Chromatographic determination of shikimate for identification of conventional soybean and glyphosate resistant soybean / Determinação cromatográfica de shikimato para identificação de soja convencional e soja resistente ao glifosato

A sensitive and reliable process was established using high-performance liquid chromatography (HPLC) to distinguish conventional varieties of glyphosate-resistant genetically modified crops via shikimate detection in soybean (Glycine max L. Merril) seeds. Glyphosate has a well-defined mechanism of action. It is the only herbicide that specifically inhibits 5-enolpiruvilshikimate-3-phosphate synthase (EPSPS E.C. 2.5.1.19), which catalyzes the condensation of shikimate with phosphoenolpyruvate. This study is based on the concept that shikimate significantly accumulates in soybean plant tissues after EPSPS inhibition by glyphosate. In plants not subjected to glyphosate, shikimate is not easily detected because it quickly metabolizes into shikimate 3-phosphate and subsequently into 5-enolpiruvilshikimate 3-phosphate through the action of EPSPS. Conversely, in non-genetically modified plants subjected to glyphosate, shikimate metabolism is impaired, resulting in its accumulation. This metabolite can be detected in extremely low quantities (in the microgram range), through HPLC. In this study, six different contrasts were analyzed, each being formed by a transgenic cultivation and its parental strain, subject or not subject to the treatment of soaking with a 0.6% glyphosate solution. Chromatographic analyses indicated shikimate accumulation only in conventional cultivars with seeds previously soaked in a 0.6% glyphosate solution. Thus, this shikimate detection method can be used as a rapid and accurate means to distinguish soybeans with glyphosate-resistant qualities.

Este estudo estabelece um processo, sensível e confiável, com aplicação de cromatografia líquida de alta eficiência (HPLC) para distinguir variedades de soja convencionais de geneticamente modificadas, resistentes ao glifosato, por detecção de chiquimato nas sementes. O mecanismo de ação doglifosato é bem definido. É o único herbicida que inibe especificamente a enzima 5-enolpiruvilchiquimato-3-fosfato sintase (EPSPS, E.C. 2.5.1.19), que catalisa a condensação do chiquimato com fosfoenolpiruvato. O trabalho está baseado na concepção do chiquimato se acumular significativamente nos tecidos vegetais de soja convencional, após a inibição da EPSP sintase pelo glifosato. Em plantas não submetidas ao glifosato, o chiquimato não é facilmente detectado, pois rapidamente é metabolizado a chiquimato 3-fosfato e, a seguir, em 5-enolpiruvilchiquimato 3-fosfato, pela ação da EPSPS. Por outro lado, em plantas não geneticamente modificadas submetidas ao glifosato, a metabolização do chiquimato é prejudicada, resultando em seu acúmulo. Este metabólito pode ser detectado em quantidades extremamente baixas (na faixa de µg), por HPLC. Neste trabalho foram analisados seis contrastes diferentes, sendo cada contraste formado por uma cultivar transgênica e sua respectiva cultivar parental convencional, submetidas ou não a embebição com solução de glifosato 0,6%. As análises cromatográficas indicaram o acúmulo de chiquimato apenas em cultivares convencionais, nas quais as sementes foram previamente embebidas em solução de glifosato 0,6%. Os resultados demonstraram que adetecção de chiquimato pode ser utilizada como um método rápido e preciso na diferenciação de soja resistente ao glifosato de soja convencional.

Control potential of Meloidogyne javanica and Ditylenchus spp. using fluorescent Pseudomonas and Bacillus spp

ABSTRACT Plant Growth Promoting Rhizobacteria (PGPR) have different mechanisms of action in the development of plants, such as growth promotion, production of phytohormones and antibiotic substances and changes in root exudates. These help to control plant diseases. In order to evaluate the potential of microorganisms in the control of Meloidogyne javanica and Ditylenchus spp., five rhizobacteria isolated from rhizosphere of garlic cultivated in the Curitibanos (SC) region were tested. Hatching chambers were set on Petri dishes, in which were added 10 mL of bacterial suspension and 1 mL of M. javanica eggs suspension, at the rate of 4500, on the filter paper of each chamber. The same procedure was performed with 300 juvenile Ditylenchus spp. The experimental design was completely randomized, with four replications. The evaluations were performed every 72 h for nine days. The antagonized population of nematodes was determined in Peters counting chamber, determining the percentage hatching (for M. javanica) and motility (for Ditylenchus spp). Isolates CBSAL02 and CBSAL05 significantly reduced the hatching of M. javanica eggs (74% and 54.77%, respectively) and the motility of Ditylenchus spp. (55.19% and 53.53%, respectively) in vitro. Isolates were identified as belonging to the genera Pseudomonas (CBSAL05) and Bacillus (CBSAL02).

Diferentes tratamentos de sementes sobre o desenvolvimento de plantas de soja / Different treatments of seeds on the development of soybean plants

A cada ano são descobertos e utilizados novos produtos para o tratamento de sementesna cultura da soja (Glycine maxL. Merril), visando aumentos de produção. Há a necessidade de conhecer a influência desses produtos sobre o desenvolvimento das plantas, a fim de obter sucesso na atividade. O objetivo do experimento foi avaliar o efeito de principais produtos aplicados via tratamento de sementes, sobre a morfologia de plantas de soja, em diferentes fases de desenvolvimento da cultura, bem como, sobre os componentes do rendimento e produtividade. Os produtos utilizados foram: tiametoxan; [abamectina+tiametoxan + fludioxonil]; [fipronil + tiofanatometilico + piraclostrobina]; [imidacloprido + tiodicarbe]; CruiserAdvanced(r); carboxamida; abamectina; [metalaxyl-M + fludioxonil]; além da testemunha sem tratamento. Na primeira etapa, avaliou-se a qualidade fisiológica das sementes tratadas. Posteriormente, avaliou-se o crescimento inicial aos 15, 30 e 45dias após a emergência. Na terceira etapa, avaliaram-se características agronômicas e componentes de rendimento. O delineamento experimental utilizado para os testes fisiológicos das sementes foi inteiramente casualizado e para as determinações de desenvolvimento inicial e de final de ciclo, blocos ao acaso. Para análise estatística, utilizou-se o programa SASM-Agri. Concluiu-se que a mistura de ingredientes ativos inseticidas e fungicidas prejudica o desenvolvimento inicial das plântulas de soja. Aos 45 dias após a emergência, há efeito positivo dos produtos tiametoxan, [abamectina + tiametoxan + fludioxonil], CruiserAdvanced(r) e [metalaxyl-M + fludioxonil] sobre o crescimento, porém este estímulo não se reflete na produção final.

Every year, new products are discovered and used for seed treatment in soybeans (Glycine max.L. Merril), aiming to increase production. There is the need to know the influence of the products on the development of plants in order to be succeed in the activity. The aim of the experiment was to evaluate the effect of products applied as seed treatment on the morphology of soybean plants at different stages of crop development, as well as the components of income and productivity. The products used were: tiametoxan; [abamectina + tiametoxan + fludioxonil]; [fipronil + tiofanatometilico + piraclostrobina]; [imidacloprido + tiodicarbe]; Cruiser Advanced(r); carboxamida; abamectina; [metalaxyl-M + fludioxonil]; and the control without treatment. In the first step, it was evaluated the physiological quality of treated seed. Subsequently, it was evaluated the initial growth at 15, 30 and 45 days after emergence. In the third stage were evaluated agronomic traits and yield components. The experimental design used for physiological tests of the seeds was randomized and the measurements of early development and late season, were randomized blocks. For statistical analysis, it was used the program SASM-Agri (CANTERI et al., 2001). It was concluded that the mixture of active ingredients insecticides and fungicides affect the early development of soybean seedlings. At 45 days after emergence, there is a positive effect of the products Tiametoxan, [abamectina + tiametoxan + fludioxonil], Cruiser Advanced(r) and [metalaxyl-M + fludioxonil] on growth, but this stimulus, is not reflected in the final production.

Mycorrhization stimulant based in formononetin associated to fungicide and doses of phosphorus in soybean in the cerrado / Estimulante de micorrização a base de formononetina associado à fungicida e doses de fósforo em soja no cerrado

Arbuscular mycorrhizal fungi (AMF) are obligatory biotrophics, which complicates the feasibility of producing inoculants. Thus, the discovery of substances capable of stimulating mycorrhizal colonization, like the isoflavone formononetin emerges as a promising alternative to explore the benefits of AMF native soil in extensive crops system. The aim of this study was evaluate the application of the isoflavone formononetin (7-hydroxy-4'-methoxy isoflavone) andfungicide via seed in Cerrado soil in no-tillage system and fertilization with two doses of phosphorus in soybean under field and controlled conditions. In the field experiment the treatments were three levels of formononetin (0, 0.5 and 1.0 mg Formononetin seed-1) in the absence and presence of fungicide (Carbendazim + Thiram commercial mix) applied to seeds with two doses of phosphorus (100 % and 33% of the recommended fertilization). The study was conducted until the maturation of soybeans. The greenhouse conditions experiment was implemented with the same treatments used in the field research and the flowering stage of the soybean plant was simulated water stress during 10 days, returning to normal irrigation until to harvest the soybean grain. Applying of formononetin stimulated the increased of mycorrhizal colonization, number of nodules, vegetative growth and soybean production in the greenhouse. In the field research the treatments contributed to attenuate the negative effect of the fungicide Carbendazim + Thiram in soybean reflecting increased soybean production and adequate supply of soil phosphorus besides high density of propagules of natives AMF that contributed to reduce the benefits of stimulating mycorrhizal (formononetin) in this research.

Os fungos micorrízicos arbusculares (FMAs) são biotróficos obrigatórios, o que dificulta a viabilidade da produção de inoculantes. Com a descoberta de substâncias capazes de estimular a colonização micorrízica, o isoflavonóide formononetina surge como uma alternativa promissora para explorar os benefícios dos FMAs nativos do solo em cultivos extensivos. O objetivo do presente estudo foi de avaliar os efeitos das aplicações do isoflavonóide formononetina (7-hydroxy-4'-methoxy isoflavone) e de fungicida nas sementes em solo do Cerrado no sistema de plantio direto com duas doses de fósforo na cultura da soja em campo e em casa de vegetação. No experimento de campo os tratamentos foram três doses de formononetina (0, 0,5 e 1,0 mg de Formononetina semente-1); na ausência e presença de fungicida (Mistura comercial Carbendazim + Thiram) aplicado via semente e duas doses de fósforo (100 % e 33 % da adubação recomendada). O experimento de casa de vegetação foi implantado com os mesmos tratamentos utilizado no estudo em campo e na fase de florescimento das plantas de soja foi simulado um estresse hídrico, por 10 dias, retornando a irrigação normal até a colheita dos grãos. A aplicação de formononetina estimulou aumento da colonização micorrízica, número de nódulos, crescimento vegetativo e a produção de grãos soja em condições de déficit hídrico simulado em casa de vegetação e no no estudo de campo contribui para amenizar o efeito negativo dos fungicidas nas plantas de soja refletindo em aumento da produção da soja.

Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses, due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and Km. Inhibitor studies with natural antioxidant molecules present in soybean revealed α-tocopherol to be the most effective inhibitor of LOX-2.

Population Dynamics and Diversity of Endophytic Bacteria Associated with Soybean (Glycine max (L) Merril).

Aim: Endophytic bacterial population and their diversity in soybean were investigated. Study Design: Endophytic population was assessed during different growth stages of soybean (CV JS 335) viz., vegetative and reproductive stages. Place and Duration of Study: Microbiology Research Laboratory, Department of Microbiology, R. A. Mahavidyalaya, Washim (MS), India, during the cultivation period of June-December 2010. Methodology: Healthy plants of soybean were screened from the different locations of Washim district (M. S., India). Samples represent each growth stage viz., vegetative (V1- V5) and reproductive (R1-R8) were collected. Population densities were expressed as log10 colony forming units (CFU) g-1 fresh weight. The isolates were identified to genus level according to Bergey’s Manual of Determinative Bacteriology on the basis morphological, cultural and biochemical characteristics. Results: The maximum endophytic population was recorded for vegetative stage at V5 and V4 (5.74 and 5.01 log10 CFU g-1 fresh weight) and for reproductive stage at R2, R1 and R3 (5.84, 5.80 and 5.74 log10 CFU g-1 fresh weight). A total of 572 (35.50 %) from vegetative growth stages and 1039 (64.50 %) from reproductive growth stages bacterial isolates were obtained. The endophytic isolates were identified as members belonging to the genera Pseudomonas, Bacillus, Enterobacter, Klebsiella, Acetobacter Burkholderia, Rhizobium and Xanthomonas. Conclusion: As soybean development progresses endophytic population increased. At maturity, the high population density was observed and thereafter the population declined.

Germination and early growth of soybean, dry bean and turnip grown UNDER Digitaria horizontalis straw / Germinação e desenvolvimento inicial de soja, feijão e nabiça cultivados sob palhada de Digitaria horizontalis

The study aimed to evaluate the possible inhibitory effects of different concentrations of crabgrass (Digitaria horizontalis Willd.) dry mass incorporated to the soil over the germination and early growth of soybean (Glycine max (L.) Merril.), dry bean (Phaseolus vulgaris L.), and turnip (Raphanus raphanistrum L.). The experimental design adopted was completely random, with four replications where, each one was consisted of a 2.5 L capacity pot. Dry mass of crabgrass at equivalent amounts of 0, 2.5, 5.0 and 10 t ha-1 were incorporated into the soil. Crops seedling emergence was checked daily, and germination, speed germination index, mean germination time, relative frequency and synchronization index of germination were computed at the final of 10 days .The height and dry mass of plants were evaluated at 35 days after sowing. The incorporation to the soil of D. horizontalis dry mass caused significant reduction of the height and dry weight of soybean, dry bean and turnip, but were not observed consistent influence over the germination of these species.

Efeitos dos danos mecânicos e fisiológicos na colheita e beneficiamento de sementes de soja / Mechanical and fisiological effects on the harvesting and processing of soybean seeds

O objetivo deste trabalho foi avaliar o efeito dos danos mecânicos e fisiológicos ocorridos na colheita e no beneficiamento de sementes de soja cv. Mosoy 8000 RR. As amostras foram retiradas nas fases de colheita manual, colheita mecânica e durante o beneficiamento (recepção, pré-limpeza, limpeza, separador espiral, classificação e mesa gravitacional). Em seguida, as sementes foram submetidas aos testes de pureza, germinação e vigor (primeira contagem da germinação, matéria seca de plântulas, envelhecimento acelerado, condutividade elétrica, tetrazólio, danos mecânicos e emergência em campo). Foi utilizado o delineamento inteiramente casualizado com nove pontos de coleta com quatro repetições, sendo as médias comparadas pelo teste de Tukey a 5% de probabilidade. Nos testes de pureza e emergência em campo foram obtidas diferenças altamente significativas entre as etapas de amostragem, sendo que esta diferença também foi verificada nos testes de vigor realizados (primeira contagem da germinação, matéria seca de plântulas, envelhecimento acelerado, condutividade elétrica), que de um modo geral apresentaram resultados menores para a colheita mecânica e o restante do processo, quando comparados à colheita manual.

The objective of this study was to evaluate the effect of mechanical damage and physiological events in harvesting and processing of soybean cv. Mosoy RR 8000. The samples were taken during harvest manual, mechanical harvesting and during processing (receipt, pre-cleaning, cleaning, spiral separator, classification and gravity table). The physiological and physical quality was analized through the purity, germination, vigor (first germination count, seedling dry matter, accelerated aging, electrical conductivity, tetrazolium, mechanical damages and seedling field emergence) tests. The statistical design used was a entirely randomized with nine treatments (9 sampling points) with 4 replications, being the means compared by the Tuckey test at 5% probability. In the purity and seedling field emergence were observed highly significative difference between the sampling process, also this differences were obtained the first germination count, seedling dry weight matter, accelerated aging and electrical conductivity which showed smaller results for the mechanical harvesting when compared with the manual harvesting. The germination was obtained differences at 5% for the manual harvesting in relation to the mechanical harvesting were obtained smaller results, being the main cause of reducing the soybean seed quality, when compared with the manual harvesting