杨酸防御反应信号传导途径。在不亲和的互作反应中,稻瘟病菌能不同程度地诱导水稻OsPR1a、OsPR2、OsPR3-1、OsPR3-2和OsPR4基因的表达,从而有效激活了防御反应系统,使水稻植株表现为抗病;而在亲和的互作反应中,多数OsPR基因的表达水平低、时间短或没有表达,水稻植株表现为感病。OsMyb基因在各种互作关系中有不同的诱导表达。说明这些防御相关基因的诱导表达可能与水稻抗稻瘟病性相关。     

转录因子MoMsn2的核定位和核输出信号序列参与调控稻瘟病菌的生长发育和致病力

 稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是水稻上最重要的真菌病害之一,每年给水稻生产造成10%~30%的产量损失。从分子水平解析该病菌的致病机理,对稻瘟病防控新途径的挖掘具有重要的理论和实践指导意义。前期研究发现,转录因子MoMsn2定位于细胞质和细胞核,通过调控一系列下游基因的表达,控制稻瘟病菌的生长发育和致病性等多个生物学过程。对MoMsn2进行结构域分析,发现其含有2个核定位信号序列NLS1和NLS2、1个核输出信号序列NES和2个锌指蛋白结构域C2H2,但这些结构域的生物学功能尚不清楚。本研究通过构建结构域缺失载体和获得互补菌株的方法对5个结构域在稻瘟病菌的功能进行了分析。结果发现,同时缺失2个C2H2对MoMsn2的功能没有影响,ΔC2H2菌株的表型与野生型和全长互补菌株一致。缺失NLS1能完全恢复ΔMomsn2突变体的营养生长、菌落色素和胁迫应答;部分恢复其产孢量和致病力缺陷。缺失NES仅能部分恢复突变体的生长缺陷;而缺失NLS2完全不能恢复突变体的缺陷,其表型与突变体一致。荧光观察发现,缺失NES和NLS2改变了MoMsn2的核定位模式,而缺失C2H2和NLS1不影响MoMsn2的亚细胞定位。上述结果表明,NLS1、NLS2和NES是MoMsn2中3个重要的结构域,对MoMsn2在稻瘟病菌中行使正常的生物学功能具有重要的调控作用。     

稻瘟病菌ABC转运蛋白基因中 SSR的分布及其功能预测

 ABC转运蛋白在真菌的致病性方面起着重要的作用。本研究查找和分析了稻瘟病菌基因组中47个ABC转运蛋白基因的外显子区、内含子区、5′-UTR和3′-UTR区中的SSR,并对编码区中SSR的扩张或收缩对蛋白结构的影响进行了预测。结果表明,SSR在这些基因的调控区和编码区分布不均一,且在基因的外显子区、内含子区、5′-UTR和3′-UTR区中SSR的组成和分布均不相同;基因的编码区中三碱基和六碱基SSR相对较多,SSR的基序大都表现为GC含量较高,编码的亲水性氨基酸出现频率远远高于疏水性氨基酸。根据稻瘟病菌自然群体中SSR的变化幅度,预测了SSR的扩张或收缩对蛋白二级结构的影响,发现SSR的扩张或收缩都可能对蛋白的结构产生影响。暗示着SSR的变异在致病相关基因的变异中可能扮演着重要角色。     

利用绿色荧光蛋白GFP研究稻瘟病菌与水稻的互作

稻瘟病菌Magnaporthe oryzae严重威胁水稻的产量与质量,明确稻瘟病菌与水稻互作过程及机理,对防治稻瘟病具有重要意义。本研究利用稻瘟病菌常用致病菌株GUY11和ZB25,构建了绿色荧光蛋白GFP的过量表达菌株,并通过荧光显微观察菌株侵染寄主水稻过程中侵染结构的形成与发育,包括孢子萌发、附着胞形成、侵染钉形成、侵染菌丝增殖、坏死斑形成及产孢。另外,通过比较过量表达菌株对稻瘟病高抗水稻和易感水稻的侵染过程,发现侵染过程的差异主要集中于侵染钉的穿透和侵染菌丝的定殖。本研究为分析稻瘟病菌对寄主水稻的定殖规律提供了一种有效工具。     

Effects of leaf blast on growth and production of a rice crop

Rice crops grown under irrigated conditions were inoculated withPyricularia oryzae during early growth stages to study the effect of leaf blast on yield formation. The inoculations led to severe epidemics of leaf blast around maximum tillering, characterized by the presence of typical blast lesions and an accelerated senescence of heavily infested leaf tissue. Leaf blast led to a prolonged tillering and a delay in flowering and maturity.Crop growth rate and leaf area formation declined sharply during establishment of the disease and continued to be reduced till maturity. This resulted in a marked reduction of total dry matter production and grain yield. Dry matter distribution was not affected. Leaf blast reduced spikelet number, 1000 grain weight, and the fraction filled grains. From this last observation it was concluded that the reduction in grain yield was exclusively source determined.Nitrogen uptake of the inoculated crops before flowering was reduced compared to the N uptake of the control crop, but shoot N content of the inoculated crops at flowering was higher. Uptake of nitrogen after flowering was negligible in both healthy and inoculated crops. Redistribution from vegetative tissue was therefore the main source of N for grain growth. The higher N content of the shoot organs in the inoculated crops during ripening led to the conclusion that the reduced N uptake was not responsible for the yield reduction observed. Consequently, the reduction in grain yield was solely determined by a reduced carbohydrate supply.     

MoRAD26参与稻瘟病菌的胁迫应答和致病过程

 稻瘟病菌引起的稻瘟病是水稻三大病害之一。DNA修复对维持基因组的完整性具有重要作用,为了解析DNA修复基因在稻瘟病菌致病过程中的功能,本试验对稻瘟病菌DNA损伤检控点蛋白MoRad26进行了功能分析。首先我们利用同源重组的方法获得了MoRAD26基因的稻瘟病菌敲除突变体。表型观察发现MoRAD26缺失突变体的致病力下降,对DNA损伤胁迫和H₂O₂胁迫更加敏感,但是对稻瘟病菌的生长、产孢以及分生孢子萌发和附着胞的形成没有明显影响。进一步分析表明,敲除MoRAD26基因降低了稻瘟病菌侵染菌丝的扩展能力进而影响稻瘟病菌的致病力,但其具体机制还有待深入研究。