Molecular mapping of genes for resistance to the bean pod weevil (Apion godmani Wagner) in common bean

The bean pod weevil (Apion godmani Wagner) is a serious insect pest of common beans (Phaseolus vulgaris L.) grown in Mexico and Central America that is best controlled by host-plant resistance available in Durango or Jalisco genotypes such as J-117. Given unreliable infestation by the insect, the use of marker-assisted selection is desirable. In the present study, we developed a set of nine molecular markers for Apion resistance and mapped them to loci on chromosomes 2, 3, 4 and 6 (linkage groups b01, b08, b07and b11, respectively) based on genetic analysis of an F _5:10 susceptible × resistant recombinant inbred line population (Jamapa × J-117) and two reference mapping populations (DOR364 × G19833 and BAT93 × JaloEEP558) for which chromosome and linkage group designations are known. All the markers were derived from randomly amplified polymorphic DNA (RAPD) bands that were identified through bulked segregant analysis and cloned for conversion to sequence tagged site (STS) markers. One of the markers was dominant while four detected polymorphism upon digestion with restriction enzymes. The other markers were mapped as RAPD fragments. Phenotypic data for the population was based on the evaluation of percentage seed damage in replicated trials conducted over four seasons in Mexico. In single point regression analysis, individual markers explained from 3.5 to 22.5% of the variance for the resistance trait with the most significant markers overall being F10-500S, U1-1400R, R20-1200S, W9-1300S and Z4-800S, all markers that mapped to chromosome 2 (b01). Two additional significant markers, B1-1400R and W6-800R, were mapped to chromosome 6 (b11) and explained from 4.3 to 10.2% of variance depending on the season. The latter of these markers was a dominant STS marker that may find immediate utility in marker-assisted selection. The association of these two loci with the Agr and Agm genes is discussed as well as the possibility of additional resistance genes on chromosome 4 (b07) and chromosome 3 (b08). These are among the first specific markers developed for tagging insect resistance in common bean and are expected to be useful for evaluating the mechanism of resistance to A. godmani.     

Inheritance of resistance to bacterial blight in common bean

Summary Inheritance of resistance to common bacterial blight in the trifoliate leaf, plant canopy, and pods was controlled by a single major gene. Additive followed by dominance effects were more important than epistatic interactions. Narrow-sense heritability values ranged from 0.18 to 0.87 for trifoliate leaf, from 0.26 to 0.76 for canopy, and from 0.11 to 0.36 for pods. Observed gains from selection for resistance were higher than expected gains. Implications of these results in breeding for resistance are discussed.     

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.     

The mechanisms of resistance in common beans to the leafhopper Empoasca kraemeri

Greenhouse and field studies were conducted to determine the mechanisms of resistance (tolerance, antibiosis and antixenosis) in common beans Phaseolus vulgaris L. to the leafhopper Empoasca kraemeri Ross & Moore (Homoptera: Cicadellidae). No antibiosis effects were detected in any of the bean lines investigated. However, in both free choice and no choice tests in the greenhouse, ovipositional antixenosis was detected, and appeared to increase with increasing plant age in resistant bean lines. The results of the field study were similar to those obtained in the greenhouse and suggested that bean line EMP 81 possessed only tolerance as a mechanism of resistance to leafhoppers, while EMP 89, EMP 94, EMP 97 and EMP 82 were non-preferred by ovipositing leafhoppers.

---

Résumé Des expériences en serres et en champ ont servi à analyser la résistance de P. vulgaris à E. kraemeri. L'importance de l'antibiose dans la résistance à E. kraemeri a été examinée en serre avec 5 variétés résistantes: EMP 81, EMP 82, EMP 89, EMP 94, EMP 97 et un témoin sensible BAT 41, 7 jours après le semis (cotylédons épanouis) et 20–25 j (2 à 3 feuilles). L'examen de la mortalité embryonnaire, du développement larvaire et du poids des larves de 5è stade n'a permis de déceler aucune manifestation d'antibiose sur les différentes variétés.Les préférences pour les lieux de ponte et d'alimentation ont été examinées avec des expériences avec des ou sans choix. Aucune variété n'a été plus rejetée que BAT 41 à 7 et 20–25 j pour l'alimentation. Par contre avec un choix libre, EMP 89 et EMP 97 ont reçu moins de pontes à 7 j que les autres variétés. A 25 j, EMP 89, EMP 94 et EMP 97 portaient moins de larves que BAT 41, EMP 81 ou EMP 82. Cette dernière cependant en l'absence de choix portait moins d'oeufs que BAT 41. L'antixénose de la ponte semble croître avec le développement de la plante pour EMP 89 et EMP 94, bien que la résistance à la ponte ait été plus stable aux fortes populations de E. kraemeri avec EMP 94 que EMP 89.Les résultats en champs, semblables à ceux des serres, ont suggéré que EMP 81 ne présente que la tolérance comme moyen de résistance à E. kraemeri tandis que EMP 89, EMP 94, EMP 97 et EMP 82 n'étaient pas préférentiellement choisis pour la ponte.

     

Inheritance of resistance to potato y viruses in Phaseolus vulgaris L

Summary Resistance to watermelon mosaic virus-2 in Phaseolus vulgaris L. is conferred by two distinct dominant alleles at independent loci. Based on segregation data one locus is designated Wmv, the other, Hsw. The dominant allele Wmv from cv. Great Northern 1140 prevents systemic spread of the virus but viral replication occurs in inoculated tissue. In contrast, Hsw confers both local and systemic resistance to WMV-2 below 30C. At higher temperatures, plants that carry this allele in the absence of modifying or epistatic factors develop systemic veinal necrosis upon inoculation with the virus that results in rapid death. Patho-type specificity has not been demonstrated for either allele; both factors confer resistance to every isolate tested. A temperature-sensitive shift in epistasis is apparent between dominant alleles at these loci. Because Hsw is very tightly linked if not identical to the following genes for hypersensitivity to potyviruses I, (bean common mosaic virus), Bcm, (blackeye cowpea mosaic virus), Cam, (cowpea aphid-borne mosaic virus) and Hss (soybean mosaic virus), parental, reciprocal dihybrid F₁ populations, and selected F₃ families were inoculated with each of these viruses and held at 35 C. F₁ populations developed vascular necrosis completely or primarily limited to inoculated tissue, while F₃ families from WMV-2-susceptible segregates were uniformly susceptible to these viruses. The relationship between Hsw, Wmv and other genes for potyvirus resistance suggest patterns in the evolution of resistance and viral pathogenicity. Characterization of the resistance spectrum associated with each factor provides an additional criterion to distinguish genes for plant virus resistance.     

Utilizing the potential for increased nitrogen fixation in common bean

Many variables affect the amount of N₂ fixation that occurs in field-grown common beans (Phaseolus vulgaris L.). When environmental conditions are optimized, genetically superior plants that are nodulated with efficient rhizobia are able to fix enough N₂ to support grain yields of up to 2000 kg ha^–1. All of the required components are available for common bean to fulfill its potential as a nitrogen-fixing food crop. However, these components must be assembled and presented to growers in a usable package that is economically attractive.     

Inoculated common beans are protected againstMacrophomina phaseolina byBurkholderia cepacia UPR 5C

Ashy stem blight (ASB), caused byMacrophomina phaseolina (Tassi) Goid., is a common severe disease affectingPhaseolus vulgaris in Puerto Rico and the Dominican Republic. The effect of inoculating seeds ofP. vulgaris withBurkholderia cepacia strain UPR 5C on the severity of ASB was studied under greenhouse conditions. Results of this study showed thatB. cepacia reduced the ASB disease severity by 71% and was compatible withRhizobium phaseoli CIAT 632.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. III. Cosegregation of phenotypically similar dominant responses to nine potyviruses

We have identified monogenic dominant resistance to azuki bean mosaic poty virus (AzMV), passionfruit woodiness potyvirus-K (PWV-K), zucchini yellow mosaic potyvirus (ZYMV), and a dominant factor that conditioned lethal necrosis to Thailand Passiflora potyvirus (ThPV), in Phaseolus vulgaris Black Turtle Soup 1. Resistance to AzMV, PWV-K, ZYMV, watermelon mosaic potyvirus, cowpea aphid-borne mosaic potyvirus, blackeye cowpea mosaic potyvirus, and lethal necrosis to soybean mosaic potyvirus and ThPV cosegregated as a unit with the I gene for resistance to bean common mosaic potyvirus.     

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

In vitro culture response of common bean explants to filtrate from Pseudomonas syringae pv. Phaseolicola and correlation with disease resistance

Summary The in vitro culture responses from different explants of a race-specific resistant cultivar (Red Mexican) and a racesusceptible cultivar (Palme?a) to halo-blight pathogen (Pseudomonas syringae pv. phaseolicola were studied. Two kinds of filtrate obtained from a phaseolotoxin producer wild type and a non-producer mutant of P. syringae pv. phaseolicola race-7 were used. Callus formation of Red Mexican was significantly reduced in the presence of phaseolotoxin. Bud-shoot growth was more sensitive than callus formation to other metabolites present in the pathogen filtrate, but the presence of phaseolotoxin in the media showed a positive correlation between resistance to halo blight race-7 pathogen and bud-shoot growth. Our results indicate that differential in vitro responses are influenced by the plant genotype and by the metabolite composition and concentration of the filtrate.     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

The accumulation of trehalose in nodules of several cultivars of common bean (Phaseolus vulgaris) and its correlation with resistance to drought stress

Nine cultivars of common bean were grown in the presence of a natural microflora without exogenous rhizobial inoculation. Nodules were harvested 30 days post planting (early flowering stage) and the presence of trehalose determined. Amounts present varied according to cultivar and were between 0.20 and 1.63 mg g⁻¹ nodule dry weight. Rhizobial strains were isolated from the nodules of three selected cultivars (Canario 101, Flor de Mayo Bajio and Flor de Mayo 38). Trehalose levels in nodules produced after either mixed strain reinfection, or after axenic homologous reinfection or after axenic cross‐reinfection could be manipulated by applying drought stress. Mixed reinfection nodules from stressed plants accumulated between two and six times the trehalose concentration found in non‐stressed control plants. After axenic cross‐reinfection up to 48‐fold increases in nodule trehalose content were recorded during drought stress. Those cultivars exhibiting high nodule trehalose levels and/or a high degree of trehalose stimulation in response to drought stress also exhibited a high leaf relative water content and were also the most drought resistant. During drought stress nodule trehalase levels rose only slightly.     

Proline metabolism in response to highest nitrogen dosages in green bean plants (Phaseolus vulgaris L. cv. Strike)

The objective of the present work was to determine what impact extremely high nitrogen dosages would have on proline metabolism in order to use this amino acid as a bioindicator of N status of green bean plants (Phaseolus vulgaris L. cv. Strike). In this effort, we identified the most favourable pathway of proline synthesis under our experimental conditions. The N was applied to the nutrient solution in the form of NH₄NO₃ at 5.4 mmol/L (N1, optimal level), 11.6 mmol/L (N2), 17.4 mmol/L (N3), and 23.2 mmol/L (N4). Our results indicate that the application of high N dosages inPhaseolus is characterized by the accumulation of NO₃⁻, NH₄⁺ and proline in root and foliar organs. However, although the enzymes in charge of proline biosynthesis, ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) and Δ¹-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) vary in behaviour depending on the N status, in our experiment, this amino acid appears to be synthesized mainly by the enzyme ornithine-δ-aminotransferase. This suggests predominance of the ornithine pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good indicator of N excess of green bean plants.     

Tolerance as a mechanism of resistance to Thrips palmi in common beans

Tolerance as a mechanism of resistance to the melon thrips, Thrips palmi Karny (Thysanoptera: Thripidae), in common beans, Phaseolus vulgaris L., was evaluated under field and greenhouse conditions. Seven resistant (Brunca, BH‐5, BH‐60, BH‐130, BH‐144, EMP 486, and FEB 115) and five susceptible (PVA 773, EMP 514, BAT 477, APN 18, and RAZ 136) bean genotypes were assessed according to adult and larval populations, visual damage and reproductive adaptation scores, and yield components in field trials. From these genotypes, four resistant (Brunca, BH‐130, EMP 486, and FEB 115) and two susceptible (APN 18 and RAZ 136) genotypes were selected for quantification of proportional plant weight and height increase changes due to thrips infestation in greenhouse tests. Under medium to high thrips infestation in the field, most resistant genotypes tended to have higher reproductive adaptation and lower yield losses, though they did not always suffer less damage, as compared to susceptible genotypes. In the greenhouse, resistant genotypes showed less reduction in plant dry weight and height increase than did some susceptible ones under the same infestation pressure. Results from both field trials and greenhouse tests suggest the possible expression of tolerance as a mechanism of resistance to T. palmi in the resistant genotype EMP 486, and confirm the existence of antixenosis in FEB 115, whereas tolerance might be combined with other resistance mechanisms in Brunca.     

Nitrate reductase and nitrogenase activities of common beans (Phaseolus vulgaris L.) from different geographic locations

Summary Cultivars ofPhaseolus vulgaris (L.) from contrasting geographic locations were cultivated under fields conditions for measurements of nitrogenase and nitrate reductase activities. A first trial with two cultivars indicated that a tropical cultivar B-789 has a higher nitrogenase activity than a temperate one Elsa. And the converse was true for the nitrate reductase activity. While where a post flowering application was made, a renewal of nitrate reductase activity occurred. Further similar comparisons of both enzymatic activities upon eight tropical and temperate cultivars of equivalent vegetative cycles indicated, on the average, that tropical cultivars have a higher level of (C₂H₂) reduction and a lower nitrate reductase activity than temperature cultivars. These observations suggest that there exists an inverse relationship between the two enzymatic activities in common beans, and there probably exists genetic variability for a possible improvement of N-fixation ability. An early application of N-fertiliser upon the Elsa and B-789 plots promotes later nitrogenase activity while a post flowering application shows obvious a renewal of nitrate reductase activity. Thus, analyses of nitrate reductase and nitrogenase activities of a common bean crop at different physiological stages may give us an indication of the best time to apply supplementary nitrogen fertilisation to common beans to increase seed yield.     

Identification of RAPD markers linked to a major rust resistance gene block in common bean

Rust in bean (Phaseolus vulgaris L.), caused byUromyces appendiculatus (Pers.) Unger var.appendiculatus [ =U. phaseoli (Reben) Wint.], is a major disease problem and production constraint in many parts of the world. The predominant form of genetic control of the pathogen is a series of major genes which necessitate the development of efficient selection strategies. Our objective was focused on the identification of RAPD (random amplified polymorphic DNA) markers linked to a major bean rust resistance gene block enabling marker-based selection and facilitating resistance gene pyramiding into susceptible bean germplasm. Using pooled DNA samples of genotyped individuals from two segregating populations, we identified two RAPD markers linked to the gene block of interest. One such RAPD, OF10₉₇₀ (generated by a 5-GGAAGCTTGG-3 decamer), was found to be closely linked (2.15±1.50 centi Morgans) in coupling with the resistance gene block. The other identified RAPD, OI19₄₆₀ (generated by a 5-AATGCGGGAG-3 decamer), was shown to be more tightly linked (also in coupling) than OF10₉₇₀ as no recombinants were detected among 97 BC₆F₂ segregating individuals in the mapping population. Analysis of a collection of resistant and susceptible cultivars and experimental lines, of both Mesoamerican and Andean origin, revealed that: (1) recombination between OF10₉₇₀ and the gene block has occurred as evidenced by the presence of the DNA fragment in several susceptible genotypes, (2) recombination between OI19₄₆₀ and the gene block has also occurred indicating that the marker is not located within the gene block itself, and (3) marker-facilitated selection using these RAPD markers, and another previously identified, will enable gene pyramiding in Andean germplasm and certain Mesoamerican bean races in which the resistance gene block does not traditionally exist. Observations of variable recombination among Mesoamerican bean races suggested suppression of recombination between introgressed segments and divergent recurrent backgrounds.Research supported by the Michigan Agricultural Research Station and the USDA-ARS. Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable     

Bean tumbling as a control measure for the common bean weevil, Acanthoscelides obtectus

Recognizing that it takes Acanthoscelides obtectus (Say)(Coleoptera: Bruchidae) larvae over 24 h to bore into a dry red kidney bean (Phaseolus vulgaris L.) and that boring can occur only at particular sites where a bean abuts some other surface, we postulated that this pest might be controlled by periodic tumbling of beans so as to place initiated holes out of register with requisite touching surfaces. Larvae repeatedly forced to initiate holes might die of exhaustion, if not smashed by tumbling beans. Indeed, brief daily tumbling of beans held in half-filled jars, buckets, and gunny sacks reduced A. obtectus populations by 97% relative to stationary controls. We recommend that small lots of beans can be protected indefinitely from the bean weevil when stored in <75% filled cylinders rolled ca 1 circumference every morning and evening. This control method should be immediately useful, particularly among subsistence families, since the only external input is knowledge. Also, this control principle might be broadly applicable to other bean and grain pests having strict spatial and temporal requirements for seed penetration.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale