Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.     

立枯丝核菌第一融合群的遗传分化*

利用12个随机引物对来自云南省不同地理环境的立枯丝核菌RhizoctoniasolaniKühn第一融合群(AG-1)的13个菌株进行遗传分化关系的研究。结果表明受试菌株被标记的DNA谱带多态性检测率为100%,即受试菌株间几乎无同质的DNA谱带。利用UPGMA法构建分子系统树分析发现,遗传距离0.5将其划分为9个遗传聚类组,遗传距离0.86将其划分为6个遗传聚类组,而遗传距离0.95可将其划分为3个遗传聚类组。结果表明受试AG1融合群的13个菌株具明显的遗传分化。     

Highly polymorphic microsatellite loci in the rice- and maize-infecting fungal pathogen Rhizoctonia solani anastomosis group 1 IA

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.     

Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.     

Genetic structure of populations of Rhizoctonia solani AG-3 on potato in eastern North Carolina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.     

山东省玉米纹枯菌融合群类型及遗传多样性

从山东省14个县市区采集的玉米纹枯病标本上分离获得103个玉米纹枯菌菌株。核荧光染色确定菌丝细胞核的数目,以及利用配对培养法确定不同菌株细胞是否融合。结果表明这些菌株分别属于多核丝核菌的AG-1-IA、AG-1-IB、AG-1-IC、AG-3、AG-4-HG-I、AG-5和WAG-Z融合群和双核丝核菌的AG-Ba融合群,其中AG-1-IA类型菌株数量占菌株总数的60.19%,为优势融合群。通过inter-simple sequence repeats（ISSR）标记技术进行菌株的遗传多样性分析,获得45个ISSR分子标记,其中91.1%的片段具有多态性,表明种群间存在丰富的遗传多样性。UPGMA聚类分析将103个菌株分成6个遗传聚类群,遗传聚类群的菌株组成说明遗传群组的划分与菌株的地理来源和菌株融合群类型均存在一定的相关性。     

Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA

A method based on restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA was developed for the rapid characterization of large populations of Rhizoctonia solani at the anastomosis group (AG) level. The restriction maps of the internal transcribed spacers (ITS) sequences were compared for 219 isolates of R. solani belonging to AG-1 to AG-12 and AG-BI, representing diverse geographic and host range origins. Four discriminant restriction enzymes (MseI, AvaII, HincII, and MunI) resolved 40 restriction fragment length polymorphism (RFLP) types among the 219 ITS sequences of R. solani. Each RFLP type could be assigned to a single AG except for two RFLP types, which were common to two AG. A fifth enzyme allowed the discrimination of AG-6 and AG-12. In addition, the combination of four enzymes allowed the discrimination of subsets within AG-1, AG-2, AG-3, and AG-4. The efficiency of the typing method was confirmed by analyzing PCR-amplified ITS sequences of 30 reference strains. Furthermore, the PCR-RFLP method was used to characterize at the AG level 307 isolates of R. solani originating from ten sugar beet fields exhibiting patches of diseased plants in France. The PCR-based procedure described in this paper provides a rapid method for AG typing in R. solani.     

High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG-1 IA (Thanatephorus cucumeris) from Texas

To date, much of the genetics of the basidiomycete Thanatephorus cucumeris (anamorph = Rhizoctonia solani) remains unknown. Here, we present a population genetics study using codominant markers to augment laboratory analyses. Seven single-copy nuclear RFLP markers were used to examine 182 isolates of Rhizoctonia solani AG-1 IA collected from six commercial rice fields in Texas. Thirty-six multilocus RFLP genotypes were identified. Population subdivision analyses indicated a high degree of gene flow/migration between the six geographic populations. Tests for Hardy-Weinberg equilibrium (HWE) among the 36 multilocus RFLP genotypes revealed that four of the seven loci did not significantly differ from HWE. Subsequent analysis demonstrated that departures from HWE at the three remaining loci were due to an excess of heterozygotes. Data presented here suggest that R. solani AG-1 IA is actively outbreeding (heterothallic). Possible explanations for heterozygote excess, which was observed at all seven RFLP loci, are discussed.     

21株马特组镰刀菌遗传多样性的ISSR分析

为明确马特组镰刀菌种间和种内的遗传差异与亲缘关系,本文利用ISSR分子标记技术对21个马特组菌株进行了遗传多样性分析.结果表明:利用筛选出的15条引物对3种供试菌株进行扩增,共扩增出239条条带,其中多态性条带230条,多态性位点比例为96.2％,平均每条引物产生条带数为15.3条.21个菌株间的遗传相似系数范围为0.494～0.933,平均为0.640.在遗传相似系数为0.593时,供试的21株马特组镰刀菌可明显分成2个ISSR类群(IG),IG-Ⅰ包括1～17号菌株,为Fusarium solani和F solani var.coeruleum;IG-Ⅱ包括18 ～21号菌株,全部为F.ventricosum.在遗传相似系数为0.933时,供试的21个菌株可被全部区分开.供试的镰刀菌基因组在SSR区域具有丰富的多态性;ISSR类群划分与菌种分类之间存在一定相关性,但与菌株的地理来源没有相关性;而同一类群中,不同菌株之间的遗传相似性与菌株的地理来源存在一定的相关性.同一地区同种寄主的相同菌种,其菌株间也存在一定的遗传差异.     

Genetic diversity of Rhizoctonia solani associated with potato tubers in France

The soilborne fungus Rhizoctonia solani is a pathogen of many plants and causes severe damage in crops around the world. Strains of R. solani from the anastomosis group (AG) 3 attack potatoes, leading to great yield losses and to the downgrading of production. The study of the genetic diversity of the strains of R. solani in France allows the structure of the populations to be determined and adapted control strategies against this pathogen to be established. The diversity of 73 French strains isolated from tubers grown in the main potato seed production areas and 31 strains isolated in nine other countries was assessed by phylogenetic analyses of (i) the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA), (ii) a part of the gene tef-1α and (iii) the total DNA fingerprints of each strain established by amplified fragment length polymorphism (AFLP). The determination of the AGs of R. solani based on the sequencing of the ITS region showed three different AGs among our collection (60 AG 3 PT, 8 AG 2-1 and 5 AG 5). Grouping of the strains belonging to the same AG was confirmed by sequencing of the gene tef-1α used for the first time to study the genetic diversity of R. solani. About 42% of ITS sequences and 72% of tef-1α sequences contained polymorphic sites, suggesting that the cells of R. solani strains contain several copies of ITS and the tef-1α gene within the same nucleus or between different nuclei. Phylogenetic trees showed a greater genetic diversity within AGs in tef-1α sequences than in ITS sequences. The AFLP analyses showed an even greater diversity among the strains demonstrating that the French strains of R. solani isolated from potatoes were not a clonal population. Moreover there was no relationship between the geographical origins of the strains or the variety from which they were isolated and their genetic diversity.     

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.     

古茶园、台地茶园遗传多样性的AFLP分析

采用AFLP-毛细管电泳法对云南省西双版纳地区4个有代表性的古茶园和 2个台地茶园(阿萨姆茶Camellia sinensis var. assamica)进行遗传多样性分析。研究表明: 阿萨姆茶变种水平的遗传多样性为: P = 92.31%, 期望杂合度He = 0.1366, Shannon多样性指数Ho = 0.2323; 古茶园居群水平是45.55％, 勐腊居群最高P = 59.11％, 勐宋居群变异度最低P = 36.44％; 而台地茶中, 有性系勐海大叶群体种P = 35.02%, 无性系云抗10号则非常低P = 13.77%, 台地茶居群水平是24.2%; 古茶园和台地茶遗传多样性相差很大, 依次是古茶园>有性系台地茶>无性系台地茶。研究还发现古茶园与台地茶园之间, 南糯山居群、勐腊易武居群与其他居群间存在多条特异谱带, 可作为南糯山居群和勐腊易武居群的分子指纹图谱, 应用于这两个居群所产晒青毛茶的鉴别。     

中间球海胆、光棘球海胆及杂交F1代（中间球海胆♀×光棘球海胆♂）群体遗传多样性AFLP分析

应用AFLP技术对中间球海胆、光棘球海胆及杂交F1代（中间球海胆♀×光棘球海胆♂）群体的遗传多样性进行了分析。结果表明,4对引物共扩增得到272个位点,其中269个多态位点,总的多态位点比例为98.89%。3个群体的香农多样性指数分别为：0.2331±0.1273、0.2005±0.1385和0.2625±0.1067。群体内遗传相似度分别为：0.6876±0.0523、0.6501±0.0548和0.6552±0.0553。分子方差分析（AMOVA）结果表明,变异来源有25.39%来自群体间,有74.61%来自群体内,群体内的遗传多样性比较丰富。尽管杂交海胆在表型上可以明显分成两种类型,但是通过AFLP统计的遗传距离进行的个体聚类却随机聚在一起,不能分成两个群体。     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

中国特有植物卧龙沙棘自然群体的RAPD分析

应用12个随机引物对卧龙沙棘全部2居群共28个个体进行了RAPD分析。结果表明,卧龙沙棘具有较丰富的遗传多样性,多态位点百分率为78.05%,Nei’s基因多样性h=0.2553,Shannon’s多态性信息指数Ι=0.3841。分布范围狭窄的卧龙沙棘在亚种水平的遗传多样性明显高于分布较广的中国沙棘。在居群水平上,卧龙沙棘同样具有很高的遗传多样性,平均多态位点百分率为63.42%,Nei’s基因多样性h=0.2193,Shannon’s多态性信息指数I=0.3287。卧龙沙棘的基因分化系数Gst=0.1425,表明遗传变异主要存在于居群内。AMOVA的结果也表明,在全部的遗传变异中,19.18%的遗传变异存在于居群之间,与Gst值基本一致。这一结果符合卧龙沙棘风媒、异交的繁育系统特点。     

瓜类枯萎病菌遗传多样性和亲缘关系的SRAP分析

尖孢镰刀菌可造成不同瓜类的枯萎病.为明确不同寄主、不同地区的瓜类枯萎病菌菌株间的遗传多样性及亲缘关系,采用相关序列扩增多态性(SRAP)分子标记技术,对来源于不同地区、不同寄主的95株尖孢镰刀菌的基因组DNA进行多态性扩增.以筛选出的19对引物共扩增出238条带,多态性比率为100%,平均每对引物扩增出12.5个位点和12.5个多态性位点;尖孢镰刀菌苦瓜专化型共扩增出166条带,其中145条为多态性条带,多态性比率为87.4%,平均每对引物扩增出8.7个位点和7.7个多态性位点,说明尖孢镰刀菌的遗传变异较为广泛.瓜类枯萎病菌株间的遗传相似系数范围为0.68～0.99,样品间的平均Nei遗传多样性指数和Shannon指数分别为0.2390和0.3718.在遗传相似系数为0.74时,可将供试的95株尖孢镰刀菌划分为苦瓜、黄瓜、西瓜、甜瓜4个专化群.在SRAP聚类树中,同一寄主的尖孢镰刀菌聚在一个分支上,其中尖孢镰刀菌苦瓜专化型菌株间的遗传相似系数范围为0.78～0.99,Nei遗传多样性指数为0.1811,平均Shannon指数为0.2750,表明尖孢镰刀菌苦瓜专化型的遗传变异较大,且菌株的聚群与地理来源存在相关性.     

Phylogeography of the Solanaceae-infecting Basidiomycota fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-3 based on sequence analysis of two nuclear DNA loci

Background  

The soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).     

Genetic diversity analysis by RAPD in Cathaya argyrophylla Chun et Kuang

Genetic diversity level of Cathaya argyrophylla was confirmed by random amplified polymorphic DNA (RAPD) markers. Seventy five samples (individuals), collected from Hunan and Sichuan provinces of China were used in the study. 21 10-mer oligonucleotide primers detected 106 sites, and 34 (32% ) of them were polymor-phic. The level of genetic variation in C. argyrophylla was lower than those of other conifers, and was considered to be associated with the complexity of habitats. The percentages of polymorphic sites (PPS) in the Hunan and Sichuan pop-ulations were 18% and 25% respectively. 7.99% of genetic variation existed between the two populations; this value was higher than the mean value (6.8%) among populations in conifers displayed by allozyme. Some subpopulations of C. argyrophylla were greatly differentiated because of site mutation and genetic drift. The highest value of genetic dif-ference between subpopulations amounted to 16. 23% . In addition, a concept of diversity coefficient(DC), a value us     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.