Quantitative aspects of Orobanche crenata infestation in faba beans as affected by abiotic factors and parasite soil seedbank

The interactions between the root parasitic weed Orobanche crenata Forsk. and its host plant faba bean ( Vicia faba L.) were quantified under controlled and field conditions at ICARDA's Tel Hadya research station. In the field experiments conducted in 1993–94 and 1994–95 faba beans were sown on two dates, in plots with 0, 50, 200 and 600 O. crenata seeds kg^–1 soil, under both limited and sufficient moisture supply. The effects of temperature on the duration of the early developmental stages of O. crenata were investigated in a growth chamber. The extent of O. crenata infestation was closely related to the number of parasite seeds in the soil. The seed-density treatment with 600 seeds kg^–1 soil resulted in complete crop failure. Furthermore, O. crenata infestation was higher under sufficient than under limiting water supply conditions, irrespective of sowing date. Only in the moderately infested plots, did shifting of the planting time of faba bean result in a significant decrease in parasite dry weight and an increase in crop seed yield. The timing of germination, attachment and further developmental stages of O. crenata was not related to faba bean growth stage and was affected primarily by soil temperature. The duration of O. crenata developmental stages was estimated using the thermal time concept. The relationship between total number of parasite attachments at the harvest of the faba bean crop and O. crenata seed density was dependent on maximum faba bean root-length density measured by the start of pod-filling in each treatment combination of sowing date and moisture supply. The results are discussed with reference to implications for the development of a dynamic simulation model for the prediction of faba bean yield losses caused by O. crenata .     

Bioassay-led isolation of Myrothecium verrucaria and verrucarin A as germination inhibitors of Orobanche crenata

A total of 188 fungal isolates was obtained from the rhizosphere of Vicia faba grown in an Egyptian soil heavily infested with Orobanche species. Agar cultures of 58 isolates inhibited the germination of conditioned seed of Orobanche crenata exposed to the germination stimulant, GR24. Filtrates of inhibitory fungi grown in liquid medium for 9–15 days were also assayed and those of five isolates, which were morphologically similar, inhibited germination even when diluted 16-fold. The fungus was identified as Myrothecium verrucaria (Alb. & Schwein.) Ditmar by its morphology and the nucleotide sequence of the ITS1 and ITS2 regions of the ribosomal repeat unit. Purification of the inhibitor to homogeneity was accomplished by solvent partitioning, flash chromatography on silica gel, semi-preparative HPLC on a reversed phase C18 column, solid phase extraction and tlc on silica gel. The inhibitor was identified as verrucarin A by nuclear magnetic resonance spectroscopy and comparison of the spectra with those of an authentic sample of the compound. A preliminary experiment demonstrated that infection of V. faba by O. crenata could be prevented by addition of spores of the fungus to soil infested by the parasite.     

Effect of sowing date and host resistance on the establishment and development of Orobanche crenata in faba bean and common vetch

The effect of early and late sowing dates on the establishment of Orobanche crenata Forsk. (crenate broomrape) in resistant and susceptible cultivars of faba bean (Vicia faba L.) and vetch (Vicia sativa L.) were studied over four seasons in southern Spain. Differences in establishment, development and shoot emergence of the parasite were compared. With resistant faba bean and vetch cultivars, there was reduced attachment and shoot emergence of O. crenata with all sowing dates. Susceptible cultivars were more severely affected by the parasite with early sowing. Orobanche crenata development was also delayed in resistant cultivars. Crop yield, estimated by the number of pods per plant, decreased with late sowings. The combined use of resistant cultivars and early sowing is a useful tool as part of an integrated control strategy. Resistant cultivars allow early sowing (with low O. crenata attack), thus avoiding yield losses due to the short crop cycle with late sowing.     

基于DNA条形码的向日葵田弯管列当种群遗传多样性研究

弯管列当是危害我国向日葵最严重的寄生杂草。为了明确我国向日葵田弯管列当种群的遗传多样性,本试验利用rbcL、matK、ITS2条形码序列对采自我国向日葵主产区的58份弯管列当样品进行PCR扩增及测序,采用Vector NTI软件对测序结果进行剪切比对,利用MEGA 6.0软件计算种内遗传距离并构建系统发育树。利用扫描电镜观察其种子的显微形态特征。结果表明,3个DNA条形码序列中仅ITS2片段扩增测序结果理想并表现出较好的聚类结果。各样品ITS2序列剪切比对后长度为453 bp,种内遗传距离为0.002～0.007,通过比较各样品ITS2序列的碱基组成和差异位点,能将不同弯管列当种群区分开。ITS2聚类结果表明58份弯管列当样品聚为3类,分别为Ⅰ型、Ⅱ型和Ⅲ型。形态分类结果表明,不同类型弯管列当在植株形态、种子形状及微观形态结构等方面存在差别。由于不同弯管列当种群的生境、寄主(向日葵栽培品种)不同,其种群遗传进化差异显著。基于ITS2条形码和扫描电镜形态学观察相结合的方法可用于弯管列当种群遗传多样性研究。     

Extent and pattern of genetic differentiation within and between European populations of Phelipanche ramosa revealed by amplified fragment length polymorphism analysis

The extent and pattern of genetic differentiation between Phelipanche ramosa populations colonising tobacco in different European regions were investigated by amplified fragment length polymorphism (AFLP) analysis, in order to determine levels of variation for tobacco resistance breeding and management programmes. Four different AFLP primer pairs amplified a total of 1050 clear and reproducible bands, of which 962 (91.62%) were polymorphic among the 35 individuals taken from four P. ramosa populations collected in Spain, Italy, Bulgaria and Germany. Cluster analysis based on the AFLP data categorised the plants into distinct groups, in line with their geographical origin, denoting clear genetic differentiation among the four populations. This differentiation was supported by both high bootstrap values and significant results of the analysis of molecular variance. The most divergent population was the one from Bulgaria. The majority of the genetic diversity was attributable to differences among populations (77.80%), as expected from the predominant autogamous behaviour of this species. Populations differed significantly in within-population diversity, as measured by Shannon's information index. The German population presented the lowest genetic diversity and the Italian population harboured the highest level of within-population genetic diversity. There are significant differences in genetic diversity level among the studied P. ramosa populations and clear population-specific genetic diversity structures. These need to be taken into account, together with the potential differences in parasite aggressiveness, when planning breeding and management strategies for P. ramosa control in tobacco cultivation.     

Host‐range studies,genetic diversity and evolutionary relationships of ACLSV isolates from ornamental,wild and cultivated Rosaceous species

A large‐scale survey was carried out to study the host range and genetic diversity of Apple chlorotic leaf spot virus (ACLSV) in various Rosaceae species, with a special emphasis on ornamentals and wild shrubs. Samples were tested by DAS‐ELISA using two different antisera, and RT‐PCR amplification of part of the CP gene. There was generally a poor correlation between the results obtained with the two sets of serological reagents and between serological and molecular detection assays. Using a nested RT‐PCR assay developed here, ACLSV was found to be widespread among cultivated, ornamental and wild species of the Rosaceae. The virus was detected for the first time in plum, wild cherry, Crataegus monogyna, Prunus spinosa and Prunus cerasifera in Greece. Sequences of a part of the CP encoding gene and the 3′ untranslated region from ACLSV isolates originating from various wild species and ornamentals were compared to those of isolates from cultivated hosts, showing similar divergence levels. Further phylogenetic analysis using the sequenced region indicated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. The possible role of different factors in the spread of ACLSV on cultivated, ornamental and wild species is discussed.     

Variability of Cuban and International Populations of Alternaria solani from Different Hosts and Localities: AFLP Genetic Analysis

As causal agent of early blight disease in tomato and potato, Alternaria solani is an internationally important horticultural pathogen. Genetic variability was surveyed by amplified fragment length polymorphism analysis in a total of 112 isolates from potato and tomato, representing pathogen populations from different Cuban provinces together with isolates from the USA, Brazil, Turkey, Greece and China. Also included in the analysis were isolates from catenulated Alternaria spp. from Brazil, Canada, Greece and Russia, along with single isolates of Alternaria porri, Alternaria alternata and a Curvularia sp. UPGMA clustering revealed a differentiation between the isolates of A. solani and all other species with the exception of A. porri which could not be distinguished from A. solani. Among the isolates of A. solani, two distinct subclusters were formed, with high genetic significance revealed by bootstrapping, corresponding to a general subdivision based on the respective solanaceous host. The results are discussed with regard to potential host specificity of A. solani on tomato and potato, and in terms of the comparative contributions of regional and international genetic variability in populations of this ubiquitous plant pathogen.     

Taxonomy,distribution and biology of lettuce powdery mildew (Golovinomyces cichoracearum sensu stricto)

This paper reviews the taxonomy, biology, importance, host–pathogen interactions and control of lettuce powdery mildew. The main causal agent of this disease, Golovinomyces cichoracearum s.s., is an important powdery mildew pathogen of many members of the family Asteraceae. The pathogen is distributed worldwide and occurs on Lactuca sativa as well as wild Lactuca spp. and related taxa (e.g. Cichorium spp.). Powdery mildew of lettuce can be a major problem in production areas with favourable environmental conditions for disease development (dry, hot weather). The fungus grows ectophytically and appears as white, powdery growth on both the upper and lower sides of leaves. There is rather limited information on the geographic distribution of powdery mildew on wild Lactuca spp. Most L. sativa cultivars have been found to be susceptible. Large variability in virulence was confirmed and existence of different races is supposed. Resistance in L. sativa and some related wild Lactuca spp. is characterized by race‐specificity, but the genetic background of resistance is poorly understood. Sources of resistance are known in L. saligna and L. virosa. Lettuce powdery mildew can be effectively controlled by common fungicides (e.g. sulphur, myclobutanil, quinoline, strobilurins, etc.) and protective compounds (e.g. extract of neem oil, Reynoutria sachaliensis extracts). However, fungicide resistance may arise. Non‐fungicidal activators of plant systemic acquired resistance (SAR) had no direct effect on the causal agent. Future issues regarding lettuce powdery mildew research are summarized.     

Exploring de novo specificity: the Pyrenophora tritici‐repentis–barley interaction

Pyrenophora tritici‐repentis (Ptr) is a destructive fungal pathogen of wheat worldwide. In addition to wheat, Ptr has been isolated from various other hosts in the family Poaceae, yet the nature of its interaction with those hosts is unknown. The Ptr–barley relationship was explored and the existence of a specific interaction between Ptr and barley is described for the first time; symptom development on several barley genotypes was evaluated in bioassays and by toxin infiltration into barley leaves. Ptr ToxB‐producing isolates of the fungus were able to cause significant damage when inoculated onto certain barley genotypes, and Ptr ToxB was able to induce chlorosis in a highly selective manner when infiltrated into those same genotypes. Ptr–barley specificity is subtle and can break with slight changes in temperature after infection. To understand the infection process in barley, a cytological analysis and in planta fungal biomass estimation using quantitative PCR were performed. The fungus penetrates through the host epidermal cells and advances to colonize the mesophyll layer intercellularly, with the infection process on barley closely resembling that on wheat. Here, evidence is provided for a specific interaction between barley and Ptr, expanding understanding of Ptr host specificity and breaking the assumption that the highest level of specificity seen with Ptr is restricted to particular genotypes of the wheat host.     

A comparative study of Turkish and Israeli populations of Didymella rabiei,the ascochyta blight pathogen of chickpea

The Fertile Crescent is the centre of domestication of chickpea (Cicer arietinum) and also the place of origin of its pathogens. Agrosystems provide different environments to natural eco‐systems, thus imposing different types of selection on pathogens. Here, the genetic structure and in vitro temperature growth response of the chickpea pathogen Didymella rabiei from domesticated chickpea (59 isolates from Turkey and 31 from Israel) and wild Cicer spp. (three isolates from Turkish C. pinnatifidum and 35 from Israeli C. judaicum) were studied. Six sequence‐tagged microsatellite site (STMS) primer pairs were used to determine the genetic structure of the 128 D. rabiei isolates. Turkish isolates exhibited the highest genetic diversity (H = 0·69). Turkish and Israeli D. rabiei from domesticated chickpea were genetically closer to each other than isolates from the wild Cicer spp. Analysis of molecular variance showed that 54% of the genetic variation resided between isolates from wild and domesticated origins. EF1‐α sequences distinguished between D. rabiei isolates from domesticated and wild Cicer spp. by four polymorphic sites. Nevertheless, a certain degree of mixing between isolates from wild and domesticated origin was demonstrated using the Bayesian algorithm as well as with principal coordinates analysis. Isolates sampled from domesticated chickpea from both countries were better adapted to temperatures typical of Levantine spring and had a significantly larger colony area at 25°C than at 15°C (typical Levantine winter temperature). These observations were in accordance to the heritability values of the temperature growth response.     

基于线粒体 COⅠ和 COⅡ基因的5种不同寄主植物 西花蓟马种群遗传多样性研究

西花蓟马Frankliniella occidentalis(Pergande)是一种分布广、危害大的世界性检疫害虫。本研究旨在探讨田间常见5种寄主植物上的西花蓟马种群的寄主专化性和遗传多样性。以采自甘肃、宁夏的辣椒、茄子、蜀葵、月季、美人蕉等5种寄主植物上的西花蓟马为对象,以线粒体COⅠ和COⅡ基因为靶标,应用Arlequin 3.5软件进行种群遗传多样性、遗传分化、基因流水平及分子变异分析,以MEGA 7.0和Network 5.0软件分别构建单倍型系统发育树和中接网状树。结果显示,当以mtDNACOⅠ和COⅡ基因为靶标时,分别检测到13种和12种单倍型,其中单倍型H_1和H_2为优势单倍型;辣椒和茄子上的单倍型数量只有3种(COⅠ基因)或2种(COⅡ基因),而蜀葵、月季和美人蕉上的单倍型数量比较多,为7~9种(COⅠ基因)或8~9种(COⅡ基因);辣椒和茄子上的种群单倍型种类、单倍型多样性、核苷酸多样性和核苷酸平均差异数等均较蜀葵、月季和美人蕉上的种群低。COⅠ和COⅡ基因总寄主种群Fu's Fs检验结果分别为2.36和4.06,种群大小整体保持稳定;总寄主种群固定指数FST和基因流Nm分析表明,西花蓟马各寄主植物种群之间基因交流充分,尚未发生明显的遗传分化;AMOVA分析显示遗传变异主要来自种群内部;基于COⅠ和COⅡ基因序列的单倍型聚类和网状树都明显分为两支,分别对应西花蓟马的温室品系Glasshouse strain和羽扇豆品系Lupin strain;其中温室品系在所有寄主植物种群中均有发生,而羽扇豆品系的单倍型主要来自多年生的蜀葵、月季和美人蕉等植物,在辣椒和茄子上并无羽扇豆品系发生。研究结果对西花蓟马种群扩张机制探讨、遗传动态分析以及有效防控措施的制定具有一定指导意义。     

Coincidence of virulence shifts and population genetic changes of Pseudoperonospora cubensis in the Czech Republic

Pseudoperonospora cubensis is an oomycete pathogen causing downy mildew disease on a variety of Cucurbitaceae, and has recently re‐emerged as a destructive disease on crops in this family, mainly on cucumber and squash. Multilocus sequence analysis (MLSA) of four mitochondrial and two nuclear DNA regions was used to detect changes in the genetic structure of P. cubensis populations occurring in the Czech Republic that might be associated with recently reported shifts in virulence. The analysed sample set contains 67 P. cubensis isolates collected from 1995 to 2012 in the Czech Republic and some other European countries. Sequence analyses revealed differences and changes in the genetic backgrounds of P. cubensis isolates. While all isolates sampled before 2009 exhibited the genotype of the subspecies of Clade II and were collected from cucumber, all samples collected from other hosts belonged to Clade I (P. cubensis sensu stricto) or were sampled from 2009 onwards. In addition, 67·16% of all post‐2009 isolates from Clade II had two heterozygous positions in their nrITS sequence, which suggests sexual reproduction and/or a mutational origin. Thus, the results indicate that, apart from the rise in prevalence of Clade I, the change in the genetic structure of P. cubensis populations may be linked with a hybridization or, less likely, a mutation event that rendered strains able to infect a broader spectrum of host species.