玉米根际球孢白僵菌群体遗传多样性的ISSR分析

为了明确玉米根际球孢白僵菌的遗传分化情况及亲缘关系,通过ISSR-PCR分子标记技术对分离自玉米根际的球孢白僵菌的遗传多样性进行了研究。从40个引物中共筛选出11个多态性高、稳定性好的引物用于正式的扩增分析,在37个菌株中共扩增出83条谱带,其中多态性条带占69条,多态性百分率为83.13%。平均每引物扩增条带在7.5条。群体的多态位点百分率(PPL)为83.13%,Nei基因多样性指数(H)为0.316 9,Shannon信息指数(I)为0.465 7。结果表明,分离自安徽省涡阳、萧县、蒙城三个地区的球孢白僵菌具有较高的遗传多样性。研究结果对进一步探讨玉米根际球孢白僵菌不同菌株的生防效果具有重要意义。     

Seed viability determination in parasitic broomrapes (Orobanche and Phelipanche) using fluorescein diacetate staining

Fluorescein diacetate (FDA) was investigated as a potential viability stain for seeds of holoparasitic broomrapes Orobanche and Phelipanche (Orobanchaceae), using four weedy and two non-weedy taxa. FDA viabilities were compared with the currently used 2,3,5-triphenyl tetrazolium chloride (TTC) test, and in vitro germination induced by the strigolactone analogue GR-24. Viable FDA-stained seeds were characterised by bright yellow–green fluorescence under 450–490 nm blue light. These viable seeds could be discriminated from non-viable seeds, in which low levels of fluorescence, similar to non-stained control seeds were observed. FDA viabilities were not significantly different from those obtained using the TTC stain; however, viability values for both stains were consistently higher than levels of artificially induced germination. Positive TTC-staining showed continuous variation that was difficult to interpret. Nevertheless, the TTC test does not involve grinding seeds, and therefore probably remains a practical alternative to FDA for screening soil samples contaminated with Orobanche and Phelipanche seeds, which indicates different applications for the two viability tests. Interestingly, the strigolactone analogue GR-24 only induced germination in O. crenata , P. ramosa and O. minor , suggesting a high specificity towards this germination inducer among the species investigated. We suggest that FDA-staining provides a potential alternative to the currently used TTC test for seed viability assays for Orobanche and Phelipanche , and other parasitic weeds which are an obstacle to crop cultivation.     

An investigation of genetic variation in Carthamus lanatus in New South Wales, Australia, using intersimple sequence repeats (ISSR) analysis

Summary Carthamus lanatus is a widespread and troublesome weed of crops and pastures in Australia. It is viewed as a target for both inundative and classical biological control. As fungal biological control agents are often specific in terms of their host range, the genetic variation within the host may be important in their successful deployment. The detection of genetic variation in populations of C. lanatus from New South Wales was studied using polymerase chain reaction (PCR)-based, intersimple sequence repeats (ISSR) analysis. DNA extracted from plant samples from 11 locations in New South Wales was compared with one sample from Western Australia and one accession of the closely related crop plant, safflower. The analysis indicated that there were two distinct groups of C. lanatus that correlated with location (northern and southern regions). Within-group genetic diversity, as determined by the Shannon genetic diversity index, was low at 0.33 in the southern group and 0.22 in the northern group. The presence of these groups may have implications for the biological control of C. lanatus using exotic fungi. In any screening of candidate agents, representatives of these populations should be included.     

Identification,characterisation and discriminatory power of microsatellite markers in the parasitic weed Orobanche cumana

Orobanche cumana is an obligate root parasite of sunflower. It represents a major agricultural problem in many countries of southern and eastern Europe. Information on O. cumana population genetics, structure and dynamics is scarce, particularly due to the lack of suitable molecular markers for such studies. The objective of this study was to identify and characterise simple sequence repeat (SSR) markers for O. cumana. Four thousand two hundred SSR‐containing candidate sequences were obtained from O. cumana using next‐generation sequencing, from which 298 SSR primer pairs were designed and 217 of them used for validation. Seventy nine SSR primers produced reproducible, high quality amplicons of the expected size that were polymorphic among 18 O. cumana populations from different geographical locations and hosts (sunflower, wild hosts from the Compositae family). The number of alleles per locus ranged from 2 to 10, with an average polymorphism information content value of 0.37. The O. cumana SSR markers were highly transferable to the closely related species Orobanche cernua. SSR markers showed high resolving power; UPGMA cluster analysis allowed proper classification of Orobanche spp. samples into species (O. cumana and O. cernua), geographical origin and host. The functional SSR markers reported in this study constitute a valuable tool for genetic analyses in O. cumana and related species and will contribute insights into the biology and genetics of this parasitic weed.     

Transformation of carrots with mutant acetolactate synthase for Orobanche (broomrape) control

Parasitic Orobanche spp are major constraints to vegetable crop production in the Mediterranean basin (to eastern Europe) and in localized places in India, China and the USA. Transgenic target-site herbicide resistance (eg, to acetolactate synthase inhibitors) allows for movement of unmetabolized herbicide through the crop to the photosynthate sink in the parasite, as well as through the soil. We report the successful engineering of a mutant acetolactate synthase (ALS) gene into carrot, allowing control of broomrape already in heterozygotes of the first back-crossed generation, by imazapyr, an imidazolinone ALS inhibitor. It is expected that homozygotes will have higher levels of resistance.     

Armillaria species on tea in Kenya identified using isozyme and DNA sequence comparisons

The aim of this study was to identify seven Armillaria isolates obtained from diseased tea bushes in Kenya using pectic enzyme profiles, PCR-RFLP and IGS-I DNA sequence data. The combination of these identification methods confirmed the presence of three distinct Armillaria groups. One of these groups resembled Zimbabwean group I ( A. fuscipes ). The second group was phylogenetically closely related to A. mellea ssp. nipponica . The third group was different from all other African isolates examined, but had isozyme patterns, especially of pectin methylesterases (PMEs), similar to those of isolates related to A. mellea ssp. nipponica. Analyses of sequence data suggested that this group is phylogenetically closely related to A. hinnulea from Australia and New Zealand.     

Orobanche crenata resistance and avoidance in pea (Pisum spp.) operate at different developmental stages of the parasite

Orobanche crenata (broomrape) is an important constraint to pea (Pisum sativum) cultivation in the Mediterranean area, because little resistance is available in commercial crop varieties. Field experiments have demonstrated that some resistance is present in a number of P. sativum and P. fulvum accessions. The goal of this work was to characterize such resistance. The Pisum–O. crenata interaction and the resistance symptoms were studied under controlled conditions by using Petri dish and polyethylene bag assays. The content of phenolics and peroxidase activity in host tissue from infected and non-infected plants were also measured. Resistance and avoidance mechanisms, acting at different developmental stages of the parasite, have been identified, including low stimulation of O. crenata seed germination, unsuccessful penetration of host roots, delay in post-attachment tubercle development and necrosis of the attached tubercles. Infection caused an increase in the content of total soluble phenolics in some Pisum genotypes. Peroxidase activity was higher in resistant than in susceptible accessions. Results obtained with different Pisum genotypes showed that resistance is the result of several mechanisms acting at different stages of the infection process. Resistance is also related to increased levels of peroxidase activity in host roots.     

Analysis of population structure of Rosellinia necatrix on Cyperus esculentus by mycelial compatibility and inter-simple sequence repeats (ISSR)

Genetic diversity was studied in a population of 24 isolates of Rosellinia necatrix obtained from Cyperus esculentus and 16 from other hosts by means of mycelial compatibility groups (MCGs) and inter-simple sequence repeat (ISSR) techniques. All isolates obtained from C. esculentus belonged to a unique MCG, irrespective of their geographical origin or year of isolation, and were incompatible with the isolates from other hosts. ISSR analysis gave results which were in agreement with the MCG determination; selected ISSR primers grouped a subset of eight representative isolates from C. esculentus in a unique cluster. The homogeneity found within the population of R. necatrix from C. esculentus in Valencia province could suggest that the pathogen was introduced recently and has spread further via infected tubers inadvertently used as propagating material, as well as by cull tubers swept away by irrigation water.     

内蒙古亚洲小车蝗种群遗传多样性和遗传分化的ISSR分析

亚洲小车蝗Oedaleus asiaticus Bei-Bienko是我国北方草原和农牧交错区的主要害虫。为评价内蒙古地区亚洲小车蝗种群的遗传多样性和遗传分化,应用ISSR标记方法对内蒙古15个亚洲小车蝗种群遗传多样性及遗传分化进行了分析。结果表明,7条引物扩增出85条ISSR条带,均为多态性条带。多态性比例（P）、Nei''s遗传多样性指数（H）和香农多样性指数（I）分别为82.59%、0.2319和0.3421,表明亚洲小车蝗种群具有较高的遗传多样性。基因流（Nm）和基因分化系数（Gst）分别为1.2298和0.3352,表明亚洲小车蝗不同地理种群具有明显的遗传分化。遗传距离与地理距离呈极显著正相关关系。表明地理距离和地形差异可能是形成亚洲小车蝗种群遗传分化的主要原因。     

An assessment of genetic diversity within and between populations of Phalaris minor using ISSR markers

Intra- and inter-sample similarities for four populations of the annual grass weed Phalaris minor from Haryana state, India, were examined using inter-simple sequence repeat (ISSR) DNA markers. Levels of polymorphism within and between populations were low in comparison with values reported from other grassy weed species. Analysis of inter-population similarities allowed a partial differentiation of the four populations and of pairs of populations classified by cropping system. Analysis of the intra-population similarity data showed a weak but consistent and statistically significant negative correlation between the molecular similarity of seedlings and the physical distance between their mother plants over distances up to 40 m (the maximum separation tested) in all four populations. The consistency of the observed relationship between molecular similarity and physical separation, and the differences in cultivation practices at the four sites, suggested that the relationship may be a result of localized out-crossing, rather than an effect of localized seed rain. The results of the analyses are discussed in relation to the potential for evolution of multiple traits in the weed in response to changes in the wheat production system in the region.     

Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain

Loquat (Eriobotrya japonica) is an important subtropical fruit crop in Spain and other Mediterranean countries. In recent years, characteristic symptoms of branch canker and dieback have been observed in the main cultivated areas of loquat in Spain. The goal of this study was to identify and characterize the species of Botryosphaeriaceae associated with these symptoms. For this, 36 affected orchards were surveyed between 2010 and 2011 in six provinces of southeastern Spain. Eighty‐four isolates belonging to the family Botryosphaeriaceae were recovered from samples with symptoms. These isolates were characterized by means of phenotypical studies, DNA sequence analyses of the internal transcribed spacer (ITS) and part of the translation elongation factor 1‐α regions, and pathogenicity tests. Ten fungal species were identified: Diplodia malorum, Diplodia olivarum, Diplodia seriata, Diplodia pseudoseriata/Diplodia alatafructa, Diplodia sp., Dothiorella sarmentorum, Neofusicoccum mediterraneum, N. parvum, Spencermartinsia plurivora and S. viticola. In addition, Diplodia eriobotryicola and Dothiorella eriobotryae are newly described. The most frequent species isolated from cankers was D. seriata and, as far as is known, this is the first report of D. malorum, and species belonging to the complex D. pseudoseriata/D. alatafructa, in Spain. All species were pathogenic to 1‐year‐old loquat plants cv. Algerie, with Diplodia sp. and S. viticola as the most virulent.     

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus , P. goodeyi , P. penetrans , P. scribneri , P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis . A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.     

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.     

瘿蚊科3种昆虫核糖体DNA的克隆及序列分析

瘿蚊科是双翅目昆虫中的重要类群?本研究对瘿蚊科3种昆虫—麦红吸浆虫?菊花瘿蚊和食蚜瘿蚊的核糖体DNA进行PCR扩增?克隆?测序及序列分析, 并对ITS-1在麦红吸浆虫4个地理种群中的遗传变异情况进行分析?结果表明, 从3种昆虫中获得的核糖体DNA序列包括:部分的18S rDNA(44 bp)?28S rDNA(37 bp), 全部的ITS-1(487~535 bp), 5.8S rDNA(121 bp)及ITS-2(336~352 bp)序列?3种昆虫ITS序列的碱基差异百分比在17.21%~29.59%之间, 共含有206个变异位点?ITS-1序列在麦红吸浆虫4个地理种群中比较保守, 只有4个变异位点, 单倍型多样性为0.311 7~0.796 5, 核苷酸多样性为0.000 6~0.002 2?本研究为今后瘿蚊科昆虫的分类鉴定?系统发育和遗传进化等相关研究提供了基础?     

Genetic diversity of Pueraria lobata (kudzu) and closely related taxa as revealed by inter-simple sequence repeat analysis

Kudzu (Pueraria lobata) is a noxious weed infesting some areas of the USA. Knowledge of its genetic variation in both native and invasive areas can lead to effective biological control measures. Inter-simple sequence repeat (ISSR) variations were studied in P. lobata and its four closely related congeneric species (P. edulis, P. montana, P. phaseoloides and P. thomsoni). ISSR results allowed a clear separation of these five species. For P. lobata, 108 plants from China and USA were analysed. The samples from the US were genetically closer to Chinese P. lobata populations than to other congeneric populations. High genetic differentiation was found for P. lobata, P. montana and P. thomsoni in Chinese samples. High genetic diversity and low population differentiation was found in P. lobata samples of the US. This supports the hypothesis of multiple introductions into the USA from different sources in Japan or China, followed by subsequent gene exchange and recombination.     

ITS—RFLP分析进行昆虫病原线虫斯氏属和异小杆属的分类鉴定

本研究通过PCR扩增和六种限制性内切酶（AluⅠ，HinfⅠ，MboⅠ，RsaⅠ，HaeⅢ和PvuⅡ）酶切，对国内害虫防治上常用的几种昆虫病原线虫，包括斯氏属S.car-pocapsae,S.feltiae和S.glaseri以及异小杆属H.bacteriophora,H.zealandica,H.indicus和H.megidis等8个品系rDNA-ITS进行分析。建立起可以区分各线虫种的标准RFLP图谱。该方法快速简便，稳定可靠，需要的样品量少。可以用于新鲜的，或冻存的样品，甚至分析单条的线虫，不仅可进行昆虫病原线虫的快速分类鉴定。而且进一步可以应用于线虫田间释放的辅助监测。实际田间感染率的测定和线虫毒力的比较。     

Study of Resistance to Orobanche ramosa in Host (Oilseed Rape and Carrot) and Non-host (Maize) Plants

Orobanche ramosa is a parasitic Angiosperm responsible for severe yield losses in several economical crops. It is a serious threat in oilseed rape in France and Morocco and is appearing in carrot crops in Morocco. In this study, several varieties of oilseed rape and carrot were screened in order to identify resistant cultivars and to characterize the resistance mechanisms involved. All the 15 oilseed rape varieties tested were susceptible. In carrot, the varieties 'Colmar à coeur rouge' and 'Nantaise demi-longue' were susceptible, whereas 'Palaiseau' and 'Buror' were resistant. In the susceptible 'Colmar à coeur rouge' carrot no defence reactions were found and the development of the parasite inhibited carrot tap root formation. In the resistant carrot varieties, the parasite germinated, became attached to the host root but became necrotic before emergence. In 'Buror' carrot, formation of a mechanical barrier was associated with the restriction to the cortex of the parasite. In maize cv. 'Vigni', a non-host of O. ramosa, thickening of xylem vessels, cell divisions in the central cylinder and formation of an encapsulation layer were observed in association with restricted development of Orobanche haustoria.