George J. Vandemark

A PCR-based assay by sequence-characterized DNA markers for the identification and detection of Aphanomyces euteiches

ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.

Quantifying Aphanomyces euteiches in Alfalfa with a Fluorescent Polymerase Chain Reaction Assay

ABSTRACT A polymerase chain reaction (PCR) assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial cultivars based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.

Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

ABSTRACT A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

A Rapid Method Using PCR-Based SCAR Markers for the Detection and Identification of Phoma sclerotioides: The Cause of Brown Root Rot Disease of Alfalfa

A rapid technique for identification and detection of Phoma sclerotioides, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from random amplified polymorphic DNA (RAPD) reactions were cloned and sequenced, and two extended primer sets were designed from the resulting data that were used to detect sequence-characterized DNA markers. A single 499-bp DNA amplification product was consistently obtained from primers PSB12499 that was specific for 19 isolates of P.sclerotioides but was not produced from Phoma medicaginis or Phoma betae, or from other soilborne pathogens including Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, Pythium ultimum, or Phytophthora infestans. A 499-bp amplification product was also produced from root tissue known to be infected with the fungus as verified by microscopic examination. A similar PCR product was obtained from soil samples collected from fields with an established infection of P. sclerotioides on alfalfa. This PCR-based assay enables detection of P. sclerotioides from alfalfa root tissue and in soil samples in a single day, including extraction of DNA, compared with standard methods that require up to 100 days for identification using agar media.

Links among Nitrification, Nitrifier Communities, and Edaphic Properties in Contrasting Soils Receiving Dairy Slurry

Soil biotic and abiotic factors strongly influence nitrogen (N) availability and increases in nitrification rates associated with the application of manure. In this study, we examine the effects of edaphic properties and a dairy (Bos taurus) slurry amendment on N availability, nitrification rates and nitrifier communities. Soils of variable texture and clay mineralogy were collected from six USDA-ARS research sites and incubated for 28 d with and without dairy slurry applied at a rate of ~300 kg N ha(-1). Periodically, subsamples were removed for analyses of 2 M KCl extractable N and nitrification potential, as well as gene copy numbers of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Spearman coefficients for nitrification potentials and AOB copy number were positively correlated with total soil C, total soil N, cation exchange capacity, and clay mineralogy in treatments with and without slurry application. Our data show that the quantity and type of clay minerals present in a soil affect nitrifier populations, nitrification rates, and the release of inorganic N. Nitrogen mineralization, nitrification potentials, and edaphic properties were positively correlated with AOB gene copy numbers. On average, AOA gene copy numbers were an order of magnitude lower than those of AOB across the six soils and did not increase with slurry application. Our research suggests that the two nitrifier communities overlap but have different optimum environmental conditions for growth and activity that are partly determined by the interaction of manure-derived ammonium with soil properties.

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.

Use of real-time PCR to examine the relationship between disease severity in pea and Aphanomyces euteiches DNA content in roots

Aphanomyces euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. Real-time fluorescent PCR assay specific for A. euteiches was used to study the relationship between disease severity and pathogen DNA content in infected peas. Five pea genotypes ranging in levels of resistance were inoculated with five isolates of A. euteiches. Plants were visually rated for disease development and the amount of pathogen DNA in roots was determined using the PCR assay. The susceptible genotypes Genie, DSP and Bolero tended to have significantly more disease and more pathogen DNA than the resistant genotypes 90-2079 and PI 180693. PI 180693 consistently had less disease, while 90-2079 had the lowest amount of pathogen DNA. The Spearman correlation between pathogen DNA quantity and disease development was positive and significant (P < 0.05) for three isolates, but was not significant for two other isolates. This suggests that the real-time PCR assay may have limited application as a selection tool for resistance in pea to A. euteiches. Its utility as a selection tool would be dependent on the correlation between disease development and pathogen DNA content for a given pathogen isolate. The accuracy and specificity of the real-time PCR assay suggests considerable application for the assay in the study of mechanisms of disease resistance and the study of microbial population dynamics in plants.

Genotyping Common Bean for the Potyvirus Resistance Alleles I and bc-1 2 with a Multiplex Real-Time Polymerase Chain Reaction Assay

ABSTRACT A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-1(2) alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F(2) population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-1(2) i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F(2) plants to one of nine genotypes. Remnant F(1) plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F(2) plants were genotyped based on results relative to the probability distributions for heterozygotes. F(2) plants also were genotyped for the I and bc-1(2) alleles by performing F(3) family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-1(2) allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.

Inheritance and Linkage Map Positions of Genes Conferring Agromorphological Traits in Lens culinaris Medik.

Agromorphological traits have immense importance in breeding lentils for higher yield and stability. We studied the genetics and identified map positions of some important agro-morphological traits including days to 50% flowering, plant height, seed diameter, 100 seed weight, cotyledon color, and growth habit in Lens culinaris. Earlier developed RILs for stemphylium blight resistance (ILL-5888 × ILL-6002), contrasted for those agro-morphological traits, were used in our study. Three QTLs for days to 50% flowering were detected with additive and epistatic effects. One QTL for days to 50% flowering, QLG483 (QTL at linkage group 4 at 83 cM position), accounted for an estimated 20.2% of the variation, while QLG124 × QLG1352 and QLG484 × QLG138 accounted for 15.6% and 24.2% of the variation, respectively. Epistatic effects accounted for most of the variation in plant height, but the main effect of one QTL, QLG84, accounted for 15.3%. For seed diameter, three QTLs were detected, and one QTL, QLG482, accounted for 32.6% of the variation. For 100 seed weight, five QTLs were identified with significant additive effects and four with significant interaction effects. The main effect of one QTL, QLG482, also accounted for 17.5% of the variation in seed diameter. QLG which appears to affect days to 50% flowering, seed diameter, and 100 seed weight is flanked by RAPD markers, UBC 34 and UBC1. Growth habit and cotyledon color are controlled by single genes with prostrate dominant to erect and red cotyledon dominant to yellow. The QTL information presented here will assist in the selection of breeding lines for early maturity, upright growth habit, and improved seed quality.