Richard C. Larsen

Potential marker-assisted selection for bc-12 resistance to bean common mosaic potyvirus in common bean

Pyramiding epistatic resistance genes to improve long term disease resistance has challenged plant breeders. Indirect selection using tightly linked markers will often facilitate the breeding of desired epistatic resistance gene combinations. In common bean, the most effective strategy for broad spectrum control of the bean common mosaic virus disease is to combine I, bc-u, bc-12, bc-22, and bc-3 genes. We describe the use of near-isogenic lines and bulked segregant analysis to identify a marker tightly linked with the bc-12 gene. The recessive bc-12 gene conditions resistance to specific strains of bean common mosaic virus and bean common mosaic necrosis virus and is masked by the bc-22 and bc-3 genes. We identified a RAPD marker completely linked (0 recombinants) with bc-12, based on 72 F3 progeny generated from a cross between the contrasting near isogenic lines (I + bc-1/I + bc-12). Segregation in this I gene background revealed that bc-12 was dominant to bc-1 in conferring resistance to top necrosis in the allelic series Bc-1 > bc-12 > bc-1. To facilitate marker-assisted selection of bc-12 across breeding programs, the RAPD was converted to a SCAR marker, designated SBD51300. Tight linkage (0 recombinants) was confirmed in a second population of 58 F2 progeny co-segregating for SBD51300 and bc-12 gene from a different source. Based on a survey of 130 genotypes, the SCAR will be useful for MAS of bc-12 in most beans of Middle American origin and snap beans, but will have very limited utility in the case of kidney and cranberry beans. The SBD51300 marker mapped on linkage group B3, revealing independence of bc-12 from the I gene on B2 and bc-3 gene on B6, which supports the opportunity to readily combine genes for broad spectrum and pyramided resistance to bean common mosaic potyviruses in a single bean cultivar.

NL-3 K Strain Is a Stable and Naturally Occurring Interspecific Recombinant Derived from Bean common mosaic necrosis virus and Bean common mosaic virus.

ABSTRACT A strain of Bean common mosaic necrosis virus (BCMNV) from Idaho was identified by enzyme-linked immunosorbent assay using monoclonal antibodies and determined to be similar to the NL-3 D strain (of Drifjhout) by reaction of differential bean cultivars. However, this BCMNV strain (designated NL-3 K) caused earlier and more severe symptoms on bean plants representing host groups 0, 4, and 5. The nucleotide sequence encoding the predicted polyprotein of NL-3 K was 9,893 nucleotides (nt) in length, yielding a peptide with a molecular size of 362.1 kDa compared with a 9,626-nt, 350.9-kDa polyprotein for NL-3 D. Sequence analysis of the putative P1 protein suggests that the NL-3 K strain is a recombinant between NL-3 D and the Russian strain (RU1) of Bean common mosaic virus. The P1 protein of NL-3 K consisted of 415 amino acids compared with 317 for NL-3 D. The first 114 predicted amino acids of the NL-3 K P1 region were 98% identical with RU1. The remaining 301 amino acids of the protein shared only 34% identity with RU1 but were 98% identical with NL-3 D. Primers were designed that flanked the recombination point in the P1 coding sequence of NL-3 K. An amplicon of the expected size was produced by reverse-transcriptase polymerase chain reaction of total nucleic acid extracts of bean plants inoculated with NL-3 K, but not from those with NL-3 D or RU1. The increased symptom severity on selected common bean lines induced by NL-3 K suggests that the P1 gene may play a significant role in pathogenicity and virulence.

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.

Generation and Molecular Mapping of a Sequence Characterized Amplified Region Marker Linked with the Bct Gene for Resistance to Beet curly top virus in Common Bean.

ABSTRACT A random amplified polymorphic DNA (RAPD) marker directly linked (0.0 cM) with a resistance gene was identified in a snap bean recombinant inbred population (Moncayo x Primo) consisting of 94 F(5:7) recombinant inbred lines that had uniform segregation for disease reaction to Beet curly top virus (BCTV) across three field locations. Resistance was conditioned by a single dominant allele tentatively designated Bct. Seven hundred and fifty decamer primers were screened to obtain the linked RAPD marker that was then converted to a sequence characterized amplified region (SCAR) marker SAS8.1550. The SCAR mapped within a cluster of resistance genes on linkage group B7 of the core map. A survey of 103 BCTV-resistant and -susceptible snap and dry bean genotypes was conducted using SAS8.1550. Results showed that the SCAR would be highly useful for marker-assisted selection of Bct in snap and dry bean originating from the Andean gene pool. Marker-assisted selection for Bct will expedite the development of BCTV-resistant cultivars and minimize the need for cumbersome pathogen tests.

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.

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.

A Strain of Clover yellow vein virus that Causes Severe Pod Necrosis Disease in Snap Bean

Soybean aphid (Aphis glycines) outbreaks occurring since 2000 have been associated with severe virus epidemics in snap bean (Phaseolus vulgaris) production in the Great Lakes region. Our objective was to identify specific viruses associated with the disease complex observed in the region and to survey bean germplasm for sources of resistance to the causal agents. The principle causal agent of the disease complex associated with extensive pod necrosis was identified as Clover yellow vein virus (ClYVV), designated ClYVV-WI. The virus alone caused severe mosaic, apical necrosis, and stunting. Putative coat protein amino acid sequence from clones of amplicons generated by reverse-transcription polymerase chain reaction was 98% identical to ClYVV strain no. 30 identified in Japan that has not been reported to cause pod necrosis. ClYVV-WI amplicons were 96% identical to a mild strain of ClYVV from Oregon. A distinguishing feature of this new strain is that it does not react with Potyvirus broad-spectrum monoclonal antibody PTY 1. A survey of common bean lines and cultivars revealed that, in addition to UI-31 and US1140 with known resistance to ClYVV, lines with the bc-3 gene for resistance to Bean common mosaic necrosis virus also were resistant to ClYVV-WI. An evaluation of 63 snap bean cultivars and breeding lines revealed just one, Roma 442, with a moderate level of tolerance to ClYVV-WI. Introgression of the bc-3 gene and resistances from UI-31 and US1140 into snap bean may offer a high level of resistance to extensive pod necrosis disease caused by ClYVV in the Great Lakes region.

Chickpea wilt incited by pea streak carlavirus

Pea streak carlavirus (PSV) incited a widespread wilting and yellowing disease of chickpea (Cicer arietinum) in commercial and experimental plantings in the Palouse region of eastern Washington and northern Idaho. Incidence of PSV usually ranged from 0.5 to 5%. Experimental host ranges of several Palouse PSV isolates were confined to the Fabaceae and one species of Amaranthaceae. Systemic necrosis developed in chickpea, lentil (Lens culinaris), pea (Pisum sativum), fenugreek (Trigonella foenum-graecum), and faba bean (Vicia faba), while alfalfa (Medicago sativa), white sweet clover (Melilotus alba), and hairy vetch (Vicia villosa subsp. villosa) were symptomless carriers of PSV

The complete nucleotide sequences of bean common mosaic necrosis virus strains NL-5, NL-8 and TN-1

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) belong to the genus Potyvirus and are probably the most economically important viruses affecting common bean (Phaseolus vulgaris L.). Of the two viruses, BCMNV has been the most important in common bean production in North America, East Africa and Europe due to significant yield losses incurred from infected fields [10]. BCMV and BCMNV are transmitted in a non-persistent manner by several aphid species. All known strains of each virus can be highly seed-transmitted in bean and are distributed worldwide, in large part as a result of movement of contaminated seed [4, 5]. Drijfhout [4] originally separated the two viruses and their associated strains into eight different pathogroups based on host reaction to infection under specific temperature regimes. Strains NL-5 and NL-3 from Africa were originally assigned to the BCMV pathogroup VI by Drijfhout [4], and NL-8 was assigned to pathogroup III according to host responses in 12 different common bean host groups. The TN-1 strain from Tanzania was later assigned to pathogroup VI by Silbernagel [12]. Prior to 1992, all strains of BCMNV were designated as BCMV, at which time Vetten et al. [14] designated stains NL-3, NL-5 and NL-8 as serogroup A (BCMNV). All other known strains were assigned to serogroup B (BCMV) based on serological reactions to two highly specific monoclonal antibodies, and this was further supported by molecular analysis and ultrastructural comparisons [14]. Currently, several isolates of strain NL-3 have been sequenced and are available in GenBank; however, the sequences of strains NL-5, NL-8 and TN-1 are unavailable. Therefore, we report here the complete genome sequences of these three strains of BCMNV. Common bean seeds (variety ‘Dubbele Witte’) infected with NL-5, NL-8 and TN-1, respectively, were germinated in pots containing a mixture of peat moss and perlite (Sun Gro Horticulture Canada Ltd.) in the greenhouse, and plants were observed for symptoms. Tissues from symptomatic seedlings were evaluated for infection with BCMNV by direct ELISA using monoclonal antibodies specific for serogroup A, and also to serogroup B [11] to verify that there was no contamination with any strain of BCMV. Total nucleic acids were extracted from plant tissues, and cDNA was produced by reverse transcription using MMLV reverse transcriptase (Promega Corp., Madison, WI). Thermocycling parameters were optimized, and a final profile was employed that consisted of a single cycle of 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, and 3 min at 72 C; and a final extension for 7 min at 72 C. The initial reactions included oligo-dT24 as the reverse primer and a degenerate forward primer, 5’-GAA YAG CAA TGC NAT AG-3’, that produced a fragment approximately 3425 bp in length. The degenerate primer begins at nucleotide 6206, located in the NIa protein region. Subsequent reactions to complete the genome sequence were carried out using custom primer combinations based on the partial nucleotide sequence data obtained. The 5’-proximal region of the genomic sequence R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov

Interactions between QTL SAP6 and SU91 on resistance to common bacterial blight in red kidney bean and pinto bean populations

Breeding efforts to improve resistance in dry bean to common bacterial blight (CBB) have focused on applying marker assisted selection strategies. We examined the interaction between two independent QTL (quantitative trait loci), SAP6 and SU91, on the expression of resistance to CBB in a pinto bean F2 population and dark red kidney bean F2 population. The disease reaction for each F2 population was scored at several time points after inoculation. The dominant SCAR markers SAP6 and SU91 were simultaneously genotyped as codominant markers using a multiplex real-time PCR assay. For both populations, at each time point plants having genotypes of (su91 su91//SAP6 SAP6); (su91 su91//SAP6 sap6), and (su91 su91//sap6 sap6) were significantly more susceptible to CBB than plants with any of the six other possible genotypes (SU91 _//_ _). Only two examples were observed across both populations and all time points in which mean disease reactions were significantly different between any of the six (SU91 _//_ _) genotypes. No significant differences were observed between SU91 SU91 and SU91 su91 genotypes at any time point for either F2 population. Similarly, no significant differences in CBB reaction were observed among the three SAP6 genotypes for plants that had the su 91/su91 genotype. The results indicate CBB resistance in these two populations is primarily conditioned by the presence of at least a single copy of the SU91 QTL. The effects of pathogen isolate and population on discerning the influence of QTL on CBB resistance are discussed.