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.     

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.     

Pathogenic and molecular diversity in highly virulent populations of the parasitic weed Orobanche cumana (sunflower broomrape) from Europe

The parasitic weed Orobanche cumana (sunflower broomrape) constrains sunflower production in eastern and southern Europe and in the Middle East. Although genetic resistance is the most effective control method, new parasite races evolve overcoming sunflower resistance. In this work, highly virulent populations of O. cumana were analysed for pathogenicity and genetic diversity. The virulence of 11 populations from Hungary, Romania, Spain and Turkey was assessed and compared after infection of sunflower inbred lines to differentiate races of the parasite under glasshouse conditions. Molecular diversity among and within 27 parasite populations was studied by RAPD‐PCR, UPGMA and amova analyses. Highly virulent race F was identified in Hungary, Spain and Turkey. The most virulent race (G) was also found in Turkey. The molecular analysis among highly virulent populations of O. cumana identified four molecular clusters, respectively, grouping populations from Central Spain, Hungary, South Spain and Turkey. The genetic homogeneity within parasite populations was confirmed, since no molecular divergences were found within them. This work constitutes the first geographical study of O. cumana together with pathogenicity and molecular traits inherent to each geographical group, and provides useful information for possible phylogenetic analyses of O. cumana. In addition, molecular markers associated with geographical origin could be developed and used as diagnostic tools to track new broomrape introductions into areas free of virulent races where they might represent a threat to sunflower production.     

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.     

Early survival of Quercus ilex subspecies from different populations after infections and co‐infections by multiple Phytophthora species

Forests in Europe are threatened by increased diversity of Phytophthora species, but effects on trees of simultaneous infections by Phytophthora and ecological consequences of their coexistence are unknown. This study explored variation in early survival of Quercus ilex to Phytophthora infections and assessed interactions between Phytophthora species when trees were co‐infected. Three Phytophthora species (P. cinnamomi, P. gonapodyides and P. quercina), seeds from 16 populations of Q. ilex (ballota and ilex subspecies) and two infection times were used as sources of variation in two experiments. The influence of Phytophthora species, Q. ilex subspecies and populations on plant germination and survival were analysed using generalized linear mixed models and survival analysis techniques. Germination rates were not influenced by Phytophthora spp. (P = 0.194) but by the subspecies and populations of Q. ilex (P < 0.001). In Phytophthora‐infested soils, Q. ilex subsp. ilex germinated at higher rates than Q. ilex subsp. ballota. Plant survival was strongly influenced by Phytophthora species (P < 0.001), not by the subspecies and populations of Q. ilex. Seedling mortality was reduced and delayed if a less virulent Phytophthora species infected plants prior to infection by a more virulent Phytophthora species. The results help to explain oak decline syndrome and the lack of natural and artificial regeneration of Q. ilex forests. Lack of interspecific variability of early survival to Phytophthora spp. discourages direct sowing for artificial reforestation programmes. Large, thick seeds, giving plants rapid growth, are advantageous traits when soils are infested with Phytophthora spp.     

Simple sequence repeat analysis of genetic diversity among Acetyl‐CoA carboxylase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola populations in Korea

Echinochloa species are amongst the most problematic weeds in rice fields of Korea. The steady reliance on the Acetyl‐CoA carboxylase (ACCase) and acetolactate synthase inhibiting herbicides for control of these weeds has led to resistance to these herbicides. The objective of this study was to assess the genetic diversity among populations of ACCase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola in Korea, to better understand their population structure and possible origins of resistance. Seven simple sequence repeat markers were applied to assess the genetic diversity between resistant and susceptible E. crus‐galli and E. oryzicola from 12 populations in Korea. Genetic diversity was slightly higher in the resistant group. The Unweighted Pair Group Method using Arithmetic algorithm (UPGMA) dendrogram generated two distinct clades. One clade consisted of Echinochloa spp. from three populations, i.e. Anmyeondo, Gimje 4 and Gongju, which are resistant and differentiated from the susceptible populations, and the other clade contained the rest of the populations. Structure modelling supported two clades of UPGMA clustering. Based on these data, we can infer that some resistant populations are greatly differentiated, whereas other resistant biotypes are still building up resistance in rice fields in Korea. Resistance traits will be fixed and continue to spread over time without proper control measures.     

Genetic differentiation and gene flow in Colletotrichum sublineolum in Ethiopia,the centre of origin and diversity of sorghum,as revealed by AFLP analysis

Isolates of Colletotrichum sublineolum were collected from different sorghum‐producing regions of Ethiopia and divided into five groups based on their geographic origin. The growth rate of 50 isolates showed considerable variation: 1·7–5·8 mm day^?1, mean 3·3 mm day^?1. However, the isolates displayed little variation in colony colour and colony margin, except for isolates from the north, which were different from the others. Amplified fragment length polymorphism analysis of 102 isolates revealed much greater variations among the different groups. Dice similarity coefficients ranged from 0·32 to 0·96 (mean 0·78). Cluster analysis and principal coordinate analysis revealed a differentiation of the isolates according to their geographic origin, and both methods clearly indicated a genetic separation between the southern, the eastern and the other isolates. Analysis of molecular variance (amova ) indicated a high level of genetic variation both among (42%) and within (58%) the C. sublineolum sampling sites in Ethiopia. The amova also indicated a high level of genetic differentiation (F_ST = 0·42) and limited gene flow (Nm = 0·343). The results of this study confirmed the presence of a highly diverse pathogen, which is in agreement with the existence of diverse host genotypes and widely ranging environmental conditions in sorghum‐producing regions of the country. Such diversity should be taken into account in future breeding programmes to achieve an effective and sustainable disease management strategy.     

Microsatellite variability of sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides (Cyperaceae) in Kinki,Japan

Schoenoplectus juncoides is one of the most harmful weeds found in East Asian paddy fields. Recent emergence of biotypes that are resistant to the herbicide sulfonylurea (SU) has made weed control difficult. To examine the effect of the evolution of this herbicide resistance on genetic diversity within local populations, we investigated microsatellite variability within and among paddy field populations of S. juncoides in Kinki, Japan. In vivo assay of acetolactate synthase activity and root elongation assay in the presence of SU revealed that of 21 populations, five were sulfonylurea‐susceptible (SU‐S) and eight were completely sulfonylurea‐resistant (SU‐R). The remaining eight populations were a mixture of SU‐S and SU‐R individuals. The average gene diversity for SU‐R populations (H_S = 0.168) was lower than those for SU‐S (H_S = 0.256) and mixed (H_S = 0.209) populations, but the difference was not significant. This indicates that positive selection for SU‐R phenotype did not cause a genome‐wide reduction in genetic diversity. Genetic differentiation among S. juncoides populations was higher than that observed for most weed species studied previously. Although populations in neighbouring paddy fields showed a high level of differentiation, Bayesian clustering analyses suggested that some level of gene flow occurs among them and that the genetic exchange or colonisation between neighbouring populations could contribute to the geographical expansion of the resistant allele.     

Genetic diversity within and between sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides in Japan

To reveal the effects of herbicide selection on genetic diversity in the outcrossing weed species Schoenoplectus juncoides, six sulfonylurea‐resistant (SU‐R) and eight sulfonylurea‐susceptible (SU‐S) populations were analysed using 40 polymorphic inter‐simple sequence repeat loci. The plants were collected from three widely separated regions: the Tohoku, Kanto and Kyushu districts of Japan. Genetic diversity values (Nei's gene diversity, h) within each SU‐S population ranged from h = 0.125 to h = 0.235. The average genetic diversity within the SU‐S populations was H_S = 0.161, and the total genetic diversity was H_T = 0.271. Although the H_S of the SU‐R populations (0.051) was lower than that of the SU‐S populations, the H_T of the SU‐R populations (0.202) was comparable with that of the SU‐S populations. Most of the genetic variation was found within the region for both the SU‐S and SU‐R populations (88% of the genetic variation respectively). Two of the SU‐R populations showed relatively high genetic diversity (h = 0.117 and 0.161), which were comparable with those of the SU‐S populations. In contrast, the genetic diversity within four SU‐R populations was much lower (from h = 0 to 0.018) than in the SU‐S populations. The results suggest that selection by sulfonylurea herbicides has decreased genetic diversity within some SU‐R populations of S. juncoides. The different level of genetic diversity in the SU‐R populations is most likely due to different levels of inbreeding in the populations.     

Dominance of a single clonal lineage in the Phytophthora infestans population from northern Shaanxi,China revealed by genetic and phenotypic diversity analysis

Late blight caused by Phytophthora infestans is the most serious disease of potato worldwide. To understand the P. infestans population structure in northern Shaanxi, an emerging potato production region in China, 125 single‐lesion isolates were randomly collected from farmers' fields in 2009 and characterized phenotypically and genotypically. A mating type assay showed that 94 isolates were A1 mating type. Virulence determination of selected isolates on a set of differential potato lines containing R1 to R11, respectively, showed the presence of two pathotypes, of which the pathotype lacking avirulence genes Avr3, Avr4 and Avr10 was dominant. Isolates lacking all avirulence factors Avr1 to Avr11 were detected but at lower frequency (13·6%). Analysis for mtDNA haplotype showed all 61 examined isolates were IIa. A total of seven multilocus genotypes were distinguished among 125 isolates, as determined with seven polymorphic microsatellite markers. The genotype SG‐1 was dominant in the population with a frequency of 75·2% and was present throughout the region. Analysis of the phenotypic and genotypic structures of P. infestans populations indicated strict clonal reproduction of the pathogen and suggested that sexual reproduction probably does not occur. Potential implications for disease management are discussed.