Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

Amino acid substitution in the coat protein of Melon necrotic spot virus causes loss of binding to the surface of Olpidium bornovanus zoospores

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidium bornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.     

New severe strains of Melon necrotic spot virus: symptomatology and sequencing

New strains of Melon necrotic spot virus (MNSV), designated MNSV-YS and MNSV-KS, caused much more severe growth retardation on melon plants than MNSV-NH, which was previously reported as the most severe strain of MNSV in Japan. MNSV-YS spread much more quickly than MNSV-NH in infected plants, and induced more severe growth retardation, even though the appearance of necrotic lesions on inoculated cotyledons was much slower. MNSV-KS had properties intermediate between those of the other two strains. The results suggest that faster-spreading strains can multiply more rapidly as a result of lower levels of activity in inducing necrotic lesions in melon plants. The complete sequences of MNSV-YS and MNSV-KS were determined, and an RT–PCR–RFLP method based on these sequences was successfully developed to detect and discriminate between the three strains.     

来源于新疆的3 个苹果褪绿叶斑病毒分离物的分子变异研究

Apple chlorotic leaf spot virus (ACLSV) isolates from Korla pear (KI-2), New pear no. 7 (XI-1) and Red Fuji apple (API-4) were collected from XinJiang and characterized by analyzing sequences of their near genomic 3忆-terminal. The RT-PCR products were cloned, and analyzed by single-strand conforma-tion polymorphism (SSCP). Eight out of 39 collected positive clones showing different SSCP patterns were sequenced. The results showed that the amplified products had sizes ranging 676 - 703 bp, including partial coat protein (CP) gene (506 bp, accounts for 87% of the complete cp gene) and 3忆-terminal non-coding re-gion (3忆NCR) sequences. The cp gene sequences from isolate KI-2 showed a high intra-isolate divergence,with 84. 8% - 85. 4% identities at the nucleotide (nt) level, and the intra-isolate identities were 99. 8 % and 92.5% - 99. 8 % for isolate XI-1 and API-4, respectively. Phylogenetic analysis on the nt sequences of cpgene showed that the analyzed ACLSV variants from three isolates fell into two different clusters. A variant KI-2-6 from KI-2 was clustered into a group with an apple isolate aclsv-c from China and a plum isolated from France, and all other variants fell into a large cluster. The 3忆NCR sequences of these variants were identical ranging 80. 6% - 100 % .     

The coat proteins and putative movement proteins of isolates of Prunus necrotic ringspot virus from different host species and geographic origins are extensively conserved

A complete sequence for the RNA 3 of Prunus necrotic ringspot virus (PNRSV) is described (Genbank Accession U57046). Primers from this sequence were used to amplify both the movement protein and coat protein genes of 3 other isolates of PNRSV originating from different host species and geographic locations. Comparisons of these sequences with those of other published sequences for PNRSV and the closely related apple mosaic virus (ApMV) showed that both the movement proteins and coat proteins of isolates of PNRSV are extensively conserved irrespective of either the original host or the geographic origin. The movement protein and coat protein of ApMV and PNRSV are sufficiently conserved to suggest that these two viruses may have evolved from a common ancestor. The amino acid sequence of the two coat proteins shows areas of similarity and difference that would explain the serological continuum reported to occur among isolates of these two viruses. Nevertheless, the movement protein and coat protein of the two viruses are sufficiently different so that ApMV and PNRSV should be considered to be distinct viruses.     

Genetic diversity of Grapevine rupestris stem pitting-associated virus isolates from Tunisian grapevine germplasm

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA-dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.     

中国不同地区亚洲韧皮杆菌遗传多样性分析

 柑桔黄龙病是最严重的柑桔病害之一,现已威胁世界柑桔产业。已被证实在中国南部该病害通过嫁接和木虱传播。因此黄龙病的流行病学研究就显得尤为重要。本研究中,基于外膜蛋白(omp)基因的PCR-RFLP的方法被用来研究中国南部各柑桔主产区的亚洲韧皮部杆菌的遗传多样性。研究采用不同的限制性内切酶消化各地区的亚洲韧皮部杆菌的omp基因产生了不同的RFLP指纹图谱,并对各地亚洲韧皮部杆菌的omp基因进行克隆测序然后构建系统发育树。结果显示:中国不同地理区域的亚洲韧皮部杆菌具有一定的遗传多样性,即便在某一特定的地区也有一定的差异。序列分析显示中国地区的亚洲韧皮部杆菌有很高的同源性。这些结果对揭示中国柑桔黄龙病的流行病学及制定科学有效的防治策略具有一定的指导意义。     

Genetic structure and diversity of Phakopsora pachyrhizi isolates from soyabean

Simple sequence repeat (SSR) markers were used to classify 116 isolates of Phakopsora pachyrhizi, the cause of soyabean rust, collected from infected soyabean leaves in four agroecological zones in Nigeria. A high degree of genetic variation was observed within the sampled populations of P. pachyrhizi. Eighty‐four distinct genotypes were identified among three of the four agroecological zones. Nei’s average genetic diversity across geographical regions was 0·22. Hierarchical analysis of molecular variance showed low genetic differentiation among all populations of P. pachyrhizi. The majority (> 90%) of the genetic diversity was distributed within each soyabean field, while approximately 6% of the genetic diversity was distributed among fields within geographic regions. Low population differentiation was indicated by the low F_ST values among populations, suggesting a wide dispersal of identical genotypes on a regional scale. Phylogenetic analysis indicated a strictly clonal structure of the populations and five main groups were observed, with group II accounting for 30% of the entire population. Because of the asexual reproduction of P. pachyrhizi, single‐step mutations in SSR genotypes are likely to account for the genetic differences within each group.     

基于外壳蛋白基因的湖南烟草黄瓜花叶病毒遗传多样性分析

为探明湖南烟草上发生的黄瓜花叶病毒Cucumber mosaic virus(CMV)的遗传多样性及分子进化特征,对来自湖南烟区的303份疑似感染病毒的烟草样品进行检测,分析CMV系统发育、遗传变异和群体结构等特征。结果表明：部分分离物的外壳蛋白(coat protein, CP)基因与NCBI上登录的CMV分离物的一致性为86.34%～98.42%;系统发育分析发现湖南烟草CMV分离物属ⅠB组,不同组间的分离物地理特征不明显,无重组现象,进化的主要驱动力是负选择;组间遗传变异比较明显,基因交流频率较低,受到遗传漂变影响,遗传多样性高,群体趋于扩张。研究结果为烟草抗CMV育种提供了理论依据,对病害防治具有重要意义。     

四川省不同地域的核盘菌遗传多样性分析

 核盘菌(Sclerotinia sclerotiorum)属于世界性分布的植物病原真菌,可以危害油菜等多种经济作物。研究不同地域核盘菌的遗传多样性对了解核盘菌的遗传演化过程和指导病害防控具有重要意义。实验采用序列相关扩增多态性(sequence-related amplified polymorphism,SRAP)标记对四川省17个不同地理来源的66株核盘菌菌株的遗传多样性进行了分析。10对检测引物共获得129个位点,其中123个为多态位点,占95.35%。UPGMA聚类结果显示,在相似性系数为0.7时,66个核盘菌菌株分为5类(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ),分别包含60、2、2、1和1个菌株。在相似性系数为0.74时,第Ⅰ类又可分为3个亚类(Ⅰ-1、Ⅰ-2、Ⅰ-3),分别包含21、37和2个菌株。聚类及组成分分析结果显示,四川省各地区的核盘菌菌株具有较高的遗传多样性,但其遗传变异与菌株地理来源无明显相关性。     

Genetic diversity in Echinochloa spp. collected from different geographic origins and within rice fields in Côte d'Ivoire

Summary Diversity studies of Echinochloa spp. are complicated by problems in taxonomy and species identification, caused by the existence of morphologically intergrading types. Six amplified fragment length polymorphism (AFLP) primer combinations and five microsatellites were used to assess variation in 24 samples morphologically identified as E. crus-galli , E. colona and E. crus-pavonis , from Bangladesh, India, Colombia, Costa Rica, Côte d'Ivoire and Philippines. Out of 909 AFLP bands generated, 775 were polymorphic. Genotype diversity for the microsatellites ranged from 0.28 to 0.72. Similarity matrices were calculated using Jaccard coefficient, and input into cluster and principal coordinates analyses. AFLP and microsatellite results were highly correlated. Echinochloa crus-pavonis and E. crus-galli were intermixed, consistent with the view that E. crus-galli occurs as numerous intergrading races in the four countries (Bangladesh, India, Côte d'Ivoire and Philippines). The E. colona samples clustered as a distinct group. In 15 samples of E. crus-pavonis collected from rice fields in a valley in Côte d'Ivoire (over a 2-km distance), four different genotypes were found in a 4 m × 4 m area. These results suggest that AFLPs and SSRs may be useful not only for discriminating genotypes and studying population structure but also for helping to resolve taxonomic relationships in Echinochloa spp.     

福建省丙环唑不同敏感性玉米小斑病菌的遗传多样性和致病性

 利用菌丝生长法、ISSR分子标记和分生孢子悬浮液喷雾接种法,探讨了福建省玉米小斑病菌对丙环唑的敏感性以及不同敏感性病菌群体的遗传多样性和致病性。敏感性测定结果表明,福建省玉米小斑病菌对丙环唑产生了抗药性,抗药性菌株的抗性倍数达到2.1~9.4倍。筛选获得的10条ISSR引物对55个菌株共检测出153个位点,其中多态性位点百分比高达93.46%。在敏感型、中间型和抗药型群体中多态性位点百分比分别为77.12%、69.93%和81.70%,在抗药型群体中,等位基因观测值、等位基因有效值、Nei’s遗传多样性指数和Shannon’s信息指数均高于敏感型群体,表明玉米小斑病菌抗药型群体的遗传多样性最丰富。聚类分析结果表明,病菌群体遗传多样性与抗药性水平和地理来源均有较高的相关性。致病性测定表明,丙环唑不同敏感性病菌群体对11个鲜食玉米品种均具有较强的致病性,但是,在9个玉米品种上,敏感型群体中强致病力菌株出现频率明显低于抗药型群体。研究结果为深入研究玉米小斑病菌群体遗传结构及其田间抗药性监测提供了理论基础。     

Sequence analyses of Wheat dwarf virus isolates from different hosts reveal low genetic diversity within the wheat strain

Wheat dwarf virus (WDV) causes disease in wheat (Triticum aestivum) and barley (Hordeum vulgare) in many parts of Europe. The host range also includes many species of the family Poaceae. WDV is only transmitted by the leafhopper Psammotettix alienus. During a five‐year period (2001–2005), grass samples were collected in central Sweden in the vicinity of fields with WDV‐infected winter wheat. Screening with ELISA and PCR identified WDV in a low number of samples (8/1098) from only three grass species: Apera spica‐venti, Avena fatua and Poa pratensis. In addition, triticale was found to be positive. Fourteen WDV isolates from Avena fatua, Apera spica‐venti, Triticum aestivum, Lolium multiflorum, Poa pratensis, triticale and the insect vector Psammotettix alienus, were partially sequenced (ca. 1200 nucleotides), providing the first published WDV sequences from the insect vector. All isolates belonged to the wheat strain of WDV and the genetic diversity was low. Phylogenetic analyses showed no clear grouping according to geographical location or host species. The results suggest that the same WDV genotypes are infecting both wheat and grasses in Sweden. Interestingly, one group of isolates (subtype B) formed a distinct clade in the phylogenetic tree. Subtype B was always found in mixed infection with the main genotype. Complete sequencing of a subtype B isolate showed that it was 98·6% identical to a typical wheat isolate from the same plant.     

山西苹果褪绿叶斑病毒的分布及遗传多样性分析

苹果褪绿叶斑病毒Apple chlorotic leaf spot virus (ACLSV) 是侵染苹果的主要潜隐性病毒之一, 在我国苹果植株上发生普遍, 严重威胁我国苹果的品质与产量。本研究从山西省12个苹果主产区随机采集360份表现褪绿和斑驳等症状的苹果叶片作为研究样本, 通过RT-PCR检测, 360份样本中有209份样本为ACLSV阳性, 对209份阳性样本的外壳蛋白(coat protein, CP)基因进行分离、测序、克隆, 得到12个新的ACLSV分离物(分别命名为 Shanxi 1~Shanxi 12)。选择17个来自不同国家的分离物与12个新的ACLSV分离物在核苷酸和氨基酸层面上进行序列一致性和系统发育分析。结果显示, 29个ACLSV分离物被划分为2个不同进化群体。进一步对2个不同ACLSV群体进行选择压分析和中性检验, 结果表明, 组Ⅰ与组Ⅱ的ACLSV群体之间存在明显的遗传差异, 其中负向选择可能是ACLSV遗传变异的原因之一。本研究较全面地分析了ACLSV的发生、危害, 并对山西苹果的ACLSV分离物进行了遗传结构分析, 为山西苹果褪绿叶斑病毒病的防治提供了理论指导。     

不同来源的柑橘衰退病毒分离物基因组5'端A、F变异区序列克隆及分析

 柑橘衰退病毒(Citrus tristeza virus,CTV)组群自然条件下存在株系分化现象。本研究利用RT-PCR技术扩增、克隆了来自我国不同地区的21个柑橘衰退病毒分离物的5'端A、F变异区。通过分析发现,不同来源的各分离物在5'端A、F区存在较大的变异。21个分离物A区序列相似性最低为85.8%,最高可达99.8%,平均为95.9%;与GenBank中9个代表性株系的平均相似性为84.2%。F区序列相似性较A区高,为98.0%;相似性最低为94.3%,最高达99.1%。结果显示不同来源的CTV分离物5'端序列A、F区变异较大。     

Genetic and morphologic diversity of <Emphasis Type="Italic">Echinochloa crus-galli</Emphasis> populations from different origins

Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass) is an annual weed that is native to Asia and found throughout the world. The broad ecological tolerance and competitive ability of E. crus-galli makes it the most important weed species in rice. Genetic studies of plants are becoming increasingly common because reliable information is necessary to better understand population dynamics, occurrence of herbicide resistance, and demographic data. Echinochloa crus-galli populations from 34 different locations in Turkey were compared with respect to morphological differences and genetic variation. For morphological variation, five seeds of each population were sown in pots and grown in a screenhouse using a randomized block design. Morphological parameters such as germination speed, flowering time, leaf area, plant height, spikelet length, above-ground biomass, root dry weight and number of seeds were measured. Distinct differences among populations with respect to hierarchical cluster analysis were observed. Genetic variations among populations were performed using random amplified polymorphic DNA (RAPD) markers. The seven RAPD primers amplified 55 bands whose molecular weight varied between 200 and 4000 bp. The percentage of polymorphic bands was 74.54%. Results showed high morphological and genetic variability among individual genotypes within geographic locations. Phenotypic and genetic variability among E. crus-galli populations would be influenced by agricultural practices, crop characteristics, geographic location and herbicide pressure. Differences between weed populations may affect response to chemical or biological control.     

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.     

Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.     

Molecular,morphological and pathogenic diversity of Sclerotinia sclerotiorum isolates from common bean (Phaseolus vulgaris) fields in Argentina

White mould, caused by Sclerotinia sclerotiorum, is one of the most threatening fungal diseases occurring across major bean production regions worldwide. In Argentina, under favourable weather conditions, up to 100% seed yield losses occur on susceptible common bean cultivars. The aim of this study was to characterize the diversity of S. sclerotiorum isolates from six dry bean fields in the main production area of Argentina by means of molecular, morphological (mycelium colour, number and pattern of sclerotia distribution) and pathogenic approaches. Among 116 isolates analysed, high genotypic and morphological variability was observed. A total of 52 mycelial compatibility groups (MCGs) and 59 URPs (universal rice primers) molecular haplotypes were found. All the MCGs were location specific, while only 12% of the URP haplotypes were shared among locations. The molecular analysis of variance revealed a significant differentiation among populations, with higher genetic variability within the populations analysed than among them. The aggressiveness of the isolates towards bean seedlings was assessed in the greenhouse. Most of the isolates were highly aggressive, while no variation among locations was observed. The information generated in the present study provides, for the first time, information on the variability of S. sclerotiorum associated with white mould in the main common bean production area in Argentina. In addition, the findings suggest the occurrence of both clonal and sexual reproduction in the populations analysed. This work contributes to the development of sustainable management strategies in bean production aimed to minimize yield losses due to white mould.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.