水稻纹枯病菌PG的分离纯化及其理化性质研究

水稻纹枯病菌产生的多聚半乳糖醛酸酶(Polygalacturonase,PG)是其重要的致病因子之一。用丙酮法提取PG粗蛋白,分别经DEAE-Sepharose Fast Flow离子交换柱、Phenyl-Sepharose 6 Fast Flow疏水柱、Sephadex G-75凝胶柱和DE52离子交换柱层析纯化得到一种具有较高活性的PG纯蛋白。该蛋白分子量为39.81 kD;等电点为4.58;含有糖基,含糖量为1.48%;含有α氨基酸,但不含芳香族氨基酸;不含脂基。这种蛋白在pH4~12范围内均具有活性,pH5时活性最大;对热不稳定,100℃下水浴20 min,活性完全丧失;对胰蛋白酶和蛋白酶K敏感,酶处理后其活性只有对照的35.0%和35.2%;对紫外线和氯仿亦敏感,处理后活性仅为对照的40.0%和51.7%。     

Purification and partial characterization of xylanase from the fungal maize pathogenHelminthosporium turcicum (Pass)

The fungal pathogenHelminthosporium turcicum was found to secret xylanase when grown on minimal medium containing xylans, wheat straw or isolated maize cell walls. The highest xylanase activity occurre when the fungus was grown on maize cell walls. When glucose was added to this medium xylanase activity was suppressed. The xylanase enzyme was purified from the culture filtrate by subsequent anion exchange chromatography, cation exchange chromatography and gel filtration. The purified xylanase gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to an apparent molecular weight of 22.5 kDa. It is determined to have a pI of 7.4, specific activity of 11300 nanokatals mg^–1, pH optimum between pH 5.5 and 6.5 and optimal temperature between 50 °C and 60 °C. The half-life of the enzyme at pH 6.0 and 50 °C was found to be 35 min. For primary structure comparison with other xylanases, the protein was digested with trypsin and the resulting peptides were separated by reversed phase chromatography and selected peptides were sequenced. The determined amino acid sequence showed high homology with xylanase fromCochliobolus carbonum and three other fungal xylanases.     

水稻条斑病菌异柠檬酸脱氢酶在致病性中的功能分析

 异柠檬酸脱氢酶(isocitrate dehydrogenase,IcdH)催化异柠檬酸转化成α-酮戊二酸,参与碳代谢途径末端的三羧酸(tricarboxylic acid,TCA)循环。然而,编码异柠檬酸脱氢酶基因是否参与水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的致病性,我们并不清楚。为了阐明IcdH的作用,通过同源重组技术获得了Xoc的icdH基因缺失突变体(RΔicdH),并对该突变体进行了相关功能研究。研究表明:该突变体不能利用苹果酸、丙酮酸和柠檬酸,在寄主水稻上的生长能力和致病力相对于野生型均显著降低,其游动性也显著减弱; 功能互补子恢复RΔicdH的上述表型至野生型水平; Real-time PCR结果显示,六碳单糖、蔗糖、苹果酸、丙酮酸与柠檬酸能显著诱导icdH基因的转录表达;与水稻细胞互作时icdH基因受诱导表达,并受HrpX和HrpG负调控。这些结果说明:icdH基因是Xoc获取碳源和在寄主水稻上具有致病性所需的。     

Molecular characterization of Diplodia seriata,a new pathogen of Prunus laurocerasus in Italy

Twig cankers and diebacks were observed on cherry laurel (Prunus laurocerasus L.) hedges located in a private garden of Perugia (Central Italy). Four fungal isolates, forming gray colonies and aerial mycelium, were obtained from cankers. For each isolate, Koch’s postulates were fulfilled by inoculating cherry laurel plants. Based on cultural characteristics, rep-PCR and phylogenetic analyses on ITS and EF1-α nucleotides sequences, the isolates were identified as Diplodia seriata De Not. To the best of our knowledge, this is the first report of D. seriata as a new pathogen of P. laurocerasus. The severe symptoms and the wide distribution of the host plant make the pathogen potentially dangerous for this ornamental species.     

稻瘟病菌激发子诱导非寄主植物的抗病效果

 Hyphal cell wall crude elicitor(CWE)of rice blast pathogen could induce hypersensitive response in tobacco and induce other nonhost plants to be resistant to other fungal pathogens.When corn was treated with CWE,they inhibited the infection of Exserohilum turcicum and Curvularia lunata,and when plants of capsicum and cucumber were treated with CWE,they inhibited the infection of Colletotrichum gloeospori-oides.CWE solution showed no bioactivity on spore germination and hyphal growth of the experiment fungi in vitro.Nonhost resistance induced by CWE to other fungal pathogens was not complete resistance.The induced resistant effect(IRE) increased as CWE concentration increased,however,IRE had somehow satura-ted concentration of CWE.Induced nonhost resistance by incompatible pathogen was quantitative to other compatible pathogens.The induced resistance was best at 2 or 3 d after CWE treatment,and then decreased.IRE was about 20 percent in 10 d after CWE treatment.     

Bis-pyrimidylpyrazolinones – a new class of acetohydroxy-acid synthase (AHAS) inhibitor

Hydroxypyrazolinones which bear two pyrimidine rings (on N-1 and C-4) were found to be potent inhibitors of acetohydroxy-acid synthase which displayed good herbicidal activity in vivo. Structure–activity relationship studies suggested the presence of a second binding niche on the enzyme for a 4,6-dimethoxypyrimidine ring.     

一种新的香蕉黑斑病病原菌鉴定及生物学特性研究

 在广西香蕉种植区发现一种引起香蕉黑斑病的新病原。通过对病原菌分离培养、致病性测定、形态学观察、分子生物学鉴定及对该病原菌生物学特性进行初步研究,表明引起该病害的病原菌为芒果球座菌(Guignardia mangiferae A.J. Roy)。该病原菌菌落为深橄榄色,产生棒状子囊、梭形子囊孢子及倒梨形分生孢子;其最适生长温度为28℃,最适pH 值为6;病原菌生长较好的碳源为果糖和阿拉伯糖,氮源为蛋白胨和酵母膏;在全光条件下,病原菌菌丝扩展速度最快;菌丝的致死温度为54℃。由G. mangiferae引起的香蕉黑斑病,在国内外为首次报道。     

Bacterial pathogens of rice in the Kingdom of Cambodia and description of a new pathogen causing a serious sheath rot disease

A study of rice diseases in Cambodia from 2005 to 2007 showed widespread occurrence of diseases caused by Acidovorax avenae subsp. avenae, Burkholderia gladioli, B. cepacia and Pantoea ananatis. This is the first report of these pathogens in Cambodia. Additionally, a pseudomonad causing a widespread disease similar to sheath brown rot (caused by Pseudomonas fuscovaginae) was isolated. The studied strains were pathogenic to rice cvs Sen Pidau and IR 66, producing similar, though slightly less severe, symptoms to those observed in the field. Based on comparative 16S rDNA gene sequence analysis, combined with cell wall fatty acid analysis and metabolic profiles, the isolated strains were allocated to the genus Pseudomonas. The novel species were differentiated from Pseudomonas fuscovaginae and P. putida by their inability to metabolize d ‐fructose, d ‐galactose, d ‐galactonic acid lactone, d ‐galacturonic acid, d ‐glucosaminic acid, d ‐glucuronic acid, p‐hydroxy phenylacetic acid, d ‐saccharic acid and urocanic acid. The major fatty acids were C_16:0, summed feature 3 (C_16:1ω7c and C_16:1ω6c) and summed feature 8 (C_18:1ω7c), representing 80% of the total. Partial 16S rRNA gene sequences (1460 bp) were identical, except for two nucleotide changes amongst the six strains. Alignment of the causal strains within type‐culture databases revealed similarities of 99·7% with Pseudomonas parafulva AJ 2129^T, 99·2% with P. fulva IAM 1592^T, 98·9% with P. plecoglossicidia FPC 951^T, and 98·1% with P. fuscovaginae MAFF 301177^T. On the basis of data from this polyphasic study, it is proposed that the unknown strains isolated from rice represent a novel species of the genus Pseudomonas.     

浙江省水稻新品种(系)对稻瘟病的抗性研究

用4个稻瘟病菌小种对浙江省280份水稻新品种(系)进行稻瘟病抗性分析,结果表明:浙江省水稻新品种(系)对稻瘟病菌具有较好的抗性,籼稻和粳稻对参试菌株的全抗率分别为21.62%和39.05%,说明粳稻具有较籼稻更广的抗谱;浙江省新品种(系)对ZB13小种具有较高的感病率,籼稻和粳稻的感病率分别为69.37%和52.07%。     

黑龙江省水稻新品种（系）抗稻瘟病性鉴定及利用

1998-2009年采用稻瘟病人工接种和自然感病鉴定2种方法,共鉴定黑龙江省水稻新品种（系）485份（次）,鉴定出抗稻瘟病新品种（系）109份,12年间先后推广了‘空育131’、‘绥粳3号’、‘龙粳12’、‘龙粳14’、‘垦稻12’等49个新品种,推广了3个超级稻品种‘龙粳14’、‘龙粳18’、‘龙粳21’,18个新品种年种植面积超过6.67万hm2,获得了较高的经济效益和社会效益。     

EK-2612, a new cyclohexane-1,3-dione possessing selectivity between rice (Oryza sativa) and barnyardgrass (Echinochloa crus-galli)

A newly synthesized experimental compound, EK-2612 is one of the class of cyclohexane-1,3-diones which are commonly known to be grasskillers. A greenhouse study was conducted to evaluate the herbicidal performances of EK-2612 on several grass species in comparison with tralkoxydim, a commercialized cyclohexanedione derivative. Like tralkoxydim, the compound EK-2612 showed excellent control efficacy on most grass weeds tested through foliar application rates between 250 and 63 g AI ha(-1). Unlike tralkoxydim, however, EK-2612 showed a good rice safety, and there was no rice damage observed at the level below 125 g AI ha(-1), while rice injury developed at the same application rates of tralkoxydim. With this rice safety, EK-2612 controlled barnyardgrass effectively up to the two-leaf stage under both submerged and dried paddy conditions. An in vitro ACCase assay indicated that EK-2612 is a strong ACCase inhibitor; however, the dose-response was not substantially different in rice and barnryardgrass, showing I50 values of 0.1 and 0.12 microM, respectively. These results suggest that the compound EK-2612 is targeting plant ACCase, but the whole-plant rice safety is not attributable to a different inhibition of the target site in rice from that in barnyardgrass.     

Phytophthora oleae,a new root pathogen of wild olives

Wild olive (Olea europaea subsp. europaea var. sylvestris) is an important component of Mediterranean forests and a key genetic source for olive improvement programmes. Since 2009, a severe decline caused by Phytophthora cryptogea and P. megasperma has been detected in a protected wild olive forest of high ecological value (Dehesa de Abajo, Seville, Spain). In this natural forest, sampling of roots and soil was carried out on 25 wild olives with symptoms in 2014 and 2015. Apart from the already known P. cryptogea A1 and P. megasperma, a third Phytophthora species was consistently isolated from wild olive rootlets with symptoms. These isolates conformed morphologically with the newly described species P. oleae and were confirmed by analysis of their ITS regions and cox1 sequences. Temperature–growth relationships showed a maximum growth at 19.9 °C on carrot agar medium, making it the lowest temperature Phytophthora species infecting wild olive roots. Pathogenicity was confirmed on 1-year-old healthy wild olive seedlings and was similar to the previously known pathogenic phytophthoras. As temperature requirements are quite different, the three Phytophthora species may be active against wild olive roots in different seasons. However, the prevalence of P. oleae infecting wild olives in recent years could be due to its introduction as a new invasive pathogen. The probable invasive nature of P. oleae, together with increasing rain episodes concentrated in short periods frequent in southern Spain, would allow the outbreak of infections in wild olive forests, and also put cultivated olive orchards at risk.     

Characterization of a new potyvirus isolated from peanut (Arachis hypogaea)

During a survey of viruses of peanuts in South Africa a mechanically transmissible virus was isolated from a plant exhibiting chlorotic ringspots and blotches on the leaves. Typical potyvirus-like flexuous particles were detected by electron microscope examination. Pinwheel-shaped and laminated inclusions in ultrathin sections, reaction with a monoclonal antibody directed to a potyvirus common epitope, a single 33 kDa coat protein and aphid transmission using Myzus persicae all confirmed that the virus was a subdivision II member of the Potyviridae. Host range studies suggested that the virus was none of the previously reported potyviruses of peanuts or of subdivision II potyviruses. The serological relationships of the virus were studied using a range of 17 antisera to potyviruses in ELISA and immunosorbent electron microscopy (ISEM). The isolate reacted weakly with antisera to plum pox virus and bean yellow mosaic virus in ISEM only. Nucleotide sequence of a 624 bp DNA product was obtained following immuno-capture with a potyvirus common epitope antiserum, cDNA synthesis and PCR amplification with potyvirus specific primers which amplify the 3' untranslated region and a part of the coat protein gene. The sequence was only distantly related to a number of potyviruses, whether amino acid or nucleotide sequences were used for comparisons. It is proposed that the virus be named peanut chlorotic blotch virus and be accepted as a new member of the genus Potyvirus in the family Potyviridae.     

Simple and rapid detection of rice false smut pathogen Ustilaginoidea virens in rice seeds

A method for simple and rapid detection of Ustilaginoidea virens in rice seeds was developed based on specific polymerase chain reaction (PCR). To design the specific primers for detection of U. virens, the comparison was made on 5.8S rDNA intra- and inter-specific variations in nucleotide sequences of U. virens and other pathogens: Fusarium verticillioides, Sclerotinia sclerotiorum, Tilletia barclayana, Fusarium graminearum and Magnaporthe oryzae. A 346-bp fragment could be amplified by using the specific primers uvr-F and uvr-R in U. virens, but not in other test fungi, indicating that the designed primers were specific for the detection of U. virens. U. virens was detected in nine of the 24 tested rice samples, indicating that 37.5% of the rice samples were contaminated with the false smut pathogen. The development of the simple and rapid detection technique using specific primers will be applicable for direct identification of U. virens in rice seeds to screen large numbers of samples.     

玉米衰老相关蛋白基因ZmSAP的克隆与表达分析

利用立枯丝核菌(Rhizoctonia solani)诱导胁迫下玉米高耐纹枯病自交系幼苗叶片所构建的cDNA文库中获得的EST序列,通过电子克隆和RT-PCR方法,结合cDNA末端快速扩增技术,从玉米叶片中分离克隆到衰老相关蛋白基因的全长cDNA序列,命名为ZmSAP(GenBank登录号:GU253311)。序列分析表明,ZmSAP全长1156bp,包含一个完整的603bp的开放阅读框(ORF),具有连续的Poly A尾和典型的加尾信号AATTAA。ProtParam程序预测表明该基因编码200个氨基酸,相对分子量为21.92kD,等电点为10.38。该氨基酸序列与豌豆、挪威云杉、寄生藻等有不同程度的同源性且在不同物种间具有一个保守结构域,染色体定位发现该基因位于玉米第1条染色体上。荧光定量PCR分析表明,在高耐纹枯病自交系R15和高感纹枯病材料478中ZmSAP基因在病菌胁迫初期呈诱导表达,可能参与了病原菌诱导的细胞程序性死亡过程,说明该基因与纹枯病菌胁迫响应机制密切相关。     

水稻稻粒黑粉病病原菌鉴定及致病性测定

为明确我国南方杂交水稻制繁种区稻粒黑粉病病原菌及其致病力情况,于2015年8月从安徽、湖南、江苏、贵州、四川遂宁、四川新津、四川温江7个主要杂交稻制繁种区采集稻粒黑粉病样本,采用冬孢子悬浮液分离法进行病原菌的分离。基于形态学、菌丝细胞核荧光染色和r DNA-ITS序列分析对病原菌进行鉴定,并应用柯赫氏法则对不同地区病样中分离的纯培养物致病性进行测定。结果表明分离到的35株病原菌菌株并无明显形态学差异,冬孢子呈球形或椭圆形,次生小孢子单细胞核,呈线状或弯曲状两种类型,与已报道的黑粉菌属Tilletia horrida形态特征相似;r DNA-ITS序列分析表明,选取的7株病原菌菌株与T.horrida DQ827699同源性达100%,说明所分离菌株为稻粒黑粉病菌;致病性试验表明,7株病原菌菌株侵染水稻稻粒均能产生稻粒黑粉病的典型症状,其中分离自江苏的菌株JS造成的单穗病粒数和产量损失最大,致病力较强,且萌芽率最高,分离自不同地区的菌株致病力具有明显差异。该7株病原菌最适生长温度为28℃,光照对其生长没有明显影响。