The chitinolytic activity of the Curtobacterium sp. isolated from field-grown soybean and analysis of its genome sequence.

Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.

Many plant pathogenic Pseudomonas savastanoi pv glycinea isolates possess an inactive quorum sensing ahlR gene via a point mutation.

Many plant bacterial pathogens monitor their group behaviour and their population density via production of N-acyl homoserine lactone signals which regulate the expression of several genes via the LuxI/R homologs. This regulatory network, termed quorum sensing (QS), is present in the soybean bacterial pathogen Pseudomonas savastanoi pv glycinea (Psg). The sequenced genomes of two strains of Psg, race 4 and B076, contain an N-acyl homoserine lactone (AHL) based LuxI/R QS system named AhlI/R. While studying the QS system of Psg strains race 4 and B076 isolated in USA, LMG5066 in New Zealand and IBSBF355 in Brazil, we found that B076, LMG5066 and IBSBF355 possess a point mutation in the ahlR gene that causes a frameshift resulting in a truncated AhlR protein. Psg race 4 does not possess the mutation in ahlR and the QS system is functional. The same mutation in the ahlR gene was found to be also present in 9 of 19 Psg strains isolated from diseased soybean in Illinois. Phenotypic analysis of strains showed that swarming motility is repressed whereas phosphate solubilisation was activated by QS in Psg. Analysing the secretome, we also found that four proteins were under QS regulation.

Phenotypic and genotypic characterization of endophytic bacteria associated with transgenic and non-transgenic soybean plants.

Endophytic bacteria isolated from non-transgenic and transgenic Roundup Ready® glyphosate-resistant (GR) soybean plants were investigated to analyze the correspondence between phenotypic and genotypic characteristics and to determine whether or not the strains could be grouped based on the source of isolation in transgenic or non-transgenic plants, respectively. Most of the strains recovered from GR plants have shown the ability for plant growth promotion (PGP) by means of IAA production and inorganic phosphate solubilization, and 100% of the strains showed great motility (swarm or swim); in addition, 90% of the strains were able to metabolize the majority of carbon sources tested. GR soybean fields showed higher endophytes abundance than non-transgenic; however, analyzing the phylogenetic trees constructed using the partial 16SrRNA gene sequences, higher diversity was observed in non-transgenic soybean fields. Overall the majority of isolated endophytes could utilize multiple patterns of carbon sources and express resistance to antibiotics, while isolates varied widely in the PGP ability. The greater pattern and frequency of utilization of carbon sources and frequency and intensity of antibiotic resistance compared with PGP ability within the soybean endophytes community suggest that carbon sources metabolism and antibiotic resistance confer a greater relative fitness benefit than PGP ability. In conclusion, cluster analysis of the phenotypes and 16SrRNA gene sequences reveals lack of correspondence between the pattern of bacterial isolates and the transgenic character of plants, and the heterogeneity of clustering suggested that various adaptive processes, such as stress response, could have contributed to generate phenotypic variability to enhance endophytes overall fitness.

Screening of bacterial endophytes as potential biocontrol agents against soybean diseases.

AIMS: This research was aimed at identifying and characterizing endophytic micro-organisms associated with soybean that have antimicrobial activity towards soybean pathogens. METHODS AND RESULTS: Soybean plants were collected from field trials in four locations of southern Brazil that were cultivated with conventional (C) and transgenic glyphosate-resistant (GR) soybeans. Endophytic bacteria isolated from roots, stems and leaves of soybeans were evaluated for their capacity to inhibit fungal and bacterial plant pathogens and 13 micro-organisms were identified with antagonistic activity. Approximately 230 bacteria were isolated and identified based on the 16S rRNA and rpoN gene sequences. Bacteria isolated from conventional and transgenic soybeans were significantly different not only in population diversity but also in their antagonistic capacity. Thirteen isolates showed in vitro antagonism against Sclerotinia sclerotiorum, Phomopsis sojae and Rhizoctonia solani. Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling bacterial and fungal pathogens in vitro. Extracts and precipitates from culture supernatants of isolates showed different patterns of inhibitory activity on growth of fungal and bacterial pathogens. CONCLUSIONS: Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling fungal pathogens in vitro, and the activity is mainly due to peptides. However, most of the studied bacteria showed the presence of antimicrobial compounds in the culture supernatant, either peptides, bacteriocins or secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be significant to develop tools for the biological control of soybean diseases. The work brought to the identification of micro-organisms such as Bacillus sp. and Burkholderia sp. that have the potential to protect crops in order to enhance a sustainable management system of crops. Furthermore, the study provides the first evidences of the influence of management as well as the genetics of glyphosate-resistant soybean on the diversity of bacterial endophytes of soybean phytobiome.

Galactinol synthase transcriptional profile in two genotypes of Coffea canephora with contrasting tolerance to drought.

Increased synthesis of galactinol and raffinose family oligosaccharides (RFOs) has been reported in vegetative tissues in response to a range of abiotic stresses. In this work, we evaluated the transcriptional profile of a Coffea canephora galactinol synthase gene (CcGolS1) in two clones that differed in tolerance to water deficit in order to assess the contribution of this gene to drought tolerance. The expression of CcGolS1 in leaves was differentially regulated by water deficit, depending on the intensity of stress and the genotype. In clone 109A (drought-susceptible), the abundance of CcGolS1 transcripts decreased upon exposure to drought, reaching minimum values during recovery from severe water deficit and stress. In contrast, CcGolS1 gene expression in clone 14 (drought-tolerant) was stimulated by water deficit. Changes in galactinol and RFO content did not correlate with variation in the steady-state transcript level. However, the magnitude of increase in RFO accumulation was higher in the tolerant cultivar, mainly under severe water deficit. The finding that the drought-tolerant coffee clone showed enhanced accumulation of CcGolS1 transcripts and RFOs under water deficit suggests the possibility of using this gene to improve drought tolerance in this important crop.

Frutificação e desenvolvimento de frutos de aceroleira no Norte do Paraná / Fruit set and development of West Indian Cherries in the North of Paraná, Brasil

O objetivo deste trabalho foi entender a frutificação e o desenvolvimento da aceroleira das cultivares "Dominga" (UEL-3), "Lígia" (UEL-4) e "Natália" (UEL-5) nas condições de Londrina (PR), em três épocas de florescimento. A porcentagem de frutificação foi obtida a partir da relação entre o número de flores marcadas, polinizadas naturalmente e o número de frutos maduros colhidos. Foi estudada a duração em dias nos estádios de botão, flor, crescimento e desenvolvimento de fruto verde e maduro. A taxa de frutificação variou de 10,7 por cento a 32,7 por cento para as cultivares "Natália" (UEL-5) no florescimento em outubro/novembro e "Dominga" (UEL-3) no florescimento em dezembro/janeiro, respectivamente. O ciclo de frutificação (da antese à colheita) foi de 19 a 32 dias, sendo maior no período outubro/novembro do que em dezembro/janeiro e janeiro/fevereiro, quando ocorreram aumento da temperatura e precipitação.

The objective of this study was to evaluate fruit set and development of West Indian Cherries. The cultivars 'Dominga' (UEL-3), 'Lígia' (UEL-4) and 'Natália' (UEL-5) were evaluated in Londrina, state of Paraná, in three flowering periods. The fruit set percentage was calculated as the ratio of the number of marked, naturally pollinated flowers to the number of harvested mature fruits. The duration of the flower-bud, flower, green fruit development, and mature fruit stages were assessed as well. The fruit set rate varied from 10.7 percent for cultivar 'Natália' (UEL-5) in the period October/November to 32.7 percent for 'Dominga' (UEL-3) in December/January, respectively. The fruit set cycle (from anthesis to harvest) lasted between 19 and 32 days. It lasted longer in the October/November period than in December/January and January/February, when the temperature and precipitation increased.

Comparative analysis of genetic diversity among the maize inbred lines (Zea mays L.) obtained by RAPD and SSR markers

The RAPD and SSR markers were used to compare the genetic diversity among the 16 maize inbred lines. Twenty-two primers were used in the RAPD reactions, resulting in the amplification of 265 fragments, while 16 pairs of SSR primers resulted in 75 fragments. The similarity based on Dice coefficient for the RAPD ranged from 53 to 84 percent and for the SSR from 11 to 82 percent. The dendrogram obtained by the RAPD showed five groups, while dendrogram obtained by the SSR showed three groups and one isolated line. The association constructed from the markers and the principal coordinate’s analysis separated lines into two groups according to endosperm color, either orange or yellow. The RAPD were effective to validate pedigree data, while the SSR were effective to recognize the differences between the quantitative characters. Because they assess the distinct regions of the genome, the selection of one or other marker would depend on the characteristics of the material used and the objectives of the project.

RAPD e SSR foram utlizados para comparar a diversidade genética entre 16 linhagens de milho. Nas reações de RAPD foram utlizados 22 primers que resultaram na amplificação de 265 fragmentos, enquanto que 16 pares de primes de SSR resultaram em 75 fragmentos. A similaridade baseada no coeficiente de Dice variou de 53 por cento a 84 por cento para o RAPD; para o SSR variou de 11 por cento a 82 por cento. O dendrograma obtido a partir do RAPD mostrou 5 grupos enquanto que o dendrograma obtido a partir do SSR mostrou 3 grupos e uma linhagem isolada. A associação construída a partir dos marcadores e a análise de coordenadas principais separaram as linhagens em dois grupos de acordo coloração de endosperma alaranjado ou amarelo, os marcadores RAPD foram eficientes para a validação dos dados de pedigree enquanto os de microssatélites para reconhecerem diferenças entre caracteres quantitativos. Por acessarem regiões distintas do genoma a escolha de um ou outro marcador vai depender das características do material utilizado e dos objetivos do trabalho.

Inheritance of a long juvenile period under short-day conditions in soybean

The long juvenile period (LJP) characteristic of soybean (Glycine max L. Merrill) cultivars delays flowering under short-day conditions. This trait may be important in increasing the adaptation range of soybean to low latitudes and provides greater flexibility for sowing times within the same latitude. The inheritance of the long juvenile period was studied in the MG/BR22 (Garimpo) soybean cultivar to provide knowledge to support the development of cultivars adapted to short day conditions. Four cultivars ('Paraná', 'Bossier', 'Bragg', and 'Davis') were crossed among each other and with MG/BR 22 ('Garimpo'). The study was conducted under short-day conditions (early sowing) in a greenhouse and in the field, in Londrina, Paraná, (23º22' south latitude). The parents and the F1, F2, and F3 populations were assessed daily for flowering. The genotype ratios of 15:1 (p > 0.95, X² test) and 8:7:1 (p = 0.193) for the F2 and F3 generations, respectively, obtained for the Paraná x Bossier cross indicated a case of digenic interaction with a duplicate recessive epistatic effect for the LJP character. Segregation of the F2 population from the MG/BR 22 (Garimpo) x Paraná cross resulted in the expected ratio of 3:1 (p = 0.166, indicating that the MG/BR 22 (Garimpo) and Paraná cultivars differed at a single locus. Similarly, in the MG/BR 22 (Garimpo) x Bossier cross, a 3:1 (p = 0.065) segregation indicated that these two cultivars differed at a single locus for number of days to flowering. The Davis cultivar had the same gene for LJP as the Paraná cultivar (aa). The F2 segregation of the classic flowering Bragg with MG/BR 22 (Garimpo) cross resulted in a 15:1 (p = 0.138) ratio, indicating that these two cultivars differed at two loci for flowering. The genotypes assigned to the cultivars were aaBB for Paraná, AAbb for Bossier and aabb for MG/BR 22 (Garimpo). A single locus in recessive homozygosis does not produce LJP