Real-Time Polymerase Chain Reaction Quantification of Phytophthora capsici in Different Pepper Genotypes

ABSTRACT Reliable and sensitive quantification of Phytophthora capsici in pepper plants is of crucial importance in managing the multiple syndromes caused by this pathogen. A real-time polymerase chain reaction (PCR) assay was developed for the determination of P. capsici in pepper tissues. DNA levels of a highly virulent and a less virulent isolate were measured in different pepper genotypes with varying degrees of resistance. Using SYBR Green and specific primers for P. capsici, the minimal amount of pathogen DNA quantified was 10 pg. Pathogen DNA was recorded as early as 8 h postinoculation. Thereafter, the increase was rapid in susceptible cultivars and slower in resistant ones. The amount of pathogen DNA quantified in each pepper genotype correlated with susceptibility to Phytophthora root rot. Likewise, there was a relationship between the virulence of the pathogen and the degree of colonization. Differences also were found in oomycete amount among pepper tissues, with maximal pathogen biomass occurring in stems. The real-time PCR technique developed in this study was sensitive and robust enough to assess both pathogen development and resistance to Phytophthora root rot in different pepper genotypes.     

Characterization of <Emphasis Type="Italic">Phytophthora clandestina</Emphasis> races on <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.     

Evaluation of Trichoderma harzianum for controlling root rot caused by Phytophthora capsici in pepper plants

The ability of Trichoderma harzianum to control the rotting of pepper ( Capsicum annuum ) plant roots caused by Phytophthora capsici was studied. Interactions between the fungi were assessed in vitro on three culture media (V8c, Czapek and 2% water agar) and in vivo in plants grown in a substrate inoculated with P. capsici and T. harzianum . Studies on mutual antagonism in vitro showed that P. capsici was inhibited by T. harzianum ; however, the intensity of inhibition differed according to the medium used, being greatest on Czapek. Analysis of the fungal populations in the plant growth substrate showed that T. harzianum consistently reduced that of P. capsici over time. This reduction in the pathogen population was associated with a reduction in root rot of between 24 and 76%, although plant growth (dry weight) was still reduced by 21.2–24.7%, compared with the uninoculated control. In the absence of T. harzianum with the same pathogen inoculum levels, the reduction in dry weight was 59.8–68.6%, suggesting that T. harzianum reduced the damage.     

新疆主要农作物疫霉菌种类鉴定

 1993~1995年,对侵染新疆主要农作物的疫霉菌进行了较全面的调查研究,共从9个地区的28种作物上采集表现根腐、根颈腐、果腐症状(包括病土)的病样1531个,结果从其中17种作物上分离到452个疫霉菌株。根据形态特征、生理生化特性、致病性和菌体可溶性蛋白电泳测定,鉴定为7个种:辣椒疫霉(Phytophthora capsici Leon.),恶疫霉[P.cactorum(Leb.et Cohn) Schroeter],掘氏疫霉(P.drechsleri Tucker),菸草疫霉(P.nicotianae van Breda de Haan),苎麻疫霉(P.boehm eriaeSaw.),柑桔褐腐疫霉[P.citrophthora (Sm.et Sm.) Leonian],隐地疫霉(P.cryptogea Pethyb.etLaff.)。这些疫菌是造成新疆茄果类、瓜类、棉花、苹果、梨、枸杞、草霉、红花、白术等幼苗及成株大量死亡及烂果的主要病原,在农业生产中具有十分重要的意义。     

Soluble phenolic acids in Capsicum annuum stems infected with Phytophthora capsici

The soluble phenolic acids of the stems of three Capsicum annuum cultivars associated with differing resistance to Phytophthora capsici showed qualitative and quantitative variation after inoculation, notably an increase in the total phenolic acid content in the resistant and intermediate varieties. The soluble phenolic acids also retarded myeelial growth of P. capsici in culture. The most pronounced inhibitory effect was produced by f-cinnamic acid, followed by p-hydroxybenzoic, vanillic and salicylic acids. Colonization and necrosis spread throughout the stem of the susceptible cultivar whereas the stem of the resistant cultivar was only partially invaded. In the latter, invasion of the tissue adjacent to necrosis occurred in only 20% of the plants.     

中国大豆疫霉根腐病和大豆种质抗病性研究

 大豆疫霉根腐病在中国的发生区域逐渐扩大,局部地区发生严重。大豆疫霉菌1号生理小种是黑龙江省的优势小种,同时也存在其它已知生理小种和新生理小种。不同大豆生态区之间大豆疫霉菌生理小种差异显著。在总体上,中国大豆种质的抗病性不强,长江流域大豆中抗病种质比率最高,其次为黄淮海流域种质,而东北地区抗病种质较少。按省归类,大豆种质抗性由强至弱依次为河南、江苏、湖北、山西、河北、辽宁、吉林、黑龙江、山东。吉林和黑龙江选育推广品种中,感病品种分别高达80%和73%;在462份大豆品系中,也表现出南方材料抗病性高于北方材料的趋势;地方品种中感病类型也高达60%以上。     

Dispersal of Phytophthora capsici and P. parasitica in furrow-irrigated rows of bell pepper, tomato and squash

Dispersal of Phytophthora capsici and P. parasitica from point sources buried near the upper end of 74 m-long irrigation furrows was studied with three annual host crops. Furrows next to tomato were inoculated with either P. capsici or P. parasitica , while furrows next to pepper and squash were inoculated with P. capsici only. Irrigation was carried out on a 14-day cycle. Dispersal of each fungus in the water was monitored by transplants in the furrows and the incidence of infected tomato fruit along furrows. Disease gradients on roots and shoots of all hosts were also measured. Repeated irrigations dispersed P. capsici and P. parasitica up to 70 m from the source. Fruit infection increased with increasing distances downstream, suggesting an accumulation of secondary inoculum with the repeated flow of water. Conversely, gradients of disease severity on roots of tomato and pepper peaked at the source and rapidly decreased to low levels up to 32 m downstream. Fruit and root infection upstream from source was negligible. Root and crown rot in squash was highly variable, being confined to locations near the initial inoculum in some units while developing to severe levels several meters downstream in other units. Unlike the other hosts, squash petioles were in contact with the irrigation water and may have served as routes of invasion by P. capsici. Transport of inoculum from the furrow water to the roots was limited. P. capsici isolates were more virulent on tomato roots and caused a higher incidence of buckeye rot on tomato fruit than did isolates of P. parasitica.     

Phytophthora species causing crown and root rot of tomato in southern Italy*

Phytophthora capsici, Phytophthora cryptogea and Phytophthora nicotianae were isolated from tomato plants with symptoms of crown and root rot in plastic‐house crops in Sicilia and Calabria (southern Italy). The species were identified primarily on the basis of morphological and cultural characteristics. The identification was confirmed using molecular methods, polyacrylamide gel electrophoresis (PAGE) of mycelial proteins and polymorphism of DNA sequences amplified by polymerase chain reaction using random primers (RAPD‐PCR). P. capsici caused significant losses in tomato crops that had succeeded capsicum crops. P. cryptogea was found to be the most frequent species causing basal stem rot of tomato, a disease of increasing importance in commercial tomato crops in plastic houses in Sicilia. P. nicotianae was common in plastic houses where poor drainage resulted in standing water.     

Race distribution of Phytophthora sojae on soybean in Hyogo, Japan

Since 1987, Phytophthora root and stem rot of soybean [Glycine max (L.) Merr. cv. Tanbakuro], caused by Phytophthora sojae Kaufman and Gerdemann, has been increasing in the Sasayama, Nishiwaki, and Kasai regions in Hyogo, the most famous soybean (cv. Tanbakuro)-producing areas in Japan. In 2002 to 2004, 51 isolates (one from each field) of P. sojae were recovered from 51 fields in Hyogo. These isolates were tested for virulence on six Japanese differential soybean cultivars used for race determination in Japan, and three additional ones containing four Rps genes used in Indiana, USA. Race E was the most prevalent from 2002 to 2004, followed by races A, C, D, and four new races (proposed as races K, L, M, and N). Interestingly, none of the new races had high virulence on the Japanese differential cultivars, compared with other races in each area. One (race N) was avirulent on all six soybean differentials. There was a difference in race distribution on each of three individual areas; race E seemed to be a major component of the P. sojae population in Sasayama, whereas race A and the new race M were the most prevalent in Nishiwaki and Kasai, respectively. Rps6 (cv. Altona) and Rps1a + Rps7 (cv. Harosoy 63) were infected by 90.2% and 33.3% of all isolates, respectively. However, Rps1d (cv. PI103091) was not susceptible to any of the 51 isolates, nor was cv. Gedenshirazu-1. These two soybean cultivars were considered to be potential sources of resistance to breed new resistant cultivars with the desirable characteristics of cv. Tanbakuro for this region.     

Virulence and aggressiveness of single-zoospore isolates of Phytophthora fragariae

A system for scoring the virulence of isolates of Phytophthora fragariae based on a scale of root rot from 0 ( no symptoms ) to 5 (76-100% roots roiled) on a series of strawberry cultivars is described. Thirty-two single-zoospore isolates from one field site were compared by subjecting their root rot scores to cluster analysis and this grouped them into two major clusters equivalent to physiologic races B66–3 and B66-11, Different sub-clusters of isolates of race B66-11 produced different degrees of rotting on the same hosts. Apart from differences in virulence between the sub-clusters there was some evidence for differences in aggressiveness between isolates within sub-clusters.
Increasing inoculum concentration by over 300-fold increased rotting by c . 25% but did not alter the rankings of different isolate/host combinations. Repeated passage of isolates through cultivars of differing susceptibilities did not affect their pathogenicity.     

Characterization of Phytophthora spp. Causing Outbreaks of Citrus Brown Rot in Florida

ABSTRACT Epidemics of citrus brown rot from 1994 to 1997 in the south-central and east-coast citrus areas of Florida were characterized and the causal Phytophthora spp. identified. Two species of Phytophthora, P. palmivora and P. nicotianae, were consistently associated with brown rot. Epidemics caused by P. palmivora appeared to be initiated on immature fruit dropped on the orchard floor. The soilborne fungus infected and sporulated on these fruit and was then disseminated to fruit above 1 m in the canopy. In contrast, infection by P. nicotianae, the common cause of root rot, was confined to the lowest 1 m of the canopy. Fruit infected by P. palmivora produced large amounts of ellipsoidal sporangia available for splash dispersal, whereas those infected by P. nicotianae produced far fewer spherical sporangia. Isolates from brown rot epidemics were compared with P. nicotianae from citrus in Florida and Texas, P. citrophthora in California, P. palmivora, and selected Phytophthora spp. from other hosts. Brown rot symptoms produced by the different pathogenic citrus isolates on inoculated fruit were indistinguishable. Morphology, mating behavior, and isozyme patterns of brown rot isolates from 1988 to 1997 matched P. palmivora from citrus roots, other host plants, and other locations, but were different from characterized isolates of P. citrophthora in California and P. nicotianae in Florida and Texas. Cellulose acetate electrophoresis of the isozyme glucose-6-phosphate isomerase rapidly identified the causal citrus pathogen from infected fruit and soil isolation plates. Although P. palmivora is an aggressive pathogen of citrus roots, bark, and fruit, populations in orchard soils were low compared with P. nicotianae.     

Asexual Reproduction of Phytophthora capsici as Affected by Extracts from Agricultural and Nonagricultural Soils

ABSTRACT Formation of sporangia and zoospores in species of Phytophthora is known to be influenced by soil microbial and chemical composition. In Phytophthora capsici, the study of the relationship of soil chemical composition to production of sporangia and zoospores has been limited. P. capsici is a soilborne pathogen of a wide array of vegetable crops, including chile pepper (Capsicum annuum) in New Mexico. Production of sporangia and zoospores by P. capsici was evaluated in extracts of soils from three different environments in New Mexico: (i) agricultural environments with a long history of chile pepper cropping and occurrence of P. capsici (CP), (ii) agricultural environments with no history of chile pepper cropping and no occurrence of P. capsici (Non-CP), and (iii) nonagricultural environments consisting of forests and rangelands (Non-Ag). There was a significant difference in production of P. capsici asexual propagules, expressed as natural log (number of sporangia x number of zoospores), among the three environments (P = 0.0298). Production of propagules was 9 to 13% greater in Non-Ag than in CP or Non-CP environments. Stepwise multiple discriminant analysis and canonical discriminant analysis identified the edaphic variables Na, pH, P, organic matter content, and asexual propagule production as contributing the most to the separation of the three environments. Two significant (P < 0.0001) canonical discriminant functions were derived with the first function, accounting for approximately 75% of the explained variance. Based on the two discriminant functions, approximately 93, 86, and 89% of observations in CP, Non-CP, and Non-Ag environments, respectively, were classified correctly. Soils from agricultural and nonagricultural environments differentially influence production of sporangia and zoospores in P. capsici, and soil samples could be effectively classified into agricultural and nonagricultural environments based on soil chemical properties and the production of asexual propagules by P. capsici in soil extracts.     

Evaluation of root damage to English walnut caused by five Phytophthora species

The pathogenicity of five species of Phytophthora to English walnut was studied in a greenhouse experiment. Phytophthora cinnamomi was the most aggressive species, causing severe root rot and seedling mortality. The other species tested, P. cambivora , P. citricola , P. cactorum and P. cryptogea , did not induce visible crown symptoms on seedlings 2 months after inoculation. Some strains of P. cambivora and P. cactorum also caused taproot damage to seedlings. All except one of the tested isolates caused significant necrosis of fine roots and a significant reduction of root weight compared with noninoculated seedlings. Reduction of above-ground plant development was not statistically significant. While P. cinnamomi is well known as an aggressive primary pathogen of English walnut, the other species of Phytophthora may act as predisposing factors to walnut decline, affecting root system development and increasing host vulnerability to environmental stress.     

Colonization of roots of subterranean clover cultivars by virulent and avirulent races of Phytophthora clandestina

The colonization of the roots of four cultivars of subterranean clover by isolates representing four races of Phytophthora clandestina was studied. There was a highly significant race × cultivar interaction in the growth of inoculated tap roots and the degree of colonization of roots by the pathogen. While all races were able to infect the roots of all cultivars tested, roots of the susceptible cultivars were colonized more rapidly and extensively than those of the resistant cultivars. In compatible combinations, fungal colonization extended for a few centimetres in the tap root and lateral roots in the moderately susceptible cultivars Trikkala and Meteora, or throughout the whole root system leading to the death of the host in the very susceptible cultivar Woogenellup. In contrast, limited fungal colonization of the tap root and lack of extension of the fungus into lateral roots was typical of incompatible combinations. In all cultivars, lateral roots were as susceptible to infection as tap roots. The number of lateral roots of Woogenellup was significantly reduced by infection. However, neither the rate of lateral root formation nor the total number of lateral roots of Seaton Park, Meteora and Trikkala was reduced by infection with virulent or avirulent races of the pathogen.     

海南省胡椒瘟病病原鉴定及发生规律

采用形态特征观察、致病性测定及rDNA-ITS序列分析等方法,对海南省主产区胡椒瘟病病原进行鉴定,通过田间实地调查对胡椒瘟病的发生规律进行了研究。结果表明,该病病原为辣椒疫霉(Phytophthora capsici),月平均降雨量对该病的发生有着显著的影响。降雨量越大,持续降雨天数越多,发病率越高。     

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(CaLecRK2.2、CaLecRK3.2、CaLecRK8.1和CaLecRK10.1)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。     

Characterization of Phytophthora capsici from Michigan Surface Irrigation Water

ABSTRACT Phytophthora capsici infects cucurbitaceous and solanaceous crops worldwide. In free water, P. capsici sporangia release zoospores that may be disseminated by moving surface water. Surface irrigation sources (river system, ponds, and ditches) in three Michigan counties with a history of P. capsici-susceptible crop production were monitored for the pathogen during four growing seasons (2002 to 2005). Pear and cucumber baits were suspended in water at monitoring sites for 3- to 7-day intervals and water temperature was recorded. Baits were washed and lesions were excised and cultured on water agar amended with rifampicin and ampicillin. P. capsici was detected at monitoring sites in multiple years, even when non-host crops were planted nearby. Recovered isolates (N = 270) were screened for sensitivity to the fungicide mefenoxam and characterized for mating type (MT). P. capsici isolates resistant to mefenoxam were common in water sources from southwest and southeast Michigan. Most monitoring sites yielded isolates of a 1:1 ratio of A1:A2 MTs. Amplified fragment length polymorphism analysis of select isolates from 2002 to 2004 indicated a lack of similarity groups persisting over time and in specific geographical locations. Data suggest that P. capsici did not overwinter in any of the surface water sources monitored. Water temperatures were correlated to positive P. capsici detection from all monitoring sites. The frequent detection of P. capsici in surface water used for irrigation in the primary vegetable growing regions in Michigan suggests that this is an important means of pathogen dissemination.     

植物内生细菌在辣椒体内的定殖动态及对辣椒疫病的防治效果

为了探讨具生防作用的植物内生细菌在辣椒体内的定殖动态与其防治辣椒疫病的关系,采用对峙培养法和盆栽苗防效法筛选生防菌株,依据菌体形态、生理生化性质和16SrDNA序列鉴定菌种,用抗利福平标记研究菌株在辣椒苗中的定殖动态,在同时接入植物内生细菌和灌根接种辣椒疫霉菌的条件下分析生防菌株的定殖数量与防效的关系。结果表明,菌株G9、R15和J13对辣椒疫病防效最好,经鉴定均为荧光假单胞菌Pseudomonasfluorescens。菌株G9和R15在辣椒根部定殖量高于菌株J13;定殖周期均在30-40d,呈“先增后减”的变化趋势;菌株G9和R15在接种第15d时定殖量最高,菌株J13在根、茎和叶中定殖量达到最高的天数分别为第9、15和15-20d,定殖数量的变化为根〉茎〉叶。菌株G9定殖量达到9．73×10^5cfu·g-1时辣椒疫病的防效达到100％,保持该数量的时间约6d;菌株R15定殖量达到6．30×10^5cfu·g-1以上时对辣椒疫病的防效达到100％,保持该数量的时间约14d。研究结果展现了植物内生细菌在辣椒疫病生物防治上的应用潜力,为制定植物内生细菌防治辣椒疫病的施用技术提供了科学依据。     

福建省大豆疫病病原鉴定及其核糖体DNA-ITS序列分析

 从福建省龙海大豆根腐病株上分离的疫霉菌株中,选取6个代表菌株,对病原菌进行了形态特征、致病性、寄主范围鉴定及核糖体DNA-ITS序列分析,结果表明,该菌为疫霉属真菌,在黑麦琼脂培养基上生长缓慢,菌丝致密、无隔,形成菌丝膨大体,近直角分支,分支处稍缢缩。水培后产生大量椭圆形孢子囊,不形成乳突,通过内层出方式产生新孢子囊,游动孢子在孢子囊内形成,同宗配合,藏卵器球形,雄器侧生;接种后可出现典型的大豆疫病症状;人工接种只侵染大豆、豇豆和菜豆等少数豆科植物。其核糖体DNA-ITS序列分析表明,分离菌株与GenBank中大豆疫霉的ITS序列的同源性均为99.8%,仅有2个碱基的差异,结合形态特征和致病性测定,将这些病原菌鉴定为Phytophthora sojae. 这是首次报道大豆疫霉菌在福建省存在。     

大豆与疫霉菌非亲和互作早期差异显示基因的表达分析

大豆疫霉根腐病是大豆的毁灭性病害。为了深入了解大豆对疫霉菌的分子抗病机制,以大豆疫霉菌1号生理小种游动孢子接种抗性品种绥农10的根部及下胚轴,通过反转录差异显示技术分离到疫霉菌侵染0、0.5、1、2和4h后大豆下胚轴和茎部的差异表达基因,其中至少有8个基因与抗病相关。接种后0.5 h开始上调表达的有肉桂酸-4-羟化酶基因、ATP合成酶β亚基基因,以及类花生泛素结合酶基因;接种后1h和2h依次开始上调表达的有尿苷二磷酸-N-乙酰基-α-D-氨基半乳糖基因和豌豆蓝铜蛋白基因;接种后4 h才上调表达的有TGA型碱性亮氨酸拉链基因、大豆环孢素基因和14-3-3蛋白基因。这8个基因中有1个基因与信号传导有关、4个基因与抗病和防御有关、2个基因与转录调控有关、1个基因与能量代谢有关。研究表明,以上8个基因在疫霉菌游动孢子萌发、侵入大豆和在大豆体内扩展过程中起着重要作用。