Global population structure and migration patterns suggest significant population differentiation among isolates of Pyrenophora tritici-repentis.

The global population structure and migration patterns of foliar wheat pathogen Pyrenophora tritici-repentis (PTR) were determined using 12 microsatellite loci. Analysis of 439 single-spore isolates of PTR from five continents (18 wheat-producing countries) showed high level of genetic diversity, and moderate to high population differentiation between continents. A high level of gene diversity (H(S)=0.31 to 0.56) was observed within each population. Allelic richness indicated the European and the North American population have a high effective population size. Bayesian analyses showed five clusters where the inferred clusters did not represent geographical populations. Corrected standardized fixation index (G(ST)(″)) estimates ranged from 0.042 to 0.265 between populations, indicating low to high genetic differentiation exists between populations. We found migration (gene flow) between old world (Europe) and new world (Americas) population; however, little migration was observed among other continents. The European population was the major source of immigrants for the North American, South American, Australian and the Asian populations. Significant (P<0.001) linkage disequilibrium (LD) was detected in the Australian and the South American populations. In contrast, non-significant (P<0.001) LD values were observed in the Asian, European and the North American populations. Overall, our findings demonstrate the population differentiation exits among the global populations and strict quarantine measures should be applied to prevent the accelerated global spread of this pathogen.

First Report of Cylindrocarpon sp. Associated with Root Rot Disease of Strawberry in North Carolina.

Strawberry (Fragaria × ananassa Duchesne) is an economically important fruit crop in North Carolina for domestic consumption and export. In April 2012, outbreaks of a destructive root disease were observed in strawberry cv. Chandler in Buncombe, New Hanover, and Roman counties, North Carolina. Samples from Rowan (ID 13175) and Buncombe (ID 13193) counties submitted to the Plant Disease and Insect Clinic of the Department of Plant Pathology, North Carolina State University, exhibited yellowing and wilting of leaves and extensive root necrosis, and disease severity based on field symptoms ranged from 20 to 30%. To identify the pathogen, five small pieces of necrotic crown and root tissues were taken from each sample, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and plated onto potato dextrose agar (PDA) with 0.5 g liter-1 of streptomycin sulfate. Colonies developing from the tissue samples were transferred to PDA. Colonies from both samples were identical, grew relatively slowly, and gradually turned yellowish to partially brownish. After about 7 days, abundant conidia were formed. These were hyaline, mostly straight with both ends rounded, predominantly three septate, and 40 to 50 × 5 to 10 µm. Based on morphological characteristics, these isolates were identified as a species of Cylindrocarpon (1) To confirm the original identification of the fungus as a species of Cylindrocarpon, genomic DNA of both isolates was extracted from mycelia using DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and analyzed using PCR (2). The internal transcribed spacers (ITS)1 and (ITS)2 flanking the 5.8S rRNA regions were amplified and sequenced using universal primers ITS1 (forward) and ITS4 (reverse). The sequences of the 421 bp (GenBank KC847090 and KC847091) of both isolates were identical. Furthermore, a BLASTn search of these sequences showed homology of 99% with the sequences of Cylidrocarpon species (AB369421.1, AM419069.1, AM419074.1, AY295332.1, JN031017.1, JN253505.1, and JQ886422.1), To fulfill Koch's postulates, inoculum of each isolate was prepared and adjusted to 1.5 × 107 conidia/ml using a hemacytometer. 'Chandler' strawberry plants were grown in 25-cm diameter plastic pots (one seedling per pot) in the greenhouse and five 6-week-old plants were injected with conidia of each isolate into the base of crown using a 5-ml syringe. The plants were covered with clear plastic for 24 h and left on the greenhouse bench with a 16-h photoperiod and 25/20°C day/night temperatures and assessed for disease development 14 days after inoculation. The inoculated plants exhibited wilting and root necrosis, consistent with the symptoms observed on strawberry plants in the field. Control plants treated with distilled water remained healthy. Isolations were made from the inoculated plants and the fungus used for inoculation was recovered from all plants. The morphology of these isolates was in agreement with published descriptions of Cylindrocarpon (1). To our knowledge, this is the first report of a Cylindrocarpon sp. causing crown and root rot on strawberry in North Carolina and effective disease management strategies need to be explored. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1996. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Identification of novel genomic regions associated with resistance to Pyrenophora tritici-repentis races 1 and 5 in spring wheat landraces using association analysis.

Tan spot, caused by Pyrenophora tritici-repentis, is a major foliar disease of wheat worldwide. Host plant resistance is the best strategy to manage this disease. Traditionally, bi-parental mapping populations have been used to identify and map quantitative trait loci (QTL) affecting tan spot resistance in wheat. The association mapping (AM) could be an alternative approach to identify QTL based on linkage disequilibrium (LD) within a diverse germplasm set. In this study, we assessed resistance to P. tritici-repentis races 1 and 5 in 567 spring wheat landraces from the USDA-ARS National Small Grains Collection (NSGC). Using 832 diversity array technology (DArT) markers, QTL for resistance to P. tritici-repentis races 1 and 5 were identified. A linear model with principal components suggests that at least seven and three DArT markers were significantly associated with resistance to P. tritici-repentis races 1 and 5, respectively. The DArT markers associated with resistance to race 1 were detected on chromosomes 1D, 2A, 2B, 2D, 4A, 5B, and 7D and explained 1.3-3.1% of the phenotypic variance, while markers associated with resistance to race 5 were distributed on 2D, 6A and 7D, and explained 2.2-5.9% of the phenotypic variance. Some of the genomic regions identified in this study correspond to previously identified loci responsible for resistance to P. tritici-repentis, offering validation for our AM approach. Other regions identified were novel and could possess genes useful for resistance breeding. Some DArT markers associated with resistance to race 1 also were localized in the same regions of wheat chromosomes where QTL for resistance to yellow rust, leaf rust and powdery mildew, have been mapped previously. This study demonstrates that AM can be a useful approach to identify and map novel genomic regions involved in resistance to P. tritici-repentis.

Identification of New Sources of Resistance in Winter Wheat to Multiple Strains of Xanthomonas translucens pv. undulosa.

Bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa, has re-emerged as an important disease of wheat (Triticum aestivum) in the United States. Planting resistant varieties is the best approach to manage BLS in the absence of effective bactericides. However, most of the wheat varieties currently grown in the Upper Midwest of the United States appeared to be susceptible to BLS. From the core subset of the USDA National Small Grain Collection (NSGC), a set of 605 winter wheat accessions of diverse origin and improvement status were initially inoculated with a virulent strain BLSW16 of X. translucens pv. undulosa from Casselton, ND on the flag leaf of each plant in a greenhouse. Disease reactions were assessed between 7 and 10 days after infiltration using a 0 to 6 rating scale, where ≤2.0 was considered resistant and >2.1 was considered susceptible. Resistance varied with geographic origin and was significantly (P ≤ 0.05) more frequent in accessions from North America compared to accessions from northern, eastern, and southern Europe and from south-central Asia. About 8.3% of accessions tested were resistant, and accessions with an improvement status of "cultivar" were significantly more likely to be resistant than were accessions classified as either landraces or breeding lines. Forty-two of the accessions exhibiting resistance in response to the strain BLSW16 in the first screening test were retested utilizing each of the two additional strains (BLS Cr25 and BLS Lb74 of X. translucens pv. undulosa) collected from Carrington and Lisbon, respectively. Nonparametric data analysis revealed 35 accessions were resistant, one accession, PI 266860, was susceptible to both strains, and six accessions showed differential responses. The majority of the BLS-resistant accessions also were resistant to at least one other important wheat disease based on the Germplasm Resources Information Network (GRIN) data. These results suggest that diverse and novel sources of resistance to BLS identified in this study can be utilized in winter wheat breeding programs.

Genetic relationships among populations of Gibberella zeae from barley, wheat, potato, and sugar beet in the upper Midwest of the United States.

Gibberella zeae, a causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically harmful pathogens of cereals in the United States. In recent years, the known host range of G. zeae has also expanded to noncereal crops. However, there is a lack of information on the population genetic structure of G. zeae associated with noncereal crops and across wheat cultivars. To test the hypothesis that G. zeae populations sampled from barley, wheat, potato, and sugar beet in the Upper Midwest of the United States are not mixtures of species or G. zeae clades, we analyzed sequence data of G. zeae, and confirmed that all populations studied were present in the same clade of G. zeae. Ten variable number tandem repeat (VNTR) markers were used to determine the genetic structure of G. zeae from the four crop populations. To examine the effect of wheat cultivars on the pathogen populations, 227 strains were sampled from 10 subpopulations according to wheat cultivar types. The VNTR markers also were used to analyze the genetic structure of these subpopulations. In all populations, gene (H = 0.453 to 0.612) and genotype diversity (GD = or >0.984) were high. There was little or no indication of linkage disequilibrium (LD) in all G. zeae populations and subpopulations. In addition, high gene flow (Nm) values were observed between cereal and noncereal populations (Nm = 10.69) and between FHB resistant and susceptible wheat cultivar subpopulations (Nm = 16.072), suggesting low population differentiation of G. zeae in this region. Analysis of molecular variance also revealed high genetic variation (>80%) among individuals within populations and subpopulations. However, low genetic variation (<5%) was observed between cereal and noncereal populations and between resistant and susceptible wheat subpopulations. Overall, these results suggest that the populations or subpopulations are likely a single large population of G. zeae affecting crops in the upper Midwest of the United States.

Genetic analysis of resistance to Pyrenophora tritici-repentis races 1 and 5 in tetraploid and hexaploid wheat.

Tan spot of wheat, caused by the fungus Pyrenophora tritici-repentis, is a destructive disease worldwide that can lead to serious losses in quality and quantity of wheat grain production. Resistance to multiple races of P. tritici-repentis was identified in a wide range of genetically diverse genotypes, including three different species Triticum aestivum (AABBDD), T. spelta (AABBDD), and T. turgidum (AABB). The major objectives of this study were to determine the genetic control of resistance to P. tritici-repentis races 1 and 5 in 12 newly identified sources of resistance. The parents, F(1), F(2), and F(2:3) or F(2:5) families of each cross were analyzed for the allelism tests and/or inheritance studies. Plants were inoculated at the two-leaf stage under controlled environmental conditions and disease reaction was assessed based on lesion-type rating scale. A single recessive gene controlled resistance to necrosis caused by P. tritici-repentis race 1 in both tetraploid and hexaploid resistant genotypes. The lack of segregation in the inter- and intra-specific crosses between the resistant tetraploid and hexaploid genotypes indicated that they possess the same genes for resistance to tan necrosis and chlorosis induced by P. tritici-repentis race 1. A single dominant gene for chlorosis in hexaploid wheat and a single recessive gene for necrosis in tetraploid wheat, controlled resistance to P. tritici-repentis race 5.

Genetic structure of Phaeosphaeria nodorum populations in the north-central and midwestern United States.

Stagonospora nodorum blotch, caused by Phaeosphaeria nodorum, is considered one of the most destructive foliar diseases of wheat in the United States. However, relatively little is known about the population biology of this fungus in the major wheat-growing regions of the central United States. To rectify this situation, 308 single-spore isolates of P. nodorum were analyzed from 12 populations, five from hard red spring wheat cultivars in Minnesota and North Dakota and seven from soft red winter wheat in Indiana and Ohio. The genetic structure of the sampled populations was determined by analyzing polymorphisms at five microsatellite or simple-sequence repeat (SSR) loci and the mating type locus. Although a few clones were identified, most P. nodorum populations had high levels of gene (H(S) = 0.175 to 0.519) and genotype (D = 0.600 to 0.972) diversity. Gene diversity was higher among isolates collected from spring wheat cultivars in North Dakota and Minnesota (mean H(S) = 0.503) than in those from winter wheat cultivars in Indiana and Ohio (H(S) = 0.269). Analyses of clone-corrected data sets showed equal frequencies of both mating types in both regional and local populations, indicating that sexual recombination may occur regularly. However, significant gametic disequilibrium occurred in three of the four populations from North Dakota, and there was genetic differentiation both within and among locations. Genetic differentiation between the hard red spring and soft red winter wheat production regions was moderate (F(ST) = 0.168), but whether this is due to differences in wheat production or to geographical variation cannot be determined. These results suggest that sexual reproduction occurs in P. nodorum populations in the major wheat-growing regions of the central United States, and that geographically separated populations can be genetically differentiated, reflecting either restrictions on gene flow or selection.

Reaction of Elite Wheat Genotypes from the Northern Great Plains of North America to Septoria Diseases.

Stagonospora nodorum blotch (SNB), caused by Phaeosphaeria nodorum, and Septoria tritici blotch (STB), caused by Mycosphaerella graminicola, are the main pathogens of the Septoria disease complex of wheat (Triticum aestivum) in North America. This study was conducted to determine the disease reaction of 126 elite hard red spring, white, and durum wheat cultivars and advanced breeding lines collected from the northern Great Plains of the United States and Canada to SNB and STB. Seedlings of the 126 wheat genotypes were evaluated for resistance to SNB and STB under controlled environmental conditions. Moreover, these 126 wheat genotypes also were infiltrated with culture filtrate of P. nodorum isolate Sn2000. Based on disease reactions, three cultivars (McNeal, Dapps, and Oklee) and 12 advanced breeding lines (CA-901-580W, 97SO254-8-1, MN03291, MN03308, WA007925, MT0245, ND756, ND801, ND803, ND808, ND809, and ND811) adapted to the northern Great Plains were found to be resistant to both Septoria diseases and insensitive to the culture filtrate. Additionally, eight genetically diverse lines and cultivars, including two tetraploid wheat genotypes, were identified to be resistant to both Septoria diseases. These results suggest that the wheat genotypes contain a broad genetic base for resistance to the Septoria diseases in the northern Great Plains of the United States and Canada, and the resistant sources identified in this study may be utilized in wheat-breeding programs.

Identification and Molecular Mapping of a Gene Conferring Resistance to Pyrenophora tritici-repentis Race 3 in Tetraploid Wheat.

ABSTRACT Race 3 of the fungus Pyrenophora tritici-repentis, causal agent of tan spot, induces differential symptoms in tetraploid and hexaploid wheat, causing necrosis and chlorosis, respectively. This study was conducted to examine the genetic control of resistance to necrosis induced by P. tritici-repentis race 3 and to map resistance genes identified in tetraploid wheat (Triticum turgidum). A mapping population of recombinant inbred lines (RILs) was developed from a cross between the resistant genotype T. tur-gidum no. 283 (PI 352519) and the susceptible durum cv. Coulter. Based on the reactions of the Langdon-T. dicoccoides (LDN[DIC]) disomic substitution lines, chromosomal location of the resistance genes was determined and further molecular mapping of the resistance genes for race 3 was conducted in 80 RILs of the cross T. turgidum no. 283/Coulter. Plants were inoculated at the two-leaf stage and disease reaction was assessed 8 days after inoculation based on lesion type. Disease reaction of the LDN(DIC) lines and molecular mapping on the T. turgidum no. 283/Coulter population indicated that the gene, designated tsn2, conditioning resistance to race 3 is located on the long arm of chromosome 3B. Genetic analysis of the F(2) generation and of the F(4:5) and F(6:7) families indicated that a single recessive gene controlled resistance to necrosis induced by race 3 in the cross studied.

Evaluation of Elite Wheat Germ Plasm for Resistance to Tan Spot.

Tan spot, caused by Pyrenophora tritici-repentis, is a serious foliar disease of wheat (Triticum aestivum) in North America. Control of tan spot through management practices and fungicide application is possible; however, the use of resistant varieties is the most effective and economical means of controlling tan spot. This study was conducted to determine the disease reaction of 126 elite hard red spring, white, and durum wheat varieties and advanced breeding lines collected from the northern Great Plains of the United States and Canada to individual races/toxins of P. tritici-repentis. Seedling evaluation of the 126 genotypes was done under controlled environmental conditions with virulent races 2, 3, and 5 of P. tritici-repentis and toxins Ptr ToxA and Ptr ToxB. Based on disease reactions, two resistant varieties and two advanced breeding lines adapted to the northern Great Plains were found to be resistant to all the races and insensitive to the toxins tested. Additionally, six genetically diverse lines/varieties were identified to be resistant to tan spot; however, these sources may not be well adapted to the northern Great Plains. These results suggest that the wheat germ plasm contains a broad genetic base for resistance to the most prevalent races of P. tritici-repentis in North America, and the resistant sources identified in this study may be utilized in wheat breeding programs to develop tan spot resistant varieties.

Molecular mapping of Stb1, a potentially durable gene for resistance to septoria tritici blotch in wheat.

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici), was the most destructive disease of wheat in Indiana and adjacent states before deployment of the resistance gene Stb1 during the early 1970s. Since then, Stb1 has provided durable protection against STB in widely grown wheat cultivars. However, its chromosomal location and allelic relationships to most other STB genes are not known, so the molecular mapping of Stb1 is of great interest. Genetic analyses and molecular mapping were performed for two mapping populations. A total of 148 F1 plants (mapping population I) were derived from a three-way cross between the resistant line P881072-75-1 and the susceptible lines P881072-75-2 and Monon, and 106 F6 recombinant-inbred lines (mapping population II) were developed from a cross between the resistant line 72626E2-12-9-1 and the susceptible cultivar Arthur. Bulked-segregant analysis with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and microsatellite or simple-sequence repeat (SSR) markers was conducted to identify those that were putatively linked to the Stb1 gene. Segregation analyses confirmed that a single dominant gene controls the resistance to M. graminicola in each mapping population. Two RAPD markers, G7(1200) and H19(520), were tightly linked to Stb1 in wheat line P881072-75-1 at distances of less than 0.68 cM and 1.4 cM, respectively. In mapping population II, the most closely linked marker was SSR Xbarc74, which was 2.8 cM proximal to Stb1 on chromosome 5BL. Microsatellite loci Xgwm335 and Xgwm213 also were proximal to Stb1 at distances of 7.4 cM and 8.3 cM, respectively. The flanking AFLP marker, EcoRI-AGC/ MseI-CTA-1, was 8.4 cM distal to Stb1. The two RAPD markers, G7(1200) and H19(520), and AFLP EcoRI-AGC/ MseI-CTA-1, were cloned and sequenced for conversion into sequence-characterized amplified region (SCAR) markers. Only RAPD allele H19(520) could be converted successfully, and none of the SCAR markers was diagnostic for the Stb1 locus. Analysis of SSR and the original RAPD primers on several 5BL deletion stocks positioned the Stb1 locus in the region delineated by chromosome breakpoints at fraction lengths 0.59 and 0.75. The molecular markers tightly linked to Stb1 could be useful for marker-assisted selection and for pyramiding of Stb1 with other genes for resistance to M. graminicola in wheat.

Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice.

Of 102 rhizoplane and endophytic bacteria isolated from rice roots and stems in California, 37% significantly (P < or = 0.05) inhibited the growth in vitro of two pathogens, Achlya klebsiana and Pythium spinosum, causing seedling disease of rice. Four endophytic strains were highly effective against seedling disease in growth pouch assays, and these were identified as Pseudomonas fluorescens (S3), Pseudomonas tolaasii (S20), Pseudomonas veronii (S21), and Sphingomonas trueperi (S12) by sequencing of amplified 16S rRNA genes. Strains S12, S20, and S21 contained the nitrogen fixation gene, nifD, but only S12 was able to reduce acetylene in pure culture. The four strains significantly enhanced plant growth in the absence of pathogens, as evidenced by increases in plant height and dry weight of inoculated rice seedlings relative to noninoculated rice. Three bacterial strains (S3, S20, and S21) were evaluated in pot bioassays and reduced disease incidence by 50%-73%. Strain S3 was as effective at suppressing disease at the lowest inoculum density (106 CFU/mL) as at higher density (10(8) CFU/mL or undiluted suspension). This study indicates that selected endophytic bacterial strains have potential for control of seedling disease of rice and for plant growth promotion.

Genotypic and Pathotypic Diversity in Xanthomonas oryzae pv. oryzae in Nepal.

ABSTRACT Among the 171 strains of Xanthomonas oryzae pv. oryzae (the bacterial blight pathogen of rice) collected from eight rice-producing zones in Nepal, 31 molecular haplotypes were distinguished using two polymerase chain reaction-based assays. Six common haplotypes represented nearly 63% of the strains, and some haplotypes were geographically dispersed. Multiple correspondence analysis divided the collection into five putative genetic lineages. Lineages 1, 2, and 4 were the most frequently detected and occurred in diverse geographic populations. Twenty-six pathotypes (virulence phenotypes) of X. oryzae pv. oryzae were identified using 11 near-iso-genic rice lines, each containing a single gene for resistance. The 26 pathotypes grouped into five clusters, and cluster 1 contained wide virulence spectrum strains from all geographic populations. Although molecular variation was greatest between strains of different virulence phenotypes, some variation was observed among strains with identical virulence. There was a weak correlation (r = 0.52) between molecular haplotypes and virulence phenotypes. There are two major groups of X. oryzae pv. oryzae in Nepal. One group consists of strains with high molecular polymorphism and many pathotypes that are either virulent to the 11 major resistance genes or avirulent only to Xa21. Strains in the second group have low molecular polymorphism and are avirulent to Xa4, xa5, Xa7, and Xa21.

Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia.

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.