First Report of Charcoal Rot Caused by Macrophomina phaseolina in Sunflower in Turkey.

Charcoal rot symptoms were observed on 2-month-old oilseed sunflower plants (Helianthus annuus L.) in the Eskisehir Province of Turkey in June 2009. The disease was observed in 70% of the fields surveyed and incidence ranged from 10 to 50%. Symptoms were first observed in plants approaching physiological maturity and consisted of silver-gray lesions girdling the stem at the soil line, reduced head diameter compared with noninfected plants, and premature plant death. Pith in the lower stem was completely absent or compressed into horizontal layers. Black, spherical microsclerotia were observed in the pith area of the lower stem, underneath the epidermis, and on the exterior of the taproot. The internal stem had a shredded appearance. Later, the vascular bundles became covered with small, black flecks or microsclerotia of the fungus. Forty plant samples were collected from 10 fields. After surface sterilization with 2% NaOCl, outer tissues sampled from diseased tissues (2 to 3 mm long) of root and stems were removed and transferred to potato dextrose agar containing 250 mg liter-1 of chloramphenicol. Petri plates were incubated for 7 days at 26 ± 2°C in the dark. Ninety-eight percent of the fungal colonies were identified as Macrophomina phaseolina (Tassi) Goidanich based on gray colony color, colony morphology, and the size of the microsclerotia, which ranged from 80 to 90 µm in diameter, from both infected sunflowers and compared with pure cultures (3). All resulting cultures produced abundant microsclerotia. The only other sunflower pathogen known to form microsclerotia is Verticillium dahliae Kleb., whose microsclerotia are irregular in shape and 15 to 50 µm in diameter. Sequence-related amplified polymorphisms technique was used for diversity of M. phaseolina since it has proven to be more informative than amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat (2). Results showed a high level of genetic diversity (60%) among the 26 isolates of M. phaseolina. Sequencing of the internal transcribed spacer region (1) showed high homology (>96%) to M. phaseolina (GenBank Accession No. HQ380051). Pathogenicity tests for 20 isolates of M. phaseolina were carried out on three commercially used cultivars, SANAY, TUNCA, and TR-3080. Groups of 10 seedlings were grown separately in an autoclaved peat/soil mixture in 30-cm-diameter plastic pots in a greenhouse at 30 ± 2°C. Soil infestation was performed 1 day before sowing. Two-week-old cultures on barley medium (4) were blended in distilled sterile water and adjusted to 105 sclerotia ml-1. Each pot received 250 ml of inoculant. Each treatment had three replications. Three pots for each cultivar were left uninoculated. Within 3 weeks, five to seven inoculated plants in each pot died. Identical disease symptoms were observed 30 days after inoculation; on the control plants no symptoms were observed. Microsclerotia were produced after 7 weeks at the stem base on 85% of the surviving plants. To our knowledge, this is the first report of M. phaseolina in sunflower in Turkey. References: (1) B. D. Babu et al. J. Plant Dis. Prot. 96:797, 2007. (2) H. Budak et al. Theor. Appl. Genet. 109:280, 2004. (3) P. Holliday and E. Punithalingam. No. 275 in: Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (4) M. R. Omar et al. J. Plant Dis. Prot. 114:196, 2007.

Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines.

Brachypodium distachyon (brachypodium) is a small grass with the biological and genomic attributes necessary to serve as a model system for all grasses including small grains and grasses being developed as energy crops (e.g., switchgrass and Miscanthus). To add natural variation to the toolkit available to plant biologists using brachypodium as a model system, it is imperative to establish extensive, well-characterized germplasm collections. The objectives of this study were to collect brachypodium accessions from throughout Turkey and then characterize the molecular (nuclear and organelle genome), morphological, and cytological variation within the collection. We collected 164 lines from 45 diverse geographic regions of Turkey and created 146 inbred lines. The majority of this material (116 of 146 inbred lines) was diploid. The similarity matrix for the diploid lines based on AFLP analysis indicated extensive diversity, with genetic distances ranging from 0.05 to 0.78. Organelle genome diversity, on the other hand, was low both among and within the lines used in this study. The geographic distribution of genotypes was not significantly correlated with either nuclear or organelle genome variation for the genotypes studied. Phenotypic characterization of the lines showed extensive variation in flowering time (7-22 weeks), seed production (4-193 seeds/plant), and biomass (15-77 g). Chromosome morphology of the collected brachypodium accessions varied from submetacentric to metacentric, except for chromosome 5, which was acrocentric. The diverse brachypodium lines developed in this study will allow experimental approaches dependent upon natural variation to be applied to this new model grass. These results will also help efforts to have a better understanding of complex large genomes (i.e., wheat, barley, and switchgrass).

Organellar genome analysis of rye (Secale cereale) representing diverse geographic regions.

Rye (Secale cereale) is an important diploid (2n = 14, RR) crop species of the Triticeae and a better understanding of its organellar genome variation can aid in its improvement. Previous genetic analyses of rye focused on the nuclear genome. In the present study, the objective was to investigate the organellar genome diversity and relationships of 96 accessions representing diverse geographic regions using chloroplast (cp) and mitochondrial (mt) DNA PCR-RFLPs. Seven cpDNA and 4 mtDNA coding and noncoding regions were amplified using universal cpDNA and mtDNA primer pairs. Each amplified fragment was digested with 13 different restriction enzymes. mtDNA analysis indicated that the number of polymorphic loci (20) was low and genetic differentiation (GST) was 0.60, excluding the outgroups (hexaploid wheat, Triticum aestivum, 2n = 6x = 42, AABBDD; triticale, xTriticosecale Wittmack, 2n = 6x = 42, AABBRR). cpDNA analysis revealed a low level of polymorphism (40%) among the accessions, and GST was 0.39. Of the 96 genotypes studied, 70 could not be differentiated using cpDNA PCR-RFLPs even though they are from different geographic regions. This is most likely due to germplasm exchange, indicating that genotypes might have a common genetic background. Two cpDNA and 3 mtDNA fragments were significantly correlated to the site of germplasm collection. However, there was no clear trend. These results indicate that the level of organellar polymorphism is low among the cultivated rye genotypes. The cpDNA and mtDNA PCR-RFLP markers used in the present study could be used as molecular markers in rye genetics and breeding programs.

Understanding ploidy complex and geographic origin of the Buchloe dactyloides genome using cytoplasmic and nuclear marker systems.

Characterizing and inferring the buffalograss [Buchloe dactyloides (Nutt.) Engelm.] genome organization and its relationship to geographic distribution are among the purposes of the buffalograss breeding and genetics program. This buffalograss study was initiated to: (1) better understand the buffalograss ploidy complex using various marker systems representing nuclear and organelle genomes; (2) determine whether the geographic distribution was related to nuclear and organelle genome variation; and (3) compare the genetic structure of accessions with different ploidy levels. The 20 buffalograss genotypes (15 individuals from each genotype) that were studied included diploid, tetraploid, pentaploid, and hexaploid using nuclear (intersimple sequence repeat (ISSRs), simple sequence repeat (SSRs), sequence related amplified polymorphism (SRAPs), and random amplified polymorphic DNA (RAPDs)) and cytoplasmic markers (mtDNA and cpDNA). There was a significant correlation between the ploidy levels and number of alleles detected using nuclear DNA (ISSR, SSR, and SRAP, r = 0.39, 0.39, and 0.41, P<0.05, respectively), but no significant correlation was detected when mitochondrial (r = 0.17, P<0.05) and chloroplast (r = 0.11, P < 0.05) DNA data sets were used. The geographic distribution of buffalograss was not correlated with nuclear and organelle genome variation for the genotypes studied. Among the total populations sampled, regression analysis indicated that geographic distance could not explain genetic differences between accessions. However, genetic distances of those populations from the southern portion of buffalograss adaptation were significantly correlated with geographic distance (r= 0.48, P<0.05). This result supports the hypothesis that genetic relationship among buffalograss populations cannot be estimated based only on geographic proximity.

The use of microsatellite markers for the detection of genetic similarity among winter bread wheat lines for chromosome 3A.

Previous studies with chromosome substitution and recombinant inbred chromosome lines identified that chromosome 3A of wheat cv. Wichita contains alleles that influence grain yield, yield components and agronomic performance traits relative to alleles on chromosome 3A of Cheyenne, a cultivar believed to be the founder parent of many Nebraska developed cultivars. This study was carried out to examine the genetic similarity among wheat cultivars based on the variation in chromosome 3A. Forty-eight cultivars, two promising lines and four substitution lines (in duplicate) were included in the study. Thirty-six chromosome 3A-specific and 12 group-3 barley simple sequence repeat (SSR) primer pairs were used. A total of 106 polymorphic bands were scored. Transferability of barley microsatellite markers to wheat was 73%. The coefficient of genetic distance (D) among the genotypes ranged from 0.40 to 0.91 and averaged D=0.66. Cluster analysis by the unweighted pair-group method with arithmetic averages showed one large and one small cluster with eight minor clusters in the large cluster. Several known pedigree relationships largely corresponded with the results of SSR clusters and principal coordinate analysis. Cluster analysis was also carried out by using 22 alleles that separate Wichita 3A from Cheyenne 3A, and three clusters were identified (a small cluster related to Cheyenne of mainly western Nebraska wheat cultivars; a larger, intermediate cluster with many modern Nebraska wheat cultivars; a large cluster related to Wichita with many modern high-yielding or Kansas wheat cultivars). Using three SSR markers that identify known agronomically important quantitative trait loci (QTL) regions, we again separated the cultivars into three main clusters that were related to Cheyenne or Wichita, or had a different 3A lineage. These results suggest that SSR markers linked to agronomically important QTLs are a valuable asset for estimating both genetic similarity for chromosome 3A and how the chromosome has been used in cultivar improvement.

Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs.

Buffalograss [ Buchloe dactyloides (Nutt.) Englem.] is the only native grass that is being used extensively as a turfgrass in the Great Plains region. Its low-growth habit, drought resistance, and low-maintenance requirement make it attractive as a turfgrass species. Our objective was to obtain an overview on the genetic relatedness among and within seeded and vegetative biotype buffalograsses using inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPDs), sequence-related amplified polymorphisms (SRAPs), and simple sequence repeats (SSRs) markers that were derived from related species (maize, pearl millet, sorghum, and sugarcane). Twenty individuals per cultivar were genotyped using 30 markers from each marker system. All buffalograss cultivars were uniquely fingerprinted by all four marker systems. Mean genetic similarities were estimated at 0.52, 0.51, 0.62, and 0.57 using SSRs, ISSRs, SRAPs, and RAPDs, respectively. Two main clusters separating the seeded-biotype from the vegetative-biotype cultivars were produced using UPGMA analysis. Further subgroupings were unequivocal. The Mantel test resulted in a very good fit (SRAP=0.92, ISSR=0.90) to good fit (RAPD=0.86, SSR=0.88) of cophenetic values. Comparing the four marker systems to each other, RAPD and SRAP similarity indices were highly correlated ( r=0.73), while Spearman's rank correlation coefficient between RAPDs and SSRs was r=0.24 and between ISSRs and SSRs was r=0.66. A genotype-assignment analytical approach might be useful for cultivar identification and property rights protection. Polymorphic SRAPs were abundant and demonstrated genetic diversity among closely related cultivars.

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers.

Buffalograss [ Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.