James R. Steadman

Bean rust biological control using bacterial agents

Abstract Over 120 bacterial strains were evaluated in a greenhouse for control of bean rust caused by Uromyces appendiculatus . The strains, found previously to be antagonistic to some fungal pathogens, were isolated from dry edible bean ( Phaseolus vulgaris ) and other hosts. Only Pantoea agglomerans B1, from a bean blossom, and Stenotrophomonas maltophilia C3, a chitinolytic strain from a Kentucky bluegrass leaf, were effective in multiple experiments in reducing bean rust severity. The addition of colloidal chitin to C3 cell suspensions and treatment with chitin broth cultures of C3 were evaluated as methods to improve biocontrol efficacy of C3. While chitin amendments increased rust control in the greenhouse as compared to C3 cells in buffer, chitin broth cultures gave the highest- and longest-lasting level of control. In four field experiments, treatments with C3 suspended in buffer, with and without chitin amendment, reduced rust severity in only one experiment. Strain B1 was not effective. In three other field experiments, C3 chitin broth cultures were comparable to multiple applications of thiophanate methyl or thiophanate methyl combined with manganese ethylenebisdithiocarbamate (maneb) in reducing bean rust severity.

Evaluation of Resistance Screening Methods for Sclerotinia Stem Rot of Soybean and Dry Bean

Three methods to identify levels of resistance to Sclerotinia sclerotiorum in soybean (Glycine max) and dry bean (Phaseolus vulgaris) were compared using multiple data analyses. The three methods were mycelial plug inoculations of cotyledons, cut stems, and detached leaves. Six S. sclerotiorum isolates of known relative aggressiveness were inoculated on each of three soybean and dry bean cultivars with varied response to S. sclerotiorum. For soybean, all three inoculation methods accurately identified isolate aggressiveness irrespective of cultivar, but identification of susceptible and partially resistant soybean cultivars was influenced by isolate. For dry bean, the cotyledon and cut stem methods accurately identified isolate aggressiveness, but identification of susceptible and partially resistant dry bean cultivars was influenced by isolate and inoculation method. The cut stem method had the smallest coefficient of variation and was more precise for detecting interactions. When considering root mean square residual error combined over species and experiments, coefficient of variation based on residual error, significance of isolate-by-cultivar interaction from ANOVA, rank correlation between pairs of methods, and sensitivity ratio for the three resistance screening methods under controlled environmental conditions, the cut stem method was statistically better than the cotyledon and detached leaf methods for evaluating resistance in soybean and dry bean cultivars.

Isolates of Uromyces appendiculatus with Specific Virulence to Landraces of Phaseolus vulgaris of Andean Origin

Five isolates of the bean rust fungus Uromyces appendiculatus were shown to be specifically virulent on bean genotypes of Andean origin. This specificity was demonstrated by the virulence of five pairs of isolates on a differential set of 30 Phaseolus vulgaris landraces. Each isolate pair was from a different country in the Americas and consisted of one Andean-specific isolate and one nonspecific isolate. Of the differential P. vulgaris landraces, 15 were of Middle American origin and 15 were of Andean origin. The Andean-specific rust isolates were highly virulent on Andean landraces but not on landraces of Middle American origin. Rust isolates with virulence to Middle American landraces were also generally virulent on Andean material; no truly Middle American-specific isolates were found. Random amplified polymorphic DNA (RAPD) analysis of the rust isolates also distinguished the two groups. Four of the Andean-specific rust isolates formed a distinct group compared to four of the nonspecific isolates. Two of the isolates, one from each of the two virulence groups, had intermediate RAPD banding patterns, suggesting that plasmagomy but not karyogamy occurred between isolates of the two groups.

Phylogenetic analysis of Rhizoctonia solani subgroups associated with web blight symptoms on common bean based on ITS-5.8S rDNA

Sixty-eight Rhizoctonia solani isolates (31 AG-1, 37 of AG-2-2) associated with web blight (WB) of common bean, Phaseolus vulgaris, were examined for sequence variations in the ITS-5.8S rDNA region. The isolates were collected in bean-growing lowland and mountainous regions in Central and South America. Sequences of these isolates were aligned with other known R. solani sequences from the NCBI GenBank and distance and parsimony analysis were used to obtain phylogenetic trees. WB isolates of AG-1 formed two clades separated from known AG-1 subgroups. WB isolates of AG-2-2 formed one clade separated from known AG-2-2 subgroups. Other isolates belonged to AG-1 IA and AG-1 IB. Based on phylogenetic analysis, we confirmed that at least five genetically different subgroups incite WB of common beans. Three new subgroups of R. solani have been identified and designated as AG-1 IE, AG-1 IF and AG-2-2 WB. DNA sequences of these isolates provided needed information to design taxon-specific primers that can be employed in ecological/epidemiological studies and seed health tests.

Phenotypic and Genotypic Characterization of Uromyces appendiculatus from Phaseolus vulgaris in the Americas

Populations of 90 Uromyces appendiculatus isolates were collected from throughout the Americas and evaluated for virulence on 19 standard bean rust differentials, and also on 12 landraces of Phaseolus vulgaris from South and Central America. The landrace differentials represented geographical centers of bean domestication. Three groups were observed. Two groups were isolates from centers of bean domestication and a third heterogeneous group comprised isolates from countries in South and Central America. Molecular analysis using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was also conducted on these isolates. Cluster analysis of the molecular profiles showed three groups that corresponded to those obtained by virulence tests. These results show a clear differentiation of the pathogen population along similar lines as its host and suggest parallel evolution in the bean rust pathosystem.

Variation in Sclerotinia sclerotiorum Bean Isolates from Multisite Resistance Screening Locations

There is no complete resistance to Sclerotinia sclerotiorum, cause of white mold in dry bean (Phaseolus vulgaris). Variable resistance expression is one problem in screening for improved white mold resistance. With no previous information in the literature, pathogen variation in multisite screening nurseries was evaluated as one cause of diverse resistance expression. In all, 10 isolates of S. sclerotiorum used in greenhouse screening and 146 isolates collected from nine white mold field screening nurseries in major bean production areas in the United States were compared using mycelial compatibility groupings (MCGs) and an aggressiveness test. These 10 greenhouse screening isolates formed six MCGs. Among 156 field and greenhouse isolates, 64 MCGs were identified and 36 of those were each composed of a single unique isolate. Significant differences in isolate aggressiveness were found between some isolates in different MCGs but the isolates within an MCG did not differ in aggressiveness. High isolate variation found within and between field locations could influence the disease phenotype of putative white mold resistant germplasm. We next compared genotype and phenotype of isolates from screening nurseries and those from producer fields. Variability found in and among screening locations did reflect variability found in the four producer fields sampled. White mold resistance screening can be improved by knowledge of isolate genotypic and phenotypic characteristics.

Contributions of the Bean/Cowpea CRSP to management of bean diseases

Abstract Diseases are a major constraint to production of beans in developing countries, reducing yields and seed quality. Contributing factors include poor disease management, lack of resistant cultivars, and the limited availability of certified disease-free seed. From the outset the Bean/Cowpea Collaborative Research Support Program (CRSP) has emphasized integrated disease management, and breeding for resistance to bean rust ( Uromyces appendiculatus ), common bacterial blight ( Xanthomonas campestris (= X. axonopodis ) pv. phaseoli ) (CBB) and web blight ( Thanatephorus cucumeris (anamorph Rhizoctonia solani )) (WB). Later in the 1990s, angular leaf spot ( Phaeoisariopsis griseola ) (ALS), and virus induced bean common mosaic (BCM) and bean common mosaic necrosis (BCMN) became more widespread and epidemic. The research included development of disease-resistant germplasm and studies of pathogenic variation and epidemiology. During the early 1990s bean golden yellow mosaic (BGYM), became a major disease of common bean in the Caribbean and Central America. No cultivar resistant to the gemini virus causing BGYM was available in the Dominican Republic (DR) and pesticides were used to reduce transmission of the virus by white flies ( Bremisia tabaci ) biotype A. A change in the bean-production system to limit reproduction of the vector through a host-free period and concentrate the bean growing season to a four month period reduced BGYM significantly. Improved resistant cultivars and disease management resulted in high yields and self-sufficiency of beans in the DR. Evidence for co-evolution of the pathogens causing ALS, CBB, and rust in the two major bean gene pools (Andean and Middle American) was found. Variation in the WB pathogen on bean indicated independent genetic populations and the presence of different anastosmosis subgroups. WB disease management practices should be designed for the sub-group of the pathogen. Seed transmission was found to be a significant source of R. solani inoculum. Specific ( Ur-9 gene) and adult-plant resistance ( Ur-12 gene) to rust was identified for PC-50 (Andean origin) in the DR, and the genes were mapped. A mobile rust nursery was developed to monitor races of rust in a region and assist in resistance-gene deployment in bean germplasm and varieties. DNA based diagnostic methods were developed to differentiate X. campestris pv. phaseoli from X. campestris pv. phaseoli var fuscans (prevalent in East Africa) and P. griseola isolates. DNA hybridization methods also were developed to identify BGYM and bean golden mosaic viruses. Research in USA and East Africa has helped define bean common mosaic virus (BCMV) and bean common mosaic necrotic virus (BCMNV) as separate viruses and has demonstrated that BCMNV appears to be indigenous to Africa. Serological tools were developed that assist in the detection and identification of potyviruses, BCMV and BCMNV. These tools are now used worldwide.

Molecular markers linked to genes for specific rust resistance and indeterminate growth habit in common bean

Bulked segregant analysis was utilized to identify random amplified polymorphic DNA (RAPD) markers linked to genes for specific resistance to a rust pathotype and indeterminate growth habit in an F2 population from the common bean cross PC-50 (resistant to rust and determinate growth habit) × Chichara 83-109 (susceptible to rust and indeterminate growth habit). Six RAPD markers were mapped in a coupling phase linkage with the gene ( Ur-9) for specific rust resistance. The linkage group spanned a distance of 41 cM. A RAPD marker OA4.1050 was the most closely linked to the Ur-9 gene at a distance of 8.6 cM. Twenty-eight RAPD markers were mapped in a coupling phase linkage with the gene ( Fin) for indeterminate growth habit. The linkage group spanned a distance of 77 cM. RAPD markers OQ3.450 and OA17.600 were linked to the Fin allele as flanking markers at a distance of 1.2 cM and 3.8 cM, respectively. The RAPD markers linked to the gene for specific rust resistance of Andean origin detected here, along with other independent rust resistance genes from other germplasm, could be utilized to pyramid the different genes into a bean cultivar for durable rust resistance.

Differential Pathogenicity of Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans Strains on Bean Genotypes with Common Blight Resistance

Both the common bacterial blight (CBB) pathogen (Xanthomonas campestris pv. phaseoli) and X. fuscans subsp. fuscans, agent of fuscous blight, cause indistinguishable symptoms in common bean, Phaseolus vulgaris. Yield losses can exceed 40%. Lack of information about the specificity between X. campestris pv. phaseoli strains and major quantitative trait loci (QTL) or alleles conferring resistance makes the task of identifying genetic changes in host-pathogen interactions and the grouping of bacterial strains difficult. This, in turn, affects the choice of pathogen isolates used for germplasm screening and complicates breeding for CBB resistance. Common bean host genotypes carrying various sources and levels of resistance to CBB were screened with 69 X. campestris pv. phaseoli and 15 X. fuscans subsp. fuscans strains from around the world. Differential pathogenicity of the CBB pathogen was identified on the 12 selected bean genotypes. The X. fuscans subsp. fuscans strains showed greater pathogenicity than X. campestris pv. phaseoli strains having the same origin. African strains were most pathogenic. The largest variation in pathogenicity came from X. campestris pv. phaseoli strains that originated in Caribbean and South American countries. Pathogenic variation was greater within X. campestris pv. phaseoli than within X. fuscans subsp. fuscans strains. Implications for breeding for CBB resistance are discussed.

Genetic Variation Among Isolates of the Web Blight Pathogen of Common Bean Based on PCR-RFLP of the ITS-rDNA Region

Variability of 45 isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris) causing web blight (WB) of common bean, Phaseolus vulgaris, was examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S subunit (5.8S) of the nuclear ribosomal DNA repeat (ITS-5.8S-rDNA). Isolates were collected from diseased bean leaves from Argentina, Costa Rica, Cuba, Dominican Republic, Honduras, Panama, and Puerto Rico. These WB isolates belong to AG-1 and AG-2 based on anastomosis reaction. Isolates of AG-1 that cause WB were separated into three distinct groups of RFLP patterns from enzymatic digestion of a 740-bp PCR fragment. Microsclerotia-producing isolates (<1 mm) were differentiated from macrosclerotia-producing isolates (5 to 20 mm) based on PCR-RFLP patterns even though they are placed in the same AG1-1B subgroup by anastomosis reaction. WB isolates of AG-2 were separated into two distinct PCR-RFLP groups as previously reported. AG-1 macrosclerotial-producing isolates were the most virulent, whereas isolates of AG-2 were the least virulent. Genetic variability of the WB pathogen may have influenced the failure or success of management practices implemented in the past in Latin America.