辣椒疫霉(Phytophthora capsici Leonian)不同发育阶段对嘧菌酯的敏感性研究

细胞色素bc1位点抑制剂包含作用于线粒体内膜外壁CoQ氧化位点抑制剂（QoIs）和作用于线粒体内膜内壁CoQ的还原位点抑制剂（QiIs）。从未使用过QoIs和QiIs的江苏省南京市和安徽省和县随机采集分离获得48个辣椒疫霉单游动孢子囊菌株，并测定了它们不同发育阶段对嘧菌酯的敏感性。结果表明，辣椒疫霉菌丝生长对嘧菌酯的敏感性最低，EC50值范围在1．225～86．100μg/mL，平均值为（21．041±14．397）μg/mL，其活性低于甲霜灵47．7倍；游动孢子囊形成和萌发对嘧菌酯的敏感性较高，EC50值范围分别在0．006～0．996μg/mL和0．053～0．334μg/mL，平均值分别为（0．161±0．126）μg/mL和（0．155±0．023）μg/mL，其活性高于甲霜灵516．8倍；游动孢子萌发对嘧菌酯的敏感性最高，EC。值范围在（0．018-0．111）μg/mL，平均值为（0．057±0．011）μg/mL，活性是甲霜灵的142．4倍。离体生物测定结果表明，水杨肟酸对嘧菌酯抑制菌丝生长的增效作用最强，对游动孢子囊形成的增效作用次之，而对游动孢子囊和游动孢子萌发过程的增效作用则不明显。

Induction and Characterization of Laboratory Mutants of Phytophthora capsici Resistant to Dimethomorph and Flumorph

Laboratory studies were conducted to evaluate the risk of Phytophthora capsici developing resistance to two morphlines, dimethomorph and flumorph. Metalaxyl, the well-known high risk of resistance fungicides, was used as reference fungicide. Resistant mutants for the three fungicides were isolated by treating mycelium with ultraviolet radiation. Metalaxyl-resistant mutants were obtained with high frequency and exhibited high level of resistance with factors more than 100 folds, while mutation frequency for dimethomorph-resistance was relatively low and the resistance factors ranged from 3.0 to 13.9 folds. Most dimethomorph-resistant mutants decreased in hyphal growth rate and the spoulation ability, which have a large impact upon the epidemic development of dimethomorph-resistant populations. These results suggested that the risk of resistant pathogen population was much lower for dimethomorph than for metalaxyl. Both the frequency of developing resistance and level of resistance （resistance factors = 1.8-14.6） to dimethomorph were similar to those of its structure analogue flumorh. Moreover, the cross-resistance were found between them, which suggested the risks of developing resistance to dimethomorph and flumorph in the pathogen were very closely related. As P. capsici can potentially develop resistance to dimethomorph and flumorph, and oomycetes usually have the high risk to develop resistance to fungicides, appropriate management against resistance development should be taken.

Spread of <i>Phytophthora capsici</i>in Black Pepper (<i>Piper nigrum</i>) in Vietnam

Black pepper is the one of most important export products in Vietnam. As the largest exporter, Vietnam’s pepper commodities account for 58% of total worldwide exporters. However, Vietnam’s pepper production is dealing with disease problems, especially foot rot/quick death infected by Phytophthora capsici. The disease results in serious and rapid spread and infection in Vietnam, with yearly reduction of about 2% of total pepper area. Disease management is recently challenging scientists and producers. Investigating characteristics of Phytophthora capsici and causes, therefore, play a significant role in treatment. This paper has indicated three main causes, which contribute to serious infection and outbreak of Phytophthora capsici;they are biological characteristics, climatic condition and cultivation. To control this disease, early detection and prevention are the best ways to manage disease. Finding new varieties, which are Phytophthora capsici tolerance or resistance, is significant in black pepper production worldwide.

Active changes of lignification-related enzymes in pepper response to Glomus intraradices and/or Phytophthora capsici

The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal coloni-zation rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was com-pletely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45 000, 53 000 and 114 000) were present in Pc-treated roots, while two major bands (53 000 and 114 000) and one minor band (45 000) were present in spectra of Gi+Pc-treated roots, the 45 000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45 000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60 000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.

Inhibitory Effect of Extracts from Helianthus tuberosus Leaves against Phytophthora capsici and Pot Verification Test

By growth rate method, the inhibitory effects of five solvent extracts from Helianthus tuberosus leaves against Phytophthora capsici were studied in the test. The results showed that different solvent extracts all had inhibitory effect against P. capsici, while 12.5 mg/mL of extracts from H. tuberosus leaves with petroleum ether and ethyl acetate as solvents had the highest inhibitory effect against P. capsici, reaching 100%. In case of various solvent extracts with different concentration gradients, ethyl acetate extract had the most significant inhibitory effect; when the concentration was 5 mg/mL, the inhibitory effect of ethyl acetate extract had reached 100% ; when the concentration reduced to 2.5 mg/mL, the inhibitory effect was still （27.91 ±2. 076） %, significantly higher than that of other solvent extracts at the same concentration. The 50 times dilution of ethyl acetate extract from H. tuberosus leaves was selected for pot test against pepper blight. , and the results showed that its control effect against pepper blight reached 100.00%, superior than that of chemical agent 25% metalaxyl WP 400 times dilution.

Genetic Diversity and Phylogenetic Relationships among Phytophthora capsici Isolates from Guizhou Province by RAPD

Ten random primers with clear amplification profile, significant and stable main band were screened from RAPD （Random Amplified Polymorphie DNAs） primers to analyze the genetic diversity among eight Phytophthora capsici isolates from Huaxi District, Wudang District and Kaiyang County of Guiyang City, and Zunyi County, Suiyang County and Luodian County of Zunyi City in Guizhou Province. A total of 70 DNA fingerprints were obtained, including 57 polymorphic bands, with a polymorphic percentage of 81.43%, suggesting abundant genetic diversity among experimental Phytophthora capsici isolates. According to the ampli- fied DNA fingerprint profiles, using genetic similarity coefficient 0.5 as the threshold, experimental Phytophthora capsici isolates were clustered into three genetic categories by UPGMA cluster analysis. The analysis result indicated that there was no direct correlation between the genetic similarity and cultivation areas of vari- ous Phytophthora caosici isolates.

南瓜疫病菌(Phytophthora capsici)的生物学特性初探

以南瓜疫病菌株ＰＣ－１为研究对象，分析其生物学特性。结果表明，不同培养基对菌丝生长影响不同，菌丝在葫萝卜琼脂（ＣＡ）培养基上生长要好于燕麦片琼脂（ＯＭＡ）培养基。孢子囊的产生需要有水分，同时也受温度、光照、培养基及菌龄的影响。在一定温度范围内，温度升高，孢子囊产生的速度加快且数量多，光照有利于孢子囊的产生，１０℃以下无论有无光照均不产生孢子囊。

辣椒疫霉(Phytophthora capsici)果胶甲基酯酶Pcpme3基因真核表达及功能研究

【目的】对辣椒疫霉果胶甲基酯酶Pcpme3基因进行真核表达,制备其特异性抗血清,为用Westernblot检测该基因在辣椒疫霉致病过程中的功能奠定基础。【方法】利用TR-PCR分离鉴定Pcpme3的成熟肽片段,克隆于pPIC9K载体。将获得的表达载体转化毕赤酵母GS115,甲醇诱导表达,对表达产物进行SDS-PAGE分析和免疫家兔制备特异性抗体。【结果】酵母转化子经MD、MM平板筛选和G418抗生素筛选与PCR鉴定,获得Mut+/His+表型高拷贝重组转化子,经1%甲醇诱导后,SDS-PAGE检测发酵上清液,检测出43kD的特异蛋白。重组蛋白免疫家兔制备特异抗血清,Westernblot检测该基因在游动孢子侵染辣椒叶片中的表达,随着病情加重,该基因的表达逐渐增强,从而证明该基因参与了病菌的侵染致病过程。【结论】利用真核表达获得的蛋白制成特异性抗体可以有效地检测Pcpme3在辣椒疫霉侵染寄主过程中的功能特性。

胡椒瘟病菌(Phytophthora capsici)孢子囊诱导及发育过程观察

孢子囊的产生和发育过程是研究胡椒瘟病菌(Phytophthora capsici)及其病害防控的重要基础。本研究分析不同诱导条件下胡椒瘟病菌孢子囊的产生情况,并通过光学显微镜和共聚焦显微镜观察孢子囊及其内部游动孢子的发育过程。结果发现:(1)3种诱导方法均能诱导出大量孢子囊,其中在V8-A平板上光照和抹伤双重诱导法获得的孢子囊数量最多,其次是在V8-A平板上光照诱导法,获得孢子囊数量最少的是V8液体光照诱导法,但仅差异最大的2个处理间达到显著水平;(2)显微镜观察发现,孢子囊由气生菌丝顶端逐步膨大形成,初始为近球形逐渐发育成倒洋梨形,孢子囊成熟后从顶端排出大量游动孢子,偶尔可见孢子囊顶端直接发育出芽管;(3)共聚焦显微镜观察发现,首先孢子囊内部原生质体被膜结构隔裂成大约数十个独立小格,然后在每个格子中积累数倍于细胞核的DNA,最后每份细胞核DNA发育成一个游动孢子。本研究从微观角度揭示P.capsici孢子囊发育外观及内部的形态特征,为胡椒瘟病菌后续致病机制研究和胡椒瘟病田间防控提供技术基础。

辣椒疫霉(Phytophthora capsici)多聚半乳糖醛酸酶PCIPG2N-糖基化突变体的构建与表达分析

为研究辣椒疫霉(Phytophthora capsici)多聚半乳糖醛酸酶PCIPG2 N-糖基化对其酶活性的影响,从基因组文库中分离克隆到Pcipg2基因,利用定点突变技术突变3个潜在的糖基化位点(N34,N76,N137);构建并表达N-糖基化突变蛋白,并对突变蛋白进行温度和缓冲液体系处理检测其活性。结果发现Pcipg2基因在辣椒疫霉侵染寄主的前期表达并起重要作用。PCIPG2蛋白在30°C,pH5.0(P<0.05)的缓冲液环境下活性最高。单个的Pcipg2糖基化位点N34、N76、N137对PCIPG2的致病性起正调控作用,而3个糖基化位点相互协调的功能抑制PCIPG2的活性,在PCIPG2表达致病过程中起负调控。N-糖基化在PCIPG2酶活性上起直接作用,使得PCIPG2酶在较低水平上保持较高的稳定性。

南瓜疫病菌(Phytophthora capsici)生理分化研究进展

文章综述了南瓜疫病菌生理分化的研究进展,包括传统的鉴别寄主技术到分子标记技术的应用,展望今后南瓜疫病菌的主要研究方向与前景,为今后南瓜抗疫病育种研究奠定理论和应用基础。

辣椒中L型凝集素类受体激酶CaLecRKs鉴定及其对辣椒疫霉的响应

L型凝集素类受体激酶(LecRKs)广泛参与植物的先天免疫过程。目前未见在辣椒Capsicum annuum中全基因组鉴定LecRKs的报道。本研究对辣椒中的CaLecRK进行了全基因组鉴定,并在接种辣椒疫霉Phytophthora capsici条件下通过基因表达分析探究其对辣椒疫霉的响应情况,旨在挖掘参与辣椒抗疫病防御反应的CaLecRK基因。研究结果表明,辣椒基因组中共鉴定出24个CaLecRK,以其构建系统发育树发现,可将24个CaLecRK分为7个分支。基因表达分析结果显示,有4个CaLecRK基因(CaLecRK 2.2、CaLecRK 3.2、CaLecRK 8.1和CaLecRK 10.1)受辣椒疫霉诱导,和接菌后0 h相比,接菌处理后12 h或36 h基因表达差异显著,推测其在辣椒抗疫病防御反应中发挥了重要作用。

A novel TIR-NBS-LRR gene regulates immune response to Phytophthora root rot in soybean

Phytophthora root rot(PRR),caused by Phytophthora sojae,is a devastating disease of soybean.The NBSLRR gene family is a class of plant genes involved in disease resistance.miRNA mediates plant response to biotic stresses by regulating the expression of target genes at the transcriptional or post-translational level.Glyma.16G135500,encoding an NBS-LRR-type protein,is a target of gma-miR1510 that responds to pathogen infections.We cloned and overexpressed Glyma.16G135500(naming it GmTNL16)and knocked down mi R1510 using short tandem target mimic technology to identify the roles of the GmTNL16/gma-mi R1510 pair in the interaction of soybean and the oomycete.By overexpressing GmTNL16 in transgenic hairy roots of soybean,we showed that biomass of P.sojae was lower in overexpressing hairy roots than in control roots.Thus,miR1510 expression was reduced upon P.sojae infection,reflecting the induced expression of GmTNL16 conferring resistance to P.sojae in soybean.Differentially expressed genes were enriched in plant-pathogen interaction,plant hormone signal transduction,and secondary metabolism by RNA sequencing analyze.In particular,jasmonate and salicylic acid pathway-associated genes,including JAZ,COI1,TGA,and PR,responded to P.sojae infection.All of these results indicate that the GmTNL16/gma-miR1510 pair participates in soybean defense response via the JA and SA pathways.

对上海郊区甜椒上6株Phytophthora capsici形态与生理生化性状的研究

作者观察了来自于上海郊区甜椒上的6株P.capsici的菌落形态、孢子囊特征。对6株病菌孢子囊的大小、最高生长温度、对孔雀石绿的敏感性、在含有酪氨酸的培养基上能否产生色素、利用硝态氮和淀粉的能力等生理生化性状进行了测定和研究。

Molecular Detection of Phytophthora colocasiae of Taro Leaf Blight Based on PCR

The present PCR assay was conducted to develop rapid and sensitive detection of Phytophthora colocasiae,in order to provide a robust and reliable tool for healthy seedling production of taro and limiting the transmission and spread of the causal organism of taro leaf blight in taro planting regions.The samples were used to extract total DNA and to be detected by PCR with P.colocasiae specific primer pairs PCSP-RL F/PCSP-RL R and PCSP-T F/PCSP-T R,respectively.Distinct fragments of about 200 bp and 240 bp were amplified by PCR using primers PCSP-RL F/PCSP-RL R and PCSP-T F/PCSP-T R,respectively.The analysis of the nucleotide sequence of the PCR products were found to be 99% identical to sequence of RAS-related protein (Ypt1) and phospho-ribosylanthranilate isomerase (TRP1) in P.colocasiae,respectively.It is concluded that rapid and sensitive developed PCR assay for detection of P.colocasiae could be used in routine diagnosis and aid in management practices to mitigate taro leaf blight.

辣椒分子连锁遗传图谱的构建及抗疫病QTL定位

以抗疫病材料perennial和感疫病材料83-60杂交得到的F2群体144个单株为试材,利用AFLP、RAPD、SSR等分子标记,采用JoinMap3.0构建辣椒分子连锁遗传图谱,该图谱包括12个连锁群,70个标记位点,其中AFLP标记54个,RAPD标记15个,SSR标记1个,覆盖长度为429.02cM,平均图距为6.13cM,连锁群长度在4.30～85.85cM之间。对F2群体进行辣椒疫病的室内人工接种,利用Windows QTL Cartographer2.0软件进行QTL分析,在第4连锁群上检测到两个QTL,解释表型差异的贡献率分别为9.5%和16.4%。

7株放线菌在辣椒根部定殖及对辣椒叶片PAL与PPO活性的影响

采用盆栽接种试验、平皿涂抹法测数及常规酶活测定法研究了7株拮抗性放线菌在辣椒根部的定殖能力及接种24 d对辣椒叶片苯丙氨酸解氨酶(PAL)与多酚氧化酶活性(PPO)的诱导效应。结果表明:(1)供试7株放线菌单独接种均不能在辣椒根内定殖,但与辣椒疫霉P3混合接种时有5株可定殖;供试放线菌在辣椒根部的定殖能力与其体外平皿试验中产生的的拮抗圈大小基本无关;可定殖放线菌的定殖密度随时间延长而降低,至40 d时均无活菌检出。(2)在放线菌单接处理中,5株菌接种后可诱导辣椒叶片PAL活性提高,全部供试菌均能诱导PPO活性提高,其中可使两种酶同步提高的有5株菌;在放线菌+P3混接处理中,有6株接种后可诱导PAL活性提高,5株菌能诱导PPO活性提高,其中可使两种酶同步提高的有4株菌;在接入放线菌时同时混接辣椒疫霉,能增强2株供试放线菌对辣椒叶片PAL活性及6株供试放线菌对辣椒叶片PPO活性的诱导作用;供试放线菌的定殖能力与辣椒叶片PAL及PPO活性变化无明显规律性关系。

球毛壳菌小麦秸秆发酵产物抑制辣椒疫霉的稳定性及机制

【目的】由辣椒疫霉(Phytophthora capsici)侵染引起的疫病给辣椒等作物产业带来了巨大的经济损失。论文旨在明确球毛壳菌(Chaetomium globosum)代谢产物抑制辣椒疫霉的稳定性和抑菌机制,为研究与开发辣椒疫霉的微生物源抑菌剂提供参考。【方法】将球毛壳菌发酵粗提物分别在不同温度(40—121℃)、pH(1—13)、光照时间(0—12 d)和储存时间(0—60 d)条件下进行处理,采用菌丝生长抑制法测定经不同处理后粗提物对辣椒疫霉的抑制率,以探究粗提物抑制辣椒疫霉的热、酸碱、光和时间稳定性。利用光学显微镜观察粗提物对辣椒疫霉菌丝形态的影响,通过多种生理生化试验探究粗提物作用于辣椒疫霉12—72 h后,对辣椒疫霉细胞壁、细胞膜、活性氧代谢、蛋白质含量、还原糖含量和致病力的影响。【结果】在所设置的处理范围内,球毛壳菌发酵粗提物(1 mg·mL^(-1))经不同光照时间和储存时间处理后对辣椒疫霉的抑制率没有显著降低,抑制率维持在93%左右;粗提物在40—70℃的热处理范围内对辣椒疫霉的抑制率没有显著降低,70℃以上的热处理会显著降低粗提物对辣椒疫霉的抑制率,但抑制率不低于70%;粗提物在pH 1—5和9—13的酸碱处理范围内会显著降低对辣椒疫霉的抑制率,但抑制率也不低于70%。粗提物处理影响辣椒疫霉菌丝形态,使菌丝发生严重的扭曲皱缩现象,并影响活性氧代谢,使菌丝中的活性氧大量积累。辣椒疫霉经粗提物处理12—72 h后,其培养液中的碱性磷酸酶活性、β-葡萄糖苷酶活性、核酸和蛋白质含量均显著升高,且其菌丝中的丙二醛和过氧化氢含量显著增加。辣椒疫霉菌丝中的过氧化氢酶活性、可溶性蛋白和还原糖含量则在粗提物处理12—72 h内显著降低,而超氧化物歧化酶、过氧化物酶、多聚半乳糖醛酸酶和β-葡萄糖苷酶活性则仅在一定时间显著降低。【结论】在本试验所设置的处理范围内,球毛壳菌的小麦秸秆发酵粗提物抑制辣椒疫霉的效果不受光照及储存时间的影响,但超过70℃的热处理和pH 1—5和9—13的酸碱处理会显著降低粗提物对辣椒疫霉的抑制效果。此外,该粗提物通过改变菌丝形态,破坏细胞壁和细胞膜,引发细胞内物质泄漏,降低菌丝中蛋白质和还原糖含量,抑制抗氧化酶活性,干扰活性氧代谢使活性氧大量积累从而抑制辣椒疫霉。

内生放线菌gCLA4对辣椒疫病的防治研究

通过皿内对峙试验从242株供试内生放线菌中筛选到辣椒疫霉病菌拮抗菌62株,抑菌带宽度≥5mm的24株,占试验总菌株的10%,分别分离自黄瓜、牛蒡等9种植物的根部、茎部和叶片。用管碟法进行抑菌活性复筛,发现其中6株的无菌滤液对辣椒疫霉病菌和大豆疫霉病菌的抑菌圈直径≥20 mm,其中gCLA4的抑菌活性最强。进一步的研究结果表明,该菌株的无菌滤液能够强烈抑制病菌孢子囊的形成及游动孢子的释放,原液对孢子囊形成及游动孢子释放的抑制率分别高达100%和96.2%,稀释50倍后的抑制率仍分别达95.3%和85.6%,并且对其菌丝生长也有明显的抑制作用。温室盆栽试验结果表明,菌株gCLA4对苗期的辣椒疫病有较好防效。接种疫霉菌前48 h2、4 h和0 h以20 mL/盆(2株/盆)浇灌gCLA4菌株无菌滤液于辣椒苗基部土壤,接种后7 d调查病株率,其防效分别达100%、92.3%和80.8%,而接种14 d后分别为77.8%、55.6%和36.1%,说明该菌株无菌滤液具有开发成生防制剂的潜力。

辣椒疫霉菌ITS及β-tubulin的扩增和序列分析

为了探索湖南省不同地区辣椒疫霉的系统发育关系与遗传多样性,从湖南芷江、宁乡等地采集辣椒疫病病株,经过分离和离体叶片接种,得到2株对辣椒叶片有强致病性的辣椒疫霉菌(Phytophthora capsici)菌株。利用PCR技术对所分离的2个菌株进行ITS以及β-tubulin扩增并测序,将测序结果与国内外报道的相关序列构建系统发育树,结果表明,离体叶片接种法能够对辣椒疫霉菌有一个初步的鉴定,PCR扩增的ITS序列长度为770bp左右,β-tubulin序列长度为780bp左右,ITS序列以及β-tubulin序列构建的系统发育树表明湖南省内辣椒疫霉的亲缘性比较近,与国外的亲缘性比较远,说明了不同地区疫霉菌株的亲缘性与地理来源有一定的相关性。