影响水稻稻瘟病菌侵染过程的生态因子研究

通过田间和室内模拟等方法研究了水稻生长阶段,品种抗性,温度,降雨量和施N量等因子对稻瘟病菌侵染过程的影响。结果表明,水稻叶表单位面积病菌孢子附着量与水稻不同生长阶段呈负相关关系。稻瘟病潜育期与温度关系密切,在10℃-33℃范围内,以28℃条件下潜育期最短,小于℃,大于28℃的潜育期相应延长;病菌孢子侵染比值与水稻生长阶段呈负相关关系;稻叶表病菌孢子附着率在孢子与叶表接触后的5h内,与降水强度和降水持续时间密切相关,5h后影响变小;在一定条件下,稻瘟病扩展性病斑与非扩展性病的比值,扩展性病斑扩展的最大面积与水稻品种抗性和当时病斑所处叶位有关。     

不同栽培条件辣椒疫霉菌ITS序列同源性分析

由疫霉菌引起的辣椒疫病是辣椒生产上的主要病害。明确设施栽培辣椒土传病害和露地栽培条件下辣椒疫病疫霉病菌基因序列和同源性,为制定病害防治措施提供依据。通过病菌形态观察、ITS基因序列测定与同源性分析开展研究。由疫霉菌侵染的辣椒病害在设施和露地不同栽培形式下发生部位略有不同,但在病菌形态上表现一致,ITS基因序列同源性极高,达到了99%以上。设施土传病害和露地栽培条件下辣椒疫病菌属于同一种,均为辣椒疫霉菌(Phytophthora capsici Leonian)。     

暖季型草坪草AMF侵染与孢子密度季节动态研究

以广州地区常见暖季型草坪草(地毯草Axonopus compressus、狗牙根Cynodon dactylon、结缕草Zoysia japonica)为研究对象,通过测定植物根系AMF侵染率、根际土壤AMF孢子密度以及土壤理化性质,探讨AMF侵染率和孢子密度的季节动态变化特征,以及与土壤理化性质之间的关系。结果表明:(1)三种草坪草的根系AMF侵染以菌丝侵染为主,总体表现为夏季最高,秋季和冬季次之,春季最低,囊泡侵染率较低,很少丛枝侵染;(2)季节对根际土壤AMF孢子密度有一定的影响,草坪草根际土壤孢子密度最大值多出现在夏季或冬季,而春季和秋季则较低;(3)对于土壤因子,AMF菌丝侵染率与土壤有机质含量成极显著正相关,而囊泡侵染率与电导率成显著正相关,与全氮和全磷成极显著负相关;对于气候因子,AMF总侵染率和菌丝侵染率均与月均气温和月均降水成极显著正相关。     

田间侵染率在大豆花叶病毒(SMV)流行中的作用及其预测

SMV田间侵染率是由田间迁飞着落的得毒蚜量决定的,是田间病株率和迁飞着落蚜量二者一定数量关系的组合。SMV流行程度取决于病害发生期田间侵染率的大小。所以应从降低初侵染水平并以一定的田间侵染率为指标推迟介体蚜虫盛发期控制SMV流行。田间SMV日侵染率超过1％病害便进入流行盛期。通过单位绿色诱盘捕获翅蚜量和当时的田间病株率实现了SMV田间日侵染率的预测。     

施用氮肥对落叶松人工林一级根外生菌根侵染及形态的影响

以落叶松人工林为研究对象,通过施N肥试验,对不同季节、不同土壤深度根系进行取样,研究了1级根外生菌根真菌侵染率和形态,及其与不同季节、土壤深度和土壤N有效性的关系.结果表明:外生菌根真菌对落叶松人工林1级根的侵染率显著受不同季节和土壤深度土壤N有效性的影响.在不同季节和土层之间,施N肥导致菌根真菌侵染率下降.与未侵染菌根真菌相比,菌根真菌侵染导致1级根形态发生明显改变,平均直径增加18.7%,平均根长缩短23.7%,比根长降低16.3%.这种根系形态变化在不同季节、不同土壤深度处理中表现明显.菌根真菌侵染改变了1级根形态,影响根系的生理生态过程.     

致腐真菌侵染对梨果实愈伤组织防御机制的影响

以梨果实愈伤组织为材料,对轮纹病原菌(Botryosphaeria berengriana f.sp.piricola,BBP)和褐腐病原菌(Monilinia fructigena Honcy,MFH)侵染后的组织细胞膜透性、丙二醛（MDA）含量和防御酶系活性进行了测定,试图从细胞水平探讨防御酶系活性变化与组织感病进程的关系及其在组织生理防卫机制中的作用.结果表明:（1）接种褐腐病菌的组织出现褐变早,腐烂扩展速度和严重程度均明显高于接种轮纹病菌的组织;接菌后其相对电导率和MDA含量均呈上升趋势,且接种褐腐病菌的组织上升程度也远高于轮纹病菌侵染的组织.（2）愈伤组织接菌后120 h内,过氧化物酶（POD）、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性均表现出先升后降的趋势,但各种酶活性高峰峰值和出现的时间不同;轮纹病菌侵染的组织内各种防御酶活性均显著高于褐腐病菌侵染的组织,这与其受侵染组织腐烂慢、褐化程度低相对应.研究发现,防御酶系在组织对病原菌侵染的生理防卫反应中发挥了重要作用,其活性变化与果实防腐能力呈正相关.     

广州地区7种菊科入侵植物丛枝菌根侵染和根际土壤孢子密度的生境差异性分析

为了解广州地区7种菊科(Compositae)入侵植物与丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)的互作共生关系,调查了这7种菊科入侵植物在4种生境中的AMF侵染和根际土壤孢子密度,并分析土壤因子对AMF的影响。结果表明,7种入侵植物根内均受到AMF侵染,根际土壤中均检测到AMF孢子;AMF侵染在宿主间差异显著,生境间的差异不显著;孢子密度在生境和宿主间的差异均显著,人工绿地、农田果园的AMF孢子密度均显著高于森林周边和滨海地带。相关性分析表明,农田果园生境的根际土壤孢子密度与土壤有机质含量呈显著负相关关系;森林周边生境的AMF总侵染率与土壤全氮呈极显著正相关关系;人工绿地的AMF总侵染率与土壤速效氮含量呈显著负相关关系;滨海地带的AMF总侵染率与土壤有效磷含量呈显著负相关关系。这些对理解菊科植物入侵机理具有非常重要的作用。     

杜鹃根际土壤肥力与菌根真菌侵染关系

以2年生杜鹃(Rhododendron simsii)苗为试材,设置杜鹃花类菌根真菌混合接菌(J1)、杜鹃花类菌根真菌和马尾松外生菌混合接菌(J2)、不接菌(J3)等3种接菌组合以及不同氮磷钾施肥组合试验,结合Pearson相关性分析与逐步回归分析统计方法,分析杜鹃苗根际菌根真菌侵染与土壤肥力指标的相关性。结果表明,接菌处理土壤侵染率和养分含量显著高于不接菌处理,各接菌处理下,施用N、P、K养分为1水平(即尿素2.2 g·株^-1、钙镁磷1.9 g·株^-1、氯化钾1.4 g·株^-1)可以促进土壤养分含量的积累,J2处理下施P、K为1/4水平及N、P、K均为1/2水平也能促进土壤中全氮、有效氮、全钾的积累。相关性分析发现,J1处理侵染率与全氮含量显著正相关,J2处理侵染率与有效钾含量极显著正相关,J3处理与有机质和有效氮含量显著正相关。逐步回归分析表明,影响杜鹃苗菌根真菌侵染率的主要肥力因子是全氮、有效氮和有效钾。     

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。     

不同林龄杉木人工林菌根侵染特征研究

丛枝菌根真菌是一种通过土壤侵染植物根系,与寄主植物互利共生的重要有益真菌。探究不同林龄杉木林中菌根侵染状况与土壤养分的变化规律,有利于深入认识丛枝菌根真菌—杉木相互作用的养分调控因素,从而为改善杉木人工林土壤肥力、促进杉木林可持续经营提供依据。分别选取10a、25a、45a杉木纯林,分析了不同林分菌根侵染率与孢子密度及部分土壤养分因子(全磷、速效磷、全钾、速效钾)的关系。结果表明:(1)菌根侵染率与孢子密度均呈现出随林龄增大而增大的趋势,pH随林龄增大而减少的趋势;(2)根际土中磷的含量总体偏低,而且受到土壤酸化流失和丛枝菌根真菌积累的双重影响呈现出先减少后增加的趋势;(3)虽然有效钾含量随林龄变化趋势不显著,但丛枝菌根真菌能促进土壤钾的积累。因此,丛枝菌根真菌能有效调控根际土的养分动力学特征,减缓土壤酸化造成的养分流失。     

疫霉侵染下辣椒幼苗消减文库的构建与初步分析

为了分离和鉴定辣椒中疫霉诱导基因,以高抗疫霉病辣椒品种L11为材料,以接种辣椒疫霉菌的幼嫩叶片为处理（tester）,以未接种自然生长的幼嫩叶片为对照(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了疫霉侵染下辣椒幼苗的消减文库。从消减文库中随机挑取30个阳性克隆,提取质粒进行PCR鉴定,显示插入片段大小大部分集中在200～1 000 bp之间,文库质量良好。随机挑取40个克隆进行测序,共获得35个有效EST序列。经Blastx分析表明：有30个EST与GenBank中其他序列有同源性,5个EST为未知功能序列。已知功能的EST序列分别编码NAC转录因子、丝氨酸/苏氨酸蛋白激酶、P450单加氧酶、叶绿素a/b结合蛋白、谷胱甘肽转移酶、几丁质酶等,这些蛋白涉及抗病信号传递、抗氧化作用、转录调控及光合作用等多种生理过程。本研究为抗病基因克隆和系统研究疫霉侵染下辣椒基因的表达奠定了重要的理论基础。     

Differential Responses to the Combined Stress of Heat and Phytophthora capsici Infection Between Resistant and Susceptible Germplasms of Pepper (Capsicum annuum L.)

Journal of Plant Growth Regulation - Stresses caused by heat and Phytophthora capsici infection reduce pepper (Capsicum annuum) yields; however, little is known about their combined effects on...     

The oomycete broad-host-range pathogen Phytophthora capsici

Phytophthora capsici is a highly dynamic and destructive pathogen of vegetables. It attacks all cucurbits, pepper, tomato and eggplant, and, more recently, snap and lima beans. The disease incidence and severity have increased significantly in recent decades and the molecular resources to study this pathogen are growing and now include a reference genome. At the population level, the epidemiology varies according to the geographical location, with populations in South America dominated by clonal reproduction, and populations in the USA and South Africa composed of many unique genotypes in which sexual reproduction is common. Just as the impact of crop loss as a result of P. capsici has increased in recent decades, there has been a similar increase in the development of new tools and resources to study this devastating pathogen. Phytophthora capsici presents an attractive model for understanding broad-host-range oomycetes, the impact of sexual recombination in field populations and the basic mechanisms of Phytophthora virulence. TAXONOMY: Kingdom Chromista; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Phytophthora; Species capsici. DISEASE SYMPTOMS: Symptoms vary considerably according to the host, plant part infected and environmental conditions. For example, in dry areas (e.g. southwestern USA and southern France), infection on tomato and bell or chilli pepper is generally on the roots and crown, and the infected plants have a distinctive black/brown lesion visible at the soil line (Fig. 1). In areas in which rainfall is more common (e.g. eastern USA), all parts of the plant are infected, including the roots, crown, foliage and fruit (Fig. 1). Root infections cause damping off in seedlings, whereas, in older plants, it is common to see stunted growth, wilting and, eventually, death. For tomatoes, it is common to see significant adventitious root growth just above an infected tap root, and the stunted plants, although severely compromised, may not die. For many cucurbit fruit, the expanding lesions produce fresh sporangia over days (or even weeks depending on the size of the fruit) and the fruit often look as if they have been dipped in white powdered confectioner's sugar (Fig. 1). Generally, hyphae do not emerge from infected plants or fruit (common with Pythium infections) and all that is visible on the surface of an infected plant is sporangia. IMPORTANCE: Phytophthora capsici presents an oomycete worst-case scenario to growers as it has a broad host range, often produces long-lived dormant sexual spores, has extensive genotypic diversity and has an explosive asexual disease cycle. It is becoming increasingly apparent that novel control strategies are needed to safeguard food production from P. capsici and other oomycetes. Considering that P. capsici is easy to grow, mate and manipulate in the laboratory and infects many plant species, this pathogen is a robust model for investigations, particularly those related to sexual reproduction, host range and virulence. USEFUL WEBSITES: Phytophthora capsici genome database: http://genome.jgi-psf.org/Phyca11/Phyca11.home.html. Molecular tools to identify Phytophthora isolates: http://phytophthora-id.org.     

Evaluation of Oospore Hyperparasites for the Control of Phytophthora Crown Rot of Pepper

Nine isolates of known oospore mycoparasites comprised of six actinomycetes (Actinoplanes missouriensis, A. philippinensis, A. utahensis, Amorphosporangium auranticolor, Ampullariella regularis, Spirillospora albida) and three fungi (Acremonium sp., Humicola fuscoatra, Verticillium chlamydosporium) were tested in the greenhouse for their ability to suppress or delay the onset of crown rot of pepper caused by Phytophthora capsici. Verticillium chlamydosporium applied as a root dip increased the number of healthy plants by more than 100% when peppers were transplanted into soil artificially infested with oospores of Phytophthora capsici, but not when peppers were transplanted into soil naturally infested with P. capsici. The other mycoparasites were ineffective in the greenhouse. All the mycoparasites tested parasitized oospores of P. capsici in vitro.     

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.     

Screening of antagonistic bacteria for biological control of nursery wilt of black pepper (Piper nigrum)

Bacterial antagonists of Phytophthora capsici were isolated from underground shoot portions of rooted cuttings of black pepper. Initially isolates were screened by dual culture on potato dextrose agar and carrot agar. Further, a screening was done on black pepper shoots for supression of lesion caused by the pathogen. Most of the antagonists showed varying levels of antagonism in the dual culture and the shoot assay. Isolate PN-026, showing the highest suppression of lesion development in the shoot assay was found to be the most efficient antagonist in reducing Phytophthora capsici induced nursery wilt of black pepper. This screening involving the host, pathogen, and the antagonist, performed on black pepper shoot (the planting material for this vegetatively propagated crop), could be used as a rapid and reliable method for the isolation of efficient bacterial antagonists of P. capsici.     

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.     

拮抗辣椒疫霉菌海洋细菌菌株SH-27的筛选鉴定及其防病促生作用

【背景】由辣椒疫霉引起的辣椒疫病是全球辣椒生产中一种毁灭性的病害。近年来生物防治因其具有对环境友好、对人畜安全的特性而倍受关注。【目的】筛选对辣椒具有防病促生作用的海洋细菌菌株SH-27并鉴定其分类地位。【方法】采用稀释分离法和平板对峙法筛选拮抗辣椒疫霉菌的海洋细菌菌株,以发酵液灌根法测定海洋细菌SH-27菌株对辣椒盆栽的防病促生作用;通过形态特征、生理生化测试及多基因序列分析对海洋细菌SH-27菌株进行鉴定。【结果】从分离的142株海洋细菌中筛选获得11株对辣椒疫霉菌具有较强抑制作用的细菌菌株,其中以来自珊瑚的SH-27菌株的抑菌作用最强、抑菌谱广;室内盆栽试验结果表明,SH-27菌株发酵液处理后的辣椒植株根长、株高、茎粗、鲜重、干重均显著高于对照处理。SH-27菌株发酵液灌根处理后,对辣椒疫病4、6和9 d的防效分别为70.81%、66.55%和48.20%。经形态特征、生理生化测试及16S rRNA、gyrA基因序列分析,鉴定SH-27菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。【结论】海洋细菌SH-27菌株对辣椒具有较好的防病促生效果,具有开发为微生物农药及菌肥的潜力。     

辣椒疫毒致病毒素

辣椒疫霉（PhytophthoracapsiciLeonian）在培养液中振荡培养时可分泌毒素,这种毒素能引起辣椒叶片形成水渍状褐腐斑,类似病原菌侵染后形成的症状。Plich’s和V8C培养液适于P.capsici的生长和产毒;生长适宜温度和pH范围分别为20～30℃与pH6～7,在25～30℃、pH5～8的条件下培养滤液毒性最强;培养15天产毒达到最高值。滤液先后经85％硫酸铰沉淀、DE52、CM32和SephadexG－75柱层析将毒素纯化。经鉴定该纯毒素为39．8kD酸性糖蛋白,并对80℃以上高温或蛋白酶敏感,而β-D-葡萄糖苷酶处理不影响其毒性,蛋白亚基为毒素的活性中心。毒素处理寄主叶片引致病理性坏死,这一作用与辣椒品种抗病性有关。     

Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici

Aim:  To isolate and identify black pepper ( Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease.
Methods and Results:  Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici . Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in green house trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa ( Pseudomonas EF568931), IISRBP 25 as P. putida ( Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium ( B. megaterium EU071712) based on 16S rDNA sequencing.
Conclusion:  Black pepper associated P. aeruginosa , P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper.
Significance and Impact of the Study:  This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.