Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Sample size for measurement of root traits on common bean by image analysis

Evaluation of root traits may be facilitated if they are assessed on samples of the root system. The objective of this work was to determine the sample size of the root system in order to estimate root traits of common bean (Phaseolus vulgaris L.) cultivars by digital image analysis. One plant was grown per pot and harvested at pod setting, with 64 and 16 pots corresponding to two and four cultivars in the first and second experiments, respectively. Root samples were scanned up to the completeness of the root system and the root area and length were estimated. Scanning a root sample demanded 21 minutes, and scanning the entire root system demanded 4 hours and 53 minutes. In the first experiment, root area and length estimated with two samples showed, respectively, a correlation of 0.977 and 0.860, with these traits measured in the entire root. In the second experiment, the correlation was 0.889 and 0.915. The increase in the correlation with more than two samples was negligible. The two samples corresponded to 13.4% and 16.9% of total root mass (excluding taproot and nodules) in the first and second experiments. Taproot stands for a high proportion of root mass and must be deducted on root trait estimations. Samples with nearly 15% of total root mass produce reliable root trait estimates.

Environmental effects on photosynthetic capacity of bean genotypes

Photosynthetic responses to daily environmental changes were studied in bean (Phaseolus vulgaris L.) genotypes 'Carioca', 'Ouro Negro', and Guarumbé. Light response curves of CO2 assimilation and stomatal conductance (g s) were also evaluated under controlled (optimum) environmental condition. Under this condition, CO2 assimilation of 'Carioca' was not saturated at 2,000 µmol m-2 s-1, whereas Guarumbé and 'Ouro Negro' exhibited different levels of light saturation. All genotypes showed dynamic photoinhibition and reversible increase in the minimum chlorophyll fluorescence yield under natural condition, as well as lower photosynthetic capacity when compared with optimum environmental condition. Since differences in g s were not observed between natural and controlled conditions for Guarumbé and 'Ouro Negro', the lower photosynthetic capacity of these genotypes under natural condition seems to be caused by high temperature effects on biochemical reactions, as suggested by increased alternative electron sinks. The highest g s values of 'Carioca' were observed at controlled condition, providing evidences that reduction of photosynthetic capacity at natural condition was due to low g s in addition to the high temperature effects on the photosynthetic apparatus. 'Carioca' exhibited the highest photosynthetic rates under optimum environmental condition, and was more affected by daily changes of air temperature and leaf-to-air vapor pressure difference.

Nitrogen fixation by groundnut and velvet bean and residual benefit to a subsequent maize crop

Chemical fertilisers are rarely avaiable to poor farmers, for whom the nitrogen (N) is often the most limiting element for cereal grain production. The objective of this study was to quantify the contribution of biological nitrogen fixation (BNF) to groundnut (Arachis hypogaea) and velvet bean (Mucuna pruriens) crops using the 15N natural abundance (delta15N) technique and to determine their residual effect and that of a natural fallow, on growth and N accumulation by two rustic maize varieties. The contribution of BNF calculated from delta15N data was 40.9, 59.6 and 30.9 kg ha-1, for groundnut, velvet bean and the natural fallow, respectively. The only legume grain harvested was from the groundnut, which yielded approximately 1.000 kg ha-1. The subsequent maize varieties ("Sol de Manhã" and "Caiana Sobralha") yielded between 1.958 and 2.971 kg ha-1, and were higher after velvet bean for both maize varieties and "Sol da Manhã" groundnut, followed by "Caiana" after groundnut and, finally, the natural fallow. For a small-holder producer the most attractive system is the groundnut followed by maize, as, in this treatment, both groundnut and maize grain harvest are possible. However, a simple N balance calculation indicated that the groundnut-maize sequence would, in the long term, deplete soil N reserves, while the velvet bean-maize sequence would lead to a build up of soil nitrogen.

Rhizobium strains competitiveness on bean nodulation in Cerrado soils

The objective of this work was to identify the most competitive and effective Rhizobium strains in order to increase common bean yield by nitrogen fixation as alternative or complementation to the nitrogen fertilization. Competitiveness tests were lead in axenic conditions, in Cerrado soil pots and in three field experiments, with native Rhizobium strains that were previously identified, according to their effectiveness and genetic variability. The identification of strains in nodules was performed using serological tests (axenic conditions) - agglutination and enzyme linked immunosorbent (Elisa) assays - and random amplified polymorfic DNA (RAPD) (Cerrado soil). Plant yield was determined using the dry weight (greenhouse conditions), total N and grain yield (field experiments). Among the analyzed Rhizobium strains, native strain SLA 2.2 and commercial strain CIAT 899 were the dominant nodules in plants of the most productive plots, presenting yield productivity similar or higher to those obtained in treatments where 20 kg ha-1 of N were applied.

Microsatellites for genetic studies and breeding programs in common bean

Twenty microsatelitte loci were identified and characterized in common bean. Microsatellites were tested in 14 genotypes. The allele number ranged from 1 to 3, and the polymorphism information content (PIC) was between 0.14 and 0.65. These polymorphic markers are available to be used for breeding programs.

Method for evaluation of root hairs of common bean genotypes

The objective of this work was to test a simple method for root hair evaluation of 21 common bean (Phaseolus vulgaris) genotypes, most of them used in breeding programs in Brazil. Hairs of basal and primary roots of 5-day old seedlings, produced on germination paper with no phosphorus addition, were visually evaluated by a rating scale after staining with 0.05% trypan blue. The method reveals variability among the genotypes, and the standard error of the mean is relatively low.

Root traits of common bean genotypes used in breeding programs for disease resistance

The objective of this work was to assess root traits of 19 common bean genotypes, used in breeding programs for disease resistance. Genotypes DOR 364 and G 19833 were used as deep and shallow basal root checks, respectively. The number of whorls and basal roots were assessed on five-day old seedlings grown in germination paper. Growth pouch studies were conducted to evaluate basal root gravitropism and lateral root length from primary roots, in seven-day old seedlings. The following root gravitropic traits were estimated: basal growth angle, shallow basal root length (localized in the top 2 cm), and relative shallow basal root growth. Number of whorls varied from 1.47 to 3.07, and number of basal roots ranged from 5.67 (genotype TO) to 12.07 (cultivar Jalo MG-65). Cultivars BRS MG Talismã, Carioca, BRS Pioneiro, and Diamante Negro exhibited shallow basal roots, while genotypes Vi-10-2-1, TU, AB 136, and México 54 showed deep basal roots. Cultivar Jalo MG-65 showed more lateral roots from the primary root than the other genotypes. Genotypes used on common bean breeding programs for disease resistance have great variability on basal and primary root traits.

New microsatellite markers developed from an enriched microsatellite common bean library

The objective of this work was to develop new microsatellite markers in common bean. Ninety nine new microsatelitte loci were developed from a microsatellite enriched library for (CT)8 and (GT)8 motifs, from CAL-143 line. The majority of microsatellite sequences (51%) was related to cellular metabolism. The remaining sequences were associated to transcription functions. Only 17.2% of the sequences presented some level of similarity with other plant species genes.

Combining ability and heterosis in common bean cultivars

The objective of this work was to determine the combining ability and heterosis, for productivity and yield components, in diallel hybrids derived from crossings between BRSMG-Talismã, IPR Uirapuru, FT Soberano, BRS Campeiro, IAC Tybatã, and IPR Juriti parent cultivars. Fifteen hybrids were generated from diallel crosses, excluding reciprocals. The general and specific combining abilities were significant for plant height, number of pods per plant, number of seeds per plant, number of seeds per pod, 50-seed weight, and grain yield, indicating the occurrence of both additive and nonadditive genetic effects. The best strategy to be adopted is the use of BRS Campeiro, FT Soberano and BRSMG-Talismã cultivars in common bean breeding programs involving selection. The most promising combinations were 'IPR Uirapuru' x 'IAC Tybatã', 'IPR Uirapuru' x 'FT Soberano', 'BRS Campeiro' x 'IPR Juriti', and 'BRS Campeiro' x 'IAC Tybatã'. The parents of these hybrids presented high estimates of specific combining abilities. Hybridization of cultivars belonging to distinguished commercial groups propitiates higher heterosis values in the segregant population.

Genetic characterization and nitrogen fixation capacity of Rhizobium strains on common bean

This study aimed to genetically characterize four new Rhizobium strains, and to evaluate their nodulation and fixation capacity compared to commercial strains and to native rhizobia population of a Brazilian Rhodic Hapludox. Two experiments were carried out in randomized blocks design, under greenhouse conditions, in 2007. In the first experiment, the nodulation and nitrogen fixation capacity of new strains were evaluated, in comparison to the commercial strains CIAT-899 and PRF-81 and to native soil population. It was carried out in plastic tubes filled with vermiculite. DNA extractions and PCR sequencing of the intergenic space were made from the isolated pure colonies, in order to genetically characterize the strains and the native rhizobia population. In the second experiment, the nodulation and productivity of common beans Perola cultivar were determined, with the use of evaluated strains, alone or in mixture with PRF-81 strain. It was carried out in pots filled with soil. The native soil population was identified as Rhizobium sp. and was inefficient in nitrogen fixation. Three different Rhizobium species were found among the four new strains. The LBMP-4BR and LBMP-12BR new strains are among the ones with greatest nodulation and fixation capacity and exhibit differential responses when mixed to PRF-81.

Toxicity of neem oil to Bemisia tabaci biotype B nymphs reared on dry bean

The objective of this work was to determine the most susceptible nymphal stage of Bemisia tabaci biotype B to neem (Azadirachta indica A. Juss.) oil applied to dry bean (Phaseolus vulgaris L.) in a screenhouse. A solution of commercial oil (Dalneem) extracted from neem seeds was sprayed directly on each nymphal instar at 0, 0.1, 0.25, 0.5, 1 and 2% concentrations for lethal concentration (LC) determination, and at 0, 0.5 and 1% concentrations for lethal time (LT) determination. The number of living and dead nymphs was recorded five days after spraying for LC determination, and daily during six days for LT determination. The LC50 estimated for fourth instar nymphs occurred at 0.56% concentration. For all instars, LC50 and LC95 were estimated at 0.32 and 2.78% concentrations, respectively. The estimated values of LT50 at 1% concentration were 2.46, 4.45, 3.02 and 6.98 days for the first to fourth instars, respectively. The LT50 occurred at five days for 0.5% and at four days for 1% concentration in all instars. A mortality rate of over 80% was observed on the 6th day for the first to third instars at 1% concentration. The first three nymphal stages were more susceptible to neem oil when compared to the fourth nymphal stage.

Quantification of the diversity among common bean accessions using Ward-MLM strategy

The present work aimed at evaluating the divergence among common bean accessions by their agronomic, morphological and molecular traits, based on the Ward-MLM procedure. A collection of 57 accessions from the gene bank of Universidade Federal do Espírito Santo was used in this study, from which: 31 were landraces belonging to the community Fortaleza, in the municipality of Muqui, ES, Brazil; 20 accessions were provided by Embrapa Trigo; and 6 were commercial cultivars. Five agronomic traits (plant cycle, number of seeds per pod, number of pods per plant, weight of 100 seeds, and grain yield), five morphological traits (growth habit, plant size, seed shape, seed color, and commercial group) and 16 microsatellite primers were evaluated. High genetic variability was detected considering morphological, agronomic and molecular traits in the 57 common bean accessions studied. The Ward-MLM procedure showed that the ideal number of groups was five, according to the pseudo F and pseudo t² criteria. The accessions from Andean origin had heavier seeds than others and formed a cluster. The Ward-MLM statistical procedure is a useful technique to detect genetic divergence and to cluster genotypes by simultaneously using morphological, agronomic and molecular data.

Potential of antimicrobial volatile organic compounds to control Sclerotinia sclerotiorum in bean seeds

The objective of this work was to evaluate the potential of an artificial mixture of volatile organic compounds (VOCs), produced by Saccharomyces cerevisiae, to control Sclerotinia sclerotiorum in vitro and in bean seeds. The phytopathogenic fungus was exposed, in polystyrene plates, to an artificial atmosphere containing a mixture of six VOCs formed by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate), in the proportions found in the atmosphere naturally produced by yeast. Bean seeds artificially contamined with the pathogen were fumigated with the mixture of VOCs in sealed glass flasks for four and seven days. In the in vitro assays, the compounds 2-methyl-1-butanol and 3-methyl-1-butanol were the most active against S. sclerotiorum, completely inhibiting its mycelial growth at 0.8 µL mL-1, followed by the ethyl acetate, at 1.2 µL mL-1. Bean seeds fumigated with the VOCs at 3.5 µL mL-1 showed a 75% reduction in S. sclerotiorum incidence after four days of fumigation. The VOCs produced by S. cerevisiae have potential to control the pathogen in stored seeds.