Multiple Effect of Different Plant Growth Promoting Microorganisms on Beans (Phaseolus vulgaris L. ) Crop

Abstract We evaluated the effect of combined Rhizobium tropici, Trichoderma asperellum and plant growth-promoting rhizobacteria (PGPR) in beans crop. The hypothesis that strains of T. asperullum, R. tropici and PGPR combined could improve growth, biomass accumulation and beans yield was tested under greenhouse and field conditions. The treatments consisted of control, mineral nitrogen application and inoculation, isolated and associated with the following microorganisms: Rhizobium tropici, Bacillus subtilis, Trichoderma asperellum and Burkholderia sp. 10N6. Results were evaluated by shoot dry weight (SDW) and root dry weight (RDW), number of nodules and yield components. In greenhouse environment all the microorganisms behaved similarly, and the treatments inoculated with Burkholderia sp. 10N6 (IBu) and R. tropici (IR) stood out regarding the production components. In field conditions the treatments IR and IRTBa presented the highest values of SDW and RDW. Our results suggest that inoculation with R. tropici, T. asperellum and PGPR may promote beans growth and bring benefits to shoot and root accumulation, increase the number of nodules as well as improve yield components, contributing to a sustainable agriculture.

Essential oils of Tahiti lemon and cinnamon bark in control of storage fungi and the physiological and sanitary quality of beans / Óleos essenciais de limão Taiti e canela em casca no controle de fungos de armazenamento e na qualidade fisiológica e sanitária de feijão

With the objective to evaluate the efficiency of essential oils of Citrus latifolia (Tahiti lemon) and Cinnamomum zeylanicum (cinnamon bark) in the control of plant pathogens Penicillium sp. and Aspergillus sp. and the quality of the bean seeds, two experiments were conducted. In the first one, the effect of essential oils of C. latifolia and C. zeylanicum was evaluated in vitro development of the fungi Penicillium sp. and Aspergillus sp. and, in the second one, the influence of essential oils on the physiological and sanitary quality of bean seeds. The variables mycelial growth, conidial germination and sporulation of Penicillium sp. and Aspergillus sp. were measured in the first experiment, while the seed germination test, first count of germination, germination speed index (GSI) and sanity test of bean seeds were measured in the second. The essential oil (EO) of C. zeylanicum was more efficient than C. latifolia in the control of Aspergillus sp. and Penicillium sp., but decreased the physiological quality of the beans seeds. The fungal diversity identified in the seed health test was composed by fungi of the genera Aspergillus, Penicillium, Cladosporium, Fusarium, Chaetomium and Rhizopus. The results indicate the potential of the use of these EOs in the seeds treatment.(AU)

Com o objetivo de avaliar a eficiência dos óleos essenciais de Citrus latifolia (limão taiti) e Cinnamomum zeylanicum (canela em casca) no controle dos fitopatógenos Penicillium sp. e Aspergillus sp. e na qualidade das sementes de feijão, foram conduzidos dois experimentos. No primeiro, avaliou-se o efeito dos óleos essenciais de C. latifolia e C. zeylanicum no desenvolvimento in vitro dos fungos Penicillium sp. e Aspergillus sp. e, no segundo, a influência dos óleos essenciais sobre a qualidade fisiológica e sanitária das sementes de feijão. As variáveis crescimento micelial, germinação de conídios e esporulação de Penicillium sp. e Aspergillus sp. foram aferidas no primeiro experimento, enquanto o teste de germinação de sementes, primeira contagem de germinação, índice de velocidade de germinação (IVG) e teste de sanidade de sementes de feijão foram aferidas no segundo. O óleo essencial (OE) de C. zeylanicum foi mais eficiente que C. latifolia no controle dos fungos Aspergillus sp. e Penicillium sp., mas diminuiu a qualidade fisiológica das sementes de feijão. A diversidade fúngica identificada no teste de sanidade de sementes foi composta por fungos dos gêneros Aspergillus, Penicillium, Cladosporium, Fusarium, Chaetomium e Rhizopus. Os resultados indicam o potencial do uso desses óleos essenciais no tratamento de sementes.(AU)

Biochar and Trichoderma harzianum for the Control of Macrophomina phaseolina

Abstract This study is based on the importance of biological control methods and the lack of information on the effect of biochar (BCH) from sewage sludge associated or not with Trichoderma harzianum on the control of Macrophomina phaseolina in the bean crop (Phaseolus vulgaris, cv. BRS Estilo). Biochar from sewage sludge, pyrolyzed at 500 ºC and used in low concentration (0.5%), has a direct effect on the in vitro control of M. phaseolina. However, higher BCH concentrations stimulated the growth of the pathogen. In culture medium with or without BCH, T. harzianum (strain 1306) inhibited the mycelial growth of M. phaseolina. The addition of BCH + T. harzianum reduced the deleterious effects caused by M. phaseolina on bean plants. This study demonstrated that joint application of BCH from sewage sludge + T. harzianum considerably increased the fresh and dry mass of bean plants, inoculated or not with M. phaseolina.

Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

Abstract This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.

Symbiotic potential and survival of native rhizobia kept on different carriers

Native rhizobia are ideal for use as commercial legume inoculants. The characteristics of the carrier used to store the inoculants are important for the survival and symbiotic potential of the rhizobia. The objective of this study was to investigate the effects of peat (PEAT), perlite sugarcane bagasse (PSB), carboxymethyl cellulose plus starch (CMCS), and yeast extract mannitol supplemented with mannitol (YEMM) on the survival, nodulation potential and N₂ fixation capacity of the native strains Sinorhizobium mexicanum ITTG R7^T and Rhizobium calliandrae LBP2-1^T and of the reference strain Rhizobium etli CFN42^T. A factorial design (4 × 3) with four repetitions was used to determine the symbiotic potential of the rhizobial strains. The survival of the strains was higher for PEAT (46% for strain LBP2-1^T, 167% for strain CFN42^T and 219% for strain ITTG R7^T) than for the other carriers after 240 days, except for CFN42^T kept on CMCS (225%). All the strains kept on the different carriers effectively nodulated common bean, with the lowest number of nodules found (5 nodules) when CFN42^T was kept on CMCS and with the highest number of nodules found (28 nodules) when ITTG R7^T was kept on PSB. The nitrogenase activity was the highest for ITTG R7^T kept on PEAT (4911 μmol C₂H₄ per fresh weight nodule h⁻¹); however, no activity was found when the strains were kept on YEMM. Thus, the survival and symbiotic potential of the rhizobia depended on the carrier used to store them.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Desarrollo de un alimento de humedad intermedia de fréjol (Phaseolus vulgaris L.)

El consumo de fréjoles se ha asociado a la reducción del riesgo de desarrollo de enfermedades crónicas no transmisibles. En general, su consumo es menor al recomendado y se realiza mayoritariamente como granos enteros. Para aumentar su consumo, hay un especial interés en su uso en otras formas. El objetivo de esta investigación fue diseñar purés de humedad intermedia (PHI) de fréjol de buena calidad química, microbiológica y sensorial. Se prepararon purés con fréjol Tórtola remojado, cocido y molido, retirando una parte importante de la testa. En la elaboración de los PHI, se estudiaron dos niveles de humedad, 25% y 30%, y dos niveles de glicerol, 10 y 15%. Se conservaron a temperatura ambiente (20 ±2 ºC) por 30 días. En los purés iniciales se determinó su composición proximal, contenido de fitatos e inhibidores de tripsina. Los PHI se analizaron el día de su preparación y luego de 15 y 30 días de almacenamiento. Se determinó su humedad, pH y actividad de agua. Se realizó recuento total de: aerobios mesófilos, de mohos y levaduras, de coliformes totales, de coliformes fecales y de Staphylococcus aureus. Se determinó calidad y aceptabilidad sensorial. Los PHI tuvieron una aw entre 0,800 y 0,844, pH entre 6,20 y 6,24 y sus recuentos microbiológicos fueron negativos o estuvieron muy por debajo de los límites permitidos para los alimentos más exigentes. Presentaron buenas calificaciones en su calidad sensorial y todos fueron bien aceptados. Los parámetros sensoriales se mantuvieron sin variación durante el almacenamiento, excepto el color que en el tratamiento con 30% de humedad y 10% de glicerina mostró un leve oscurecimiento. Es posible elaborar purés de fréjol de humedad intermedia, con bajos niveles de compuestos antinutricionales, de buena calidad y aceptabilidad sensorial y que ofrecen condiciones adversas para el desarrollo de los microorganismos de mayor significación para la inocuidad alimentaria.

Development of an intermediate moisture food from bean (Phaseolus vulgaris L.). Beans consumption has been associated to reduction on chronic, non transmissible, diseases development. Generally, its consumption is less to the recommended and it is done principally as whole grain dishes. To increase the consumption, there is an especial interest in the use of new products. The main goal of this research was to design intermediate moisture bean purees (IMP) using soaked, cooked and grained Tortola bean, removing an important testa portion. In preparation of IMP two moisture levels, 25 and 30%, and two glycerol levels, 10 and 15%, were studied. Purees were preserved at ambient temperature (20 ±2 ºC) for 30 days. Proximal analysis, phytate content and trypsin inhibitors were determined in the original non processed purees. IMPs were analyzed at preparation time and after 15 and 30 days of storage for moisture content, pH and water activity. Besides, microbiological analysis for total plate count on aerobic mesophile microorganisms, molds and yeast, total colifoms, fecal coliforms and Staphylococcus aureus, was carried out. Global quality and sensory acceptance was also determined. IMPs presented aw values between 0.800 and 0.844, a pH between 6.20 and 6.24 and the microbiological counting was negative or far below the accepted limits for more strict foods. IMPs presented good qualifications for global quality and were well accepted. Sensory parameters maintain their values during storage, except for color that in treatment with 30% moisture and 10% glycerol showed a light darkening. It is possible to elaborate intermediate moisture bean puree, with low level of antinutritional compounds, good quality and sensory acceptance and with adverse conditions for the development of food safety importance microorganisms.

Desenvolvimento de Zabrotes subfasciatus (Boh.) (Coleoptera: Chrysomelidae, Bruchinae) em genótipos de Phaseolus vulgaris L. (Fabaceae) cultivados no Estado do Paraná e contendo arcelina / Development of Zabrotes subfasciatus (Boh.) (Coleoptera: Chrysomelidae, Bruchinae) in genotypes of Phaseolus vulgaris L. (Fabaceae) cultivated in the State of Parana and containing arcelin

O presente trabalho objetivou avaliar o desenvolvimento de Zabrotes subfasciatus (Boh.), praga de grãos armazenados, em genótipos de feijoeiro (Phaseolus vulgaris L.) comumente cultivados no Paraná e com arcelina, e a possível resistência desses materiais ao bruquíneo. Foram realizados testes em laboratório (27°C, 50 ± 10 por cento UR, fotofase 12h) com os genótipos TPS-Bionobre, IAC-Una, IPR-Uirapuru, IAPAR 44, IPR Juriti, IAPAR 81, Pérola, Carioca e Bolinha, além de dois outros contendo arcelina, Arc 1 e Arc 2. Os genótipos com os alelos Arc 1 e 2 sugerem resistência do tipo antibiose, com elevada mortalidade das fases imaturas, e Arc 1, com período de desenvolvimento prolongado e drástica redução de peso de machos e fêmeas. A resistência do tipo não-preferência para oviposição não foi observada para estes dois genótipos. Dentre os genótipos sem arcelina, IAPAR 44 destacou-se como o mais resistente a Z. subfasciatus, pois foi o menos preferido para oviposição, promovendo baixo percentual de ovos viáveis, longo período de desenvolvimento e reduzido peso de machos e fêmeas. As cultivares Pérola, IPR Juriti e Bolinha foram os mais suscetíveis, com alto número de ovos e ovos viáveis, além de baixa porcentagem de mortalidade de fases imaturas.

This research intended to evaluate the development of Zabrotes subfasciatus (Boh.), a stored-grain pest, on bean genotypes (Phaseolus vulgaris L.) commonly cultivated in the State of Parana and containing arcelin, and the possible resistance of these genotypes to the bruchine. Tests were performed under laboratory conditions (27°C, fotophase 12h, 50 ± 10 percent RH) with the genotypes TPS-Bionobre, IAC-Una, IPR-Uirapuru, IAPAR 44, IPR Juriti, IAPAR 81, Pérola, Carioca, Bolinha, and two others containing arcelin, Arc 1 and Arc 2. The genotypes with Arc 1 and 2 alleles caused higher mortality of immature stages; in Arc 1 developmental period was prolonged and the male and female dry weights were the lowest, suggesting an antibiosis mechanism of resistance. Non-preference for oviposition was not observed for these two genotypes. Among varieties without arcelin, IAPAR 44 was the most resistant to the bruchid, being the least preferred for oviposition, and promoting low percentage of viable eggs, long developmental period and reduced male and female adult dry weight. Perola, IPR Juriti and Bolinha with high number of eggs and viable eggs, low mortality of immature stages, were the most susceptible.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Influência dos tricomas na preferência para oviposição de Bemisia tabaci (Genn) biótipo B (Hemiptera: Aleyrodidae) em genótipos de feijoeiro / Influence of trichomes on ovipositional preference of Bemisia tabaci (Genn) biotype B (Hemiptera: Aleyrodidae) for bean genotypes

Foi testada a preferência para oviposição (com chance de escolha) de Bemisia tabaci (Genn.) biótipo B em 20 genótipos de feijoeiro. Os materiais foram divididos em dois lotes, utilizando-se em cada um deles delineamento em blocos ao acaso, com 20 repetições. Folhas recém-expandidas de cada genótipo foram mantidas em tubos de vidro contendo água destilada, colocadas em uma gaiola acrílica, onde foram liberados cerca de 500 insetos. Após 24h, contou-se o número total de ovos na superfície abaxial e o número de tricomas aciculares e unciformes em duas áreas de 4,9 mm² da superfície abaxial. Mediu-se também a área foliar dos feijoeiros e o comprimento máximo e mínimo dos tricomas, que foram classificados em curto, médio ou longo. Não foram constatadas correlações significativas entre o número de ovos e a densidade de tricomas total e de tricomas aciculares e unciformes. Foram observadas correlações significativas entre o número de ovos e a densidade de tricomas aciculares longos tipo A1 (r = 2,85) e a densidade de tricomas unciformes curtos tipo B3 (r = -2,44). Assim, pode-se inferir que a preferência para oviposição da mosca-branca pela cultivar Bolinha (4,8 ovos/cm²) pode estar relacionada ao grande número de tricomas aciculares longos neste material tipo A1 (31,7 tricomas/4,9 mm²). O contrário pode ser considerado quanto ao genótipo menos preferido, G13028 (1,1 ovos/cm²), que possui principalmente tricomas unciformes curtos tipo B3 (64,1 tricomas/4,9 mm²) em suas folhas.

The oviposition preference (free-choice tests) of Bemisia tabaci (Genn.) Biotype B for 20 bean genotypes was tested. The genotypes were divided into two plots and set up in a block-randomized design with 20 replications. New expanded bean leaves were sectioned and placed in glass tubes containing distilled water and disposed in acrylic cages containing approximately 500 insects. After 24h, the total number of eggs laid on the abaxial surface of leaves and the density of straight and hooked trichomes in two areas of 4.9 mm² on the abaxial surface were counted. The leaf area was measured as well as the maximum and minimum length of hooked and straight trichomes, which were classified in short, medium or long. No significant correlation was found between the number of eggs and the density of total trichomes (straight + hooked) or between hooked or straight trichomes. Significant correlation was detected between the number of eggs and long straight trichomes type A1 (r = 2.85) and short hooked trichomes type B3 (r = -2.44). In this way, the ovipositional preference of B. tabaci for Bolinha genotype (4.8 eggs/cm²) in a free-choice test could be associated to the larger number of long straight trichomes type A1 (31.7 trichomes/4.9 mm²) on its leaves surface. In addition, the whitefly ovipositional nonpreference for G13028 wild genotype (1.1 eggs/cm²) could be related with the numerous short hooked trichomes type B3 (64.1 trichomes/4.9 mm²) on its leaves surface.

Atratividade e não-preferência para oviposição de Bemisia tabaci (Genn) biótipo B (Hemiptera: Aleyrodidae) em genótipos de feijoeiro / Atractiveness and oviposition nonpreference of Bemisia tabaci (Genn) biotype B (Hemiptera: Aleyrodidae) for bean genotypes

A mosca-branca é um dos insetos mais prejudiciais à cultura do feijoeiro, devido principalmente à intensa sucção de seiva elaborada e à transmissão do vírus do mosaico dourado. O uso de cultivares resistentes a esse inseto é uma ferramenta importante no seu controle. Foi testada a preferência para oviposição de Bemisia tabaci (Genn.) biótipo B em 20 genótipos de feijoeiro. Nos ensaios com chance de escolha, os materiais foram divididos em dois lotes de 10 genótipos, com 10 repetições de uma planta/vaso, dispostas em gaiolas de 80 x 50 x 50 cm, cobertas com voil e infestadas com 1.000 moscas-brancas durante quatro dias. Nos ensaios sem chance de escolha, realizaram-se 20 repetições de um folíolo (genótipos em melhoramento e cultivares) ou de um trifolíolo (genótipos selvagens) recém-expandido por gaiola e infestados com 25 casais de moscas-brancas durante 48h. Nos ensaios com chance de escolha, verificou-se que os genótipos G13028, Arc 5s, Arc 1s, G11056, Arc 3s apresentaram as menores oviposições e os menores índices de preferência, variando de -91,9 a -78,6, enquanto Arc 1 (+33,0) teve o maior índice. Nos ensaios sem chance de escolha, os menores números de ovos/folha foram observados nos genótipos G13028 (28,2), Arc 3s (37,6) e G23425E (39,0), enquanto que o genótipo mais preferido teve 105,0 ovos/folha (Bolinha). O genótipo selvagem G13028 foi altamente resistente (não-preferência para oviposição) a B. tabaci biótipo B. Destacaram-se também como resistentes, os genótipos Arc 3s, Arc 5s e G11056; já Bolinha, Arc 1 e Arc 4 mostraram-se altamente suscetíveis à mosca-branca.

Whitefly is one of the most harmful pests that attack bean crops, mainly for extracting large quantities of phloem sap and transmitting the bean golden mosaic virus. Resistant germoplasm plants can be an important method for controlling this pest. The oviposition preference of Bemisia tabaci biotype B for 20 bean genotypes was tested. In a free-choice test, the genotypes were divided into two plots of 10 replicates. The bean plant pots were set up in a randomized design in a cage (80 x 50 x 50 cm) covered by nylon netting and infested with 1.000 adults during four days. No-choice test was carried out with 20 replicates where one newly expanded leaflet from bred genotypes and cultivars or one leaf from wild genotypes, was placed in a cage infested by 25 pairs of whiteflies during 48h. In free-choice tests, the genotypes G13028, Arc 5s, Arc 1s, G11056, Arc 3s presented the smaller oviposition and preference index, varying from -91.9 to -78.6, while Arc 1 (+33.0) had greater index. In no-choice tests, smaller number of eggs/leaflet were observed in genotypes G13028 (28.2), Arc 3s (37.6) and G23425E (39.0), whereas the most preferred had 105.0 eggs/leaflet (Bolinha). The wild genotype G13028 was highly resistant (oviposition nonpreference) toward B. tabaci B biotype. The wild genotypes Arc 3s, Arc 5s and G11056 were also resistant. The Bolinha, Arc 1 and Arc 4 genotypes were highly susceptible to whitefly oviposition.