Two butylated aminooligosaccharides isolated from the culture filtrate of Streptomyces luteogriseus

Two novel aminooligosaccharides, butytatins M03 and M13 were isolated and purified from the culture filtrate of Streptomyces luteogriseus. Analysis by liquid chromatography coupled to electrospray ionization mass spectrometry indicated their resemblance to isovalertatin, with a four-carbon acyl group. Their structures were established by NMR as aminooligosaccharide derivatives possessing a butylated side chain.     

Segregation following interspecific transfer of isolated nuclei between Phytophthora parasitica and P. capsici

Nuclei isolated from metalaxyl-resistant (MR) protoplasts of Phytophthora parasitica were transferred into chloroneb-resistant (CnR) protoplasts of Phytophthora capsici and vice versa, with an average success rate of 2.6 x 10(-4) (protoplasts with donor nuclei/regenerated protoplasts), using a selective medium containing only the fungicide tolerated by the nuclear donor. No colonies appeared when self-fusion products of donor nuclei or recipient protoplasts were exposed to the selective medium. Colonies produced by the nuclear transfer formed sectors commonly, and differed from the parental types in appearance. All the zoospores produced by the nuclear hybrids were of normal size, and one-fifth of them contained both MR and CnR genes. Since zoospores are mostly uninucleate, these results indicated the occurrence of chromosome re-assortment or mitotic crossing-over following the production of transitory tetraploids, followed by diploidization during zoosporogenesis, thus suggesting the completion of events leading to a parasexual cycle. Hyphal fragment cultures from a nuclear hybrid tested showed considerable variation in growth rate, mycelial morphology, and level of resistance to metalaxyl, indicating uneven distribution and continuous segregation of different types of nuclei in mycelia during vegetative growth.     

Genetic transformation of the plant pathogens Phytophthora capsici and Phytophthora parasitica.

Phytophthora capsici and P.parasitica were transformed to hygromycin B resistance using plasmids pCM54 and pHL1, which contain the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Ustilago maydis heat shock hsp70 gene. Enzymes Driselase and Novozyme 234 were used to generate protoplasts which were then transformed following exposure to plasmid DNA and polyethylene glycol 6000. Transformation frequencies of over 500 transformants per micrograms of DNA per 1 x 10(6) protoplasts were obtained. Plasmid pCM54 appears to be transmitted in Phytophthora spp. as an extra-chromosomal element through replication, as shown by Southern blot hybridization and by the loss of plasmid methylation. In addition, transformed strains retained their capacity of infecting Serrano pepper seedlings and Mc. Intosh apple fruits, the host plants for P.capsici and P.parasitica, respectively.     

Characterisation of Phytophthora capsici isolates from black pepper in Vietnam

Phytophthora foot rot of black pepper caused by Phytophthora capsici is a major disease of black pepper (Piper nigrum) throughout Vietnam. To understand the population structure of P. capsici, a large collection of P. capsici isolates from black pepper was studied on the basis of mating type, random amplified microsatellites (RAMS) and repetitive extragenic palindromic (REP) fingerprinting. Two mating types A1 and A2 were detected in four provinces in two climatic regions, with A1:A2 ratios ranging from 1:3 to 1:5. In several instances A1 and A2 mating types were found to co-exist in the same farm or black pepper pole, suggesting the potential for sexual reproduction of P. capsici in the field in Vietnam although its contribution to disease epidemics is uncertain. RAMS and REP DNA fingerprinting analysis of 118 isolates of P. capsici from black pepper showed that the population was genetically more diverse where two mating types were found, although the overall genetic diversity was low with most of the isolates belonging to one clonal group. The implication of these findings is discussed. The low diversity among isolates suggests that the P. capsici population may have originated from a single source. There was no genetic differentiation of isolates from different climatic regions. In addition to the large clonal group, several isolates with unique RAMS/REP phenotypes were also detected. Most of these unique phenotypes belonged to the minority A1 mating type. This may have significant implications for a gradual increase in overall genetic diversity.     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )     

辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶

CRN(crinkling and necrosis-inducing protein)为疫霉菌在与寄主互作过程中分泌的一类特有胞质效应因子,干扰寄主细胞正常的生理代谢和功能。采用PCR法从辣椒疫霉LT1534菌株c DNA中克隆PcCRN20-C基因。该基因序列长783bp,编码261个氨基酸。构建重组表达载体,并转化大肠杆菌BL21(DE3)。在优化条件下诱导表达重组蛋白,利用Ni-NTA金属螯合层析、离子交换层析、分子筛层析和胰蛋白酶酶解技术获得高纯目的蛋白,SDS-PAGE分析表明,蛋白质分子量约为25kDa。采用座滴气相扩散法进行晶体制备和筛选,成功获得了蛋白质晶体,并通过X-射线衍射仪收集了晶体衍射花样。结合蛋白质晶体学方法,获得了有衍射的辣椒疫霉PcCRN20-C蛋白晶体,为进一步研究CRN蛋白的结构与病原菌致病机制提供参考资料。     

贵州地区木霉菌分离鉴定及对辣椒疫霉的拮抗作用

【背景】辣椒疫霉是一种毁灭性的土传病害,当前主要使用化学合成杀菌剂防治,但容易导致环境污染和食品安全等问题。【目的】筛选可拮抗辣椒疫霉的候选菌株,探究分离菌株拮抗辣椒疫霉的生理生化作用机制。【方法】综合应用形态学、核糖体RNA (rRNA)基因非转录区ITS序列相似性方法鉴定分离菌株,通过对峙实验筛选抑菌效果较高的拮抗菌株,基于比色法测定分离菌株发酵液粗提物对辣椒疫霉菌丝脂质过氧化、纤维素酶、β-葡萄糖苷酶(β-GC)和多聚半乳糖醛酸酶(PG)活性的影响。【结果】从腐木和土壤样品中分离得到11株木霉,分属于绿色木霉(Trichodermavirens)、哈茨木霉(Trichoderma harzianum)、钩状木霉(Trichoderma hamatum)和棘孢木霉(Trichoderma asperellum) 4个种。11株木霉对辣椒疫霉均有一定的抑制作用,抑制率达到90%以上的菌株包括:绿色木霉Tv-1(92.68%)、Tv-2 (95.12%),哈茨木霉Thz-2 (92.68%),钩状木霉Tha-1 (90.24%)。以4株高效木霉的发酵液粗提物处理辣椒疫霉菌丝5 d后,因脂质过氧化产生的丙二醛含量显著增加,分别达到1.20、1.48、2.69和3.16 nmol/g,显著高于对照处理的0.77 nmol/g;与对照组相比,β-GC、PG酶活性显著下降,分别降低了12.28%-64.91%、7.2%-15.5%;同时纤维素酶活性呈上升趋势,最显著组为2.647 U/mL,相对于对照组增加了0.831U/mL。【结论】分离得到4株明显抑制辣椒疫霉菌生长的高效木霉菌,主要通过破坏细胞壁结构、降低致病因子酶活力和增强脂质过氧化等方式起拮抗作用,可为辣椒疫病的生物防治提供理论依据和技术支持。     

Structural investigations and biological activity of inositol sphingophospholipids from Phytophthora capsici

Inositol sphingophospholipids that protect pepper (Capsicum annuum c.v. Yolo Wonder) against pathogen have been isolated by chromatographic methods from the mycelium of Phytophthora capsici. The structure of the major compound was determined by chemical methods and mass spectrometry. Phosphodiester bond cleavage of the phospholipid by mild alkaline hydrolysis liberated a ceramide which contained a C16-sphingosine. This long-chain base was identified by gas chromatography and mass spectrometry of its trimethylsilyl derivative. One of the amide-linked fatty acids was found to be 4-hydroxy-2 docosenoic acid. Fast-atom-bombardment mass spectrometry and fast-atom-bombardment collison-induced tandem mass spectrometry were used to characterize the ceramide as N(4-hydroxy-2-docosenoyl)C16-sphingosine. These sphingolipids have a protective effect on cotyledons of young peppers against necrotic lesions induced by the pathogen P. capsici.     

抗甲霜灵辣椒疫霉菌的环境适合度

摘要:【目的】研究抗甲霜灵辣椒疫霉菌的环境适合度,对于评估甲霜灵防治辣椒疫霉的抗药性风险具有重要意义。【方法】以室内药剂驯化的抗甲霜灵辣椒疫霉菌株Pc2-3为研究对象,分析比较其与原始敏感菌株Pc2的主要生物学特性、生长竞争力、致病力及土壤适合度等环境适合度指标。【结果】Pc2-3孢子囊产生量(3 d后)、孢子囊释放率(24 h后)和游动孢子萌发率(8 h后)分别是Pc2的0.44、0.09和0.54倍。Pc2-3可生长温度和pH范围及最适生长温度与Pc2基本一致,但菌丝生长速率低于Pc2。竞争力测定显示,在胡萝卜(CA)平板培养条件下,Pc2-3生长极显著弱于Pc2。盆栽致病试验显示,Pc2-3对辣椒植株的致病率为14.3%,明显低于Pc2(88.6%)。两者等量混合后接种,辣椒植株的发病率为75.7%,接近单独接种Pc2时的发病率,且所有病株分离出的辣椒疫霉菌均为甲霜灵敏感型。分别将Pc2-3和Pc2游动孢子加入自然土壤培养20 d后,实时定量PCR检测显示Pc2-3数量是Pc2的0.28倍,当土壤中含有300 mg/kg干土的甲霜灵,则前者为后者的0.42倍。此外,2个菌株最适存活土壤温度和湿度基本一致,当土壤温度和湿度利于辣椒疫霉存活时,Pc2-3土壤适合度显著低于Pc2,不利于辣椒疫霉存活时,Pc2-3土壤适合度略低于Pc2。【结论】抗甲霜灵菌株Pc2-3环境适合度弱于原始敏感菌株Pc2。     

土壤中辣椒疫霉分离方法的研究与量化测定

从杭州、西安、广州及武汉等辣椒病田分别采集土样 ,室内晾干研碎后 ,用选择性培养基 ,采用土壤稀释平板法和组织诱饵法分离辣椒疫霉 (PhytophthoracapsiciLeonian) ,并对土壤中辣椒疫霉的密度进行量化处理。结果表明 ,利用选择性燕麦培养基 ,采用土壤稀释平板法可分离获得大量的辣椒疫霉菌株 ,而且辣椒连作田的辣椒疫霉菌密度高于轮作田。组织诱饵法试验结果表明 ,辣椒叶片诱集效果最好 ,其次是辣椒果实。     

Functional analysis of Pcipg2 from the straminopilous plant pathogen Phytophthora capsici

Phytophthora capsici causes serious diseases in numerous crop plants. Polygalacturonases (PGs) are cell wall‐degrading enzymes that play an important role in pathogenesis in straminopilous pathogens. To understand PGs as they relate to the virulence of P. capsici, Pcipg2 was identified from a genomic library of a highly virulent P. capsici strain. Pcipg2 was strongly expressed during symptom development after the inoculation of pepper leaves with P. capsici. The wild protein (PCIPGII) was obtained from the expression of pcipg2 and found that increasing activity of PGs in PCIPGII‐treated pepper leaves was consistent with increasing symptom development. Asp residues in active sites within pcipg2 affected PCIPGII activity or its virulence on pepper leaves. Results show that pcipg2 is an important gene among pcipg genes, and illustrate the benefit of analyzing mechanisms of pathogenicity during the period of host/parasite interaction. genesis 47:535–544, 2009. © 2009 Wiley‐Liss, Inc.     

The oxidative processes induced in cell suspensions of Solanum species by culture filtrate of Phytophthora infestans

Solanum genotypes that differ in the level of polygenic resistance to the oomycete plant pathogen Phytophthora infestans were studied for their oxidative response to culture filtrate (CF) of the pathogen. Reactive oxygen species (ROS) production, peroxidase activity and lipid peroxidation have been studied in the CF-treated cell suspensions derived from leaves of the resistant S. nigrum (nonhost) and S. tuberosum cv. Bzura as well as from the susceptible S. tuberosum cv. Tarpan and clone H-8105. In both the resistant and susceptible cells the CF induced similar processes, but these varied with respect to the kinetics and intensity. In all cells probably the membrane-bound NADPH oxidase, was responsible for the ROS production. This process was more intensive and prolonged in the susceptible cells than in the resistant ones. The CF treatment slightly affected peroxidase activity in all cells studied. Lipid peroxidation that occurred as a consequence of the ROS accumulation was pronounced mainly in the susceptible cells. We suggest that lack of stringent control of the oxidative processes and sensitivity to the pathogen toxins may be decisive for limited polygenic resistance in potato.     

Crystal structure and expression patterns of prolyl 4-hydroxylases from Phytophthora capsici

Prolyl 4-hydroxylases (P4Hs) are members of the Fe²⁺ and 2-oxoglutarate- dependent oxygenases family, which play central roles in the collagen stabilization, hypoxia sensing, and translational regulation in eukaryotes. Thus far, nothing is known about the role of P4Hs in development and pathogenesis in oomycetes. Here we show that the Phytophthora capsici genome contains five putative prolyl 4-hydroxylases. In mycelia, all P4Hs were downregulated in response to hypoxia, but the expression of PcP4H1 was most affected. Strikingly, Pc4H1 was upregulated more than 110 fold at the onset of infection, and Pc4H5 was upregulated seven fold, while the expression of other P4H's were unchanged. Similar to well-characterized P4H proteins, the crystallographic structure of PcP4H1 contains a highly conserved double-stranded β-helix core fold and catalytic residues. However, the binding affinity of 2-oxoglutarate to PcP4H1 is very low. The extended C-terminal α-helix bundle and longer β2-β3 disordered substrate binding loop may help in confirming the peptide target of this enzyme.     

Genetic diversity of Phytophthora capsici isolates from pepper and pumpkin in Argentina

Phytophthora capsici is a soilborne oomycete plant pathogen that limits pepper production worldwide. The population structure varies significantly depending on the location (e.g. Peru vs. USA) and little is known about the diversity of P. capsici in Argentina. Our objective was to assess the diversity of P. capsici in Argentina at key pepper production areas. Forty isolates were recovered 2006-2009 from pepper and one isolate from pumpkin at 11 locations. Isolates were assessed for mating type, mefenoxam sensitivity and multilocus single nucleotide polymorphism (SNP) genotype profiles. Ten isolates with identical SNP profiles also were genotyped with amplified fragment length polymorphism (AFLP) markers. All 41 isolates had the A1 mating type and were sensitive to mefenoxam. Genotypic analysis using eight polymorphic SNP markers indicated 87% of the isolates had the same multilocus genotype, which is fixed for heterozygosity at seven of the eight SNP sites. AFLP analyses confirmed these findings, and overall it appears that clonal reproduction drives the population structure of P. capsici in Argentina. The implications for breeding resistant peppers and overall disease management are discussed.     

辣椒疫霉菌果胶裂解酶PL101基因的克隆及生物信息学分析

【背景】辣椒疫病是一种世界性土传病害,严重影响世界各国辣椒生产,并带来巨大经济损失。果胶裂解酶(pectate lyase,PL)作为一类重要的细胞壁降解酶类是该病的重要致病因子。【目的】对果胶裂解酶基因进行克隆,并对其生物信息学特性进行相关分析,进一步阐明该酶的作用机制。【方法】根据辣椒疫霉菌全基因组序列,以高致病菌株SD33为模板扩增PL101基因的全长cDNA序列,并对其理化性质、跨膜区、亲疏水性、结构域等生物学特性进行分析。【结果】除获得PL101相关生物学特性信息外,还对PL101进行三维结构建模,获得可信度较高的蛋白结构,并确定PL101可能的催化位点为Asp183、Arg212、Arg272三个氨基酸。【结论】对PL101基因的克隆及相关生物学特性的分析为进一步阐明PL功能特性提供参考。     

A cytokinin from the culture filtrate of Pseudomonas syringae pv. savastanoi

The structure of a new cytokinin, isolated from the culture filtrate of Pseudomonas syringae pv. savastanoi, is assigned on the basis of spectroscopic data including its tetracetyl derivative and comparison with related adenine derivatives. It was identified as 6-(4-hydroxy-1,3-dimethylbut-trans-2-enylamino-9-β-D-ribofuranosyl)purine.     

优质抗疫病甜椒种质资源的选育

本文利用国外引进的2个抗疫病的商业品种,通过系谱法选择,获得了6个园艺性状普遍优于茄门、对疫病达到抗病和高抗水平的株系,其中有4个株系兼中抗CMV和TMV,另有1个株系兼抗CMV.其中20079-0-3-1-27和20080-0-1-3-29综合表现尤其突出.开展优质抗疫病甜椒种质资源的选育,将为国内抗疫病的甜椒育种起到积极的作用.