Bactérias endofíticas associadas à cultura de tecidos e às folhas de Plinia peruviana

The objective of this work was to isolate endophytic bacteria from tissue culture and leaves of jaboticaba (Plinia peruviana) and to evaluate their potential as plant growth-promoting bacteria. The bacteria were isolated from nodal segments grown in vitro and from leaves from a tree under natural conditions, totaling 11 and 54 isolates, respectively. The isolates were characterized by colony morphology. The indolic compounds produced by the isolates, in the presence or absence of 100 mg L-1 tryptophan, were quantified. The greatest producers of these compounds were identified by sequencing the 16S rRNA gene and were inoculated on jaboticaba seeds, using Azospirillum brasilense (Ab-V6) as a positive control. The sensitivity of bacteria to eight antibiotics was also evaluated. All assessed bacteria produced indolic compounds, especially Bacillus sp., with a content of 27.41 µg mL-1. The germination rate of the seeds inoculated with Stenotrophomonas sp. was high – 97.34% compared with that of 74.67% of the negative control. Bacillus sp. and Stenotrophomonas sp. also sped up germination. Chloramphenicol limited the growth of 82% of the isolates, followed by amoxicillin, gentamicin, levofloxacin, and tetracycline, which limited 70%; erythromycin was only effective against 35%. The endophytic bacteria isolated from jaboticaba show characteristics of plant growth-promoting bacteria, and Bacillus sp. and Stenotrophomonas sp., obtained from tissue culture, are capable of enhancing jaboticaba seed germination.O objetivo deste trabalho foi isolar bactérias endofíticas de cultura de tecidos e de folhas de jabuticabeira (Plinia peruviana) e avaliar seu potencial como bactérias promotoras de crescimento de plantas. As bactérias foram isoladas a partir de segmentos nodais cultivados in vitro e de folhas de árvore sob condições naturais, tendo totalizado 11 e 54 isolados, respectivamente. Os isolados foram caracterizados pela morfologia da colônia. Foram quantificados os compostos indólicos produzidos pelos isolados, na presença ou na ausência de 100 mg L-1 de triptofano. Os maiores produtores destes compostos foram identificados pelo sequenciamento do gene 16S rRNA e foram inoculados em sementes de jabuticaba, tendo-se utilizado Azospirillum brasilense (Ab-V6) como controle positivo. Também avaliou-se a sensibilidade das bactérias a oito antibióticos. Todas as bactérias avaliadas produziram compostos indólicos, especialmente Bacillus sp., com quantidade de 27,41 µg mL-1. A taxa de germinação das sementes inoculadas com Stenotrophomonas sp. foi elevada – 97,34% em comparação à de 74,67% do controle negativo. Bacillus sp. e Stenotrophomonas sp. também aumentaram a velocidade de germinação. O cloranfenicol limitou o crescimento de 82% dos isolados, seguido pelas amoxicilina, gentamicina, levofloxacina e tetraciclina, que limitaram 70%; a eritromicina foi eficaz apenas contra 35%. As bactérias endofíticas isoladas da jabuticaba apresentam características de bactérias promotoras de crescimento de plantas, e Bacillus sp. e Stenotrophomonas sp., provenientes da cultura de tecido, são capazes de aprimorar a germinação de sementes de jabuticaba

Bactérias endofíticas promotoras de crescimento de plantas em mudas de pupunheira

The objective of this work was to isolate endophytic bacteria from peach palm (Bactris gasipaes var. gasipaes) plants and to evaluate the effects of their inoculation on the plant seedlings. Bacteria were isolated from the leaves and roots of the seedlings and from the meristems of peach palm plants in vitro. The isolates were characterized phenotypically and, then, 15 of them, representing different phenotypic groups, were selected and identified by partial sequencing of the 16S rRNA gene. Afterward, these isolates and two commercial strains of Azospirillum brasilense (Ab-V5 and Ab-V6) were inoculated in the peach palm seedlings. After 76 days, the seedlings were evaluated for plant development. The following six genera were identified based on the sequencing: Pseudomonas, Enterobacter, Rhizobium, Stenotrophomonas, Klebsiella, and Erwinia. Out of the 15 inoculated isolates, 9 had a positive effect on the root dry mass of palm peach, with CNPF 77 (Enterobacter sp.), CNPF 100 (Rhizobium sp.), and CNP 179 and CNPF 277 (Stenotrophomonas sp.) standing out. Peach palm seedlings harbor endophytic bacteria which are able to increase root dry matter.O objetivo deste trabalho foi isolar bactérias endofíticas de pupunheira (Bactris gasipaes var. gasipaes) e avaliar os efeitos da inoculação delas em mudas da planta. As bactérias foram isoladas de folhas e raízes das mudas e de meristemas de pupunheira in vitro. Os isolados foram caracterizados fenotipicamente, e, depois, 15 deles, representando grupos fenotípicos distintos, foram selecionados e identificados por meio do sequenciamento parcial do gene 16S rRNA. Em seguida, esses isolados e duas estirpes comerciais de Azospirillum brasilense (Ab-V5 e Ab-V6) foram inoculados em plântulas de pupunheira. Após 76 dias, as mudas foram avaliadas quanto ao desenvolvimento vegetal. Foram identificados os seis seguintes gêneros com base no sequenciamento: Pseudomonas, Enterobacter, Rhizobium, Stenotrophomonas, Klebsiella e Erwinia. Dos 15 isolados inoculados, 9 tiveram efeito positivo sobre a massa de matéria seca de raízes, com destaque para CNPF 77 (Enterobacter sp.), CNPF 100 (Rhizobium sp.), e CNP 179 e CNPF 277 (Stenotrophomonas sp.). Mudas de pupunheira abrigam bactérias endofíticas capazes de aumentar a matéria seca das raízes

Sesbania virgata estimula a ocorrência de seu microssimbionte nos solos, mas não inibe os microssimbiontes de outras espécies

The legume species Sesbania virgata establishes a specific and efficient symbiosis with Azorhizobium doebereinerae. Previous studies have shown that A. doebereinerae occurrence correlates to the presence of S. virgata. This work aimed to evaluate the occurrence of A. doebereinerae and of other nitrogen-fixing Leguminosae-nodulating bacteria (NFLNB) in soil samples collected adjacent to and 10 m away from the stems of five S. virgata plants in pasture areas. Symbiotic characteristics of isolates from these NFLNB populations were also studied. S. virgata and the four promiscuous legume species Leucaena leucocephala, Macroptilium atropurpureum, Phaseolus vulgaris and Vigna unguiculata were inoculated with soil samples to trap A. doebereinerae and other NFLNB. NFLNB capable of inducing nodulation in at least one of these legumes were found in all samples. M. atropurpureum was the most promiscuous species, as it trapped the highest number of NFLNB cultural types from soil suspensions. The other species were less promiscuous in the following order: V. unguiculata, P. vulgaris, and L. leucocephala. Isolates of the promiscuous legumes were classified into seven cultural groups. One of these groups, isolated from all promiscuous species, showed fast-growth alkali-reaction in culture medium (like Azorhizobium); it was identified as Cupriavidus. This is the first report of symbiosis of Cupriavidus with Papilionoideae species. The symbiotic efficiency of promiscuous hosts with NFLNB varied, but it was always less than that of controls with mineral nitrogen or an inoculant strain. S. virgata was efficiently nodulated only by A. doebereinerae, which occurred mainly in samples collected close to the plant stem, corroborating a high stimulus by its host species. A high diversity of NFLNB occurs as saprophytes close to the S. virgata root system.A espécie de leguminosa Sesbania virgata estabelece uma simbiose especifica e eficiente com Azorhizobium doebereinerae. Estudos prévios indicam que a ocorrência de A. doebereinerae esta relacionada à presença de S. virgata. Avaliou-se a ocorrência de A. doebereinerae e de outras Bactérias Fixadoras de Nitrogênio Nodulíferas em Leguminosas (BFNNL) em amostras de solos coletadas próximo e a 10 metros do caule de cinco plantas de S. virgata em áreas de pastagem. As características simbióticas de isolados das populações dessas BFNNL foram também estudadas. Para captura de A. doebereinerae e de outras BFNNL, essas amostras de solos foram inoculadas em S. virgata e nas leguminosas promíscuas Leucaena leucocephala, Macroptilium atropurpureum, Phaseolus vulgaris e Vigna unguiculata. Todas as amostras de solos apresentaram BFNNL capazes de nodular, pelo menos, uma espécie. M. atropurpureum foi a espécie mais promíscua capturando um grande número de tipos culturais de BFNNL das suspensões de solo. As outras espécies foram menos promíscuas na seguinte ordem: V. unguiculata, P. vulgaris, e L. leucocephala. Os isolados das espécies hospedeiras consideradas promíscuas foram agrupados em sete tipos culturais. Um desses grupos, isolado de todas as espécies promiscuas, apresentou crescimento rápido e reação alcalina em meio de cultura (como Azorhizobium) e foi identificado como Cupriavidus. Este é o primeiro relato da simbiose de Cupriavidus com espécies de Papilionoideae. A simbiose de BFNNL com as demais espécies teve eficiência variável, mas foi sempre menor que a da adubação nitrogenada e da estirpe recomendada como inoculante. S. virgata formou simbiose somente com A. doebereinerae, a qual foi mais freqüente nas amostras de solos coletadas próximo ao caule da planta, corroborando alto estímulo deste microsimbionte por sua espécie hospedeira. Uma alta diversidade de BFNNL ocorre saprofiticamente próximo ao sistema radicular de S. virgata

Seasonal variation of arbuscular mycorrhizal fungi in ecotone forests of the northern region of Brazilian Amazonia

The ecotone forests in the northern region of Brazilian Amazonia are important areas representing transition zones between forest and non-forest ecosystems. These areas have soils nutrient-poor that poorly drain. Under these environmental conditions, Peltogyne gracilipes (Leguminoseae), an endemic tree species, can form natural monodominant forests. Here, we assessed the arbuscular mycorrhizal fungi (AMF) community in three forest types on the eastern side of Maracá Island and the relationship of these microorganisms with the monodominance of P. gracilipes. In this study, soil samples were collected in two seasons (dry and rainy). The samples were collected in 9 plots, in rich areas, poor areas and areas without P. gracilipes. Soil samples were evaluated for chemical and particle size analysis, spore density and morphology, and identification of AMF. AMF species were identified using two approaches: spores collected in the field and trap cultures. Eighteen and 13 AMF species were identified in the dry and rainy seasons, respectively, for spores extracted from the field. Six total species were detected exclusively in trap cultures in the dry season. AMF communities were co-dominated by members of the Gigasporaceae, Acaulosporaceae and Glomeraceae families. Redundancy analyses indicated that several soil attributes, such as pH, Fe, Mg, and sand content associated with the AMF species richness in both seasons. We conclude that the ecotone forests in the eastern region of Maracá Island are home to important richness and diversity of AMF species and that various soil factors influence the composition of the AMF community in this ecosystem

Ambientes para a produção de mudas e nodulação por rizóbios em Tachigali vulgaris

This study had as objectives to determine the best shading condition and the type of substrate to produce seedlings and the establishment of a spontaneous symbiosis by rhizobia in taxi-branco (Tachigali vulgaris). A randomized block design with four replications and treatments arranged in a split-plot scheme was installed. The plot consisted of two shading levels (50% and 75%) and subplots of four types of substrates: soil; soil + sand (1:1); soil + sand + sawdust (2:1:1); and soil + vermiculite (1:1). Each experimental plot consisted of fve seedlings. The seedlings were evaluated for a set of shoot and root morphological and physiological traits. At the end of the experiment, the isolation and the phenotypic characterization of bacteria from nodules collected in the seedlings were also carried out. The characterization was performed by the morphological evaluation of the colonies. The shading of 50% is indicated for seedling productions, while in relation to the substrate, that one composed by soil + sand is what produces seedlings of better quality. Although the number of isolates was not influenced by the substrate type and the shading level, taxi-branco seedlings establish symbiosis with a great diversity of rhizobia. Most of the isolates have very fast growth rate and acid pH of the culture medium.O presente estudo teve como objetivos determinar a melhor condição de sombreamento e o tipo de substrato para a produção de mudas e estabelecimento da simbiose espontânea por rizóbios em taxi-branco (Tachigali vulgaris). O experimento foi instalado no delineamento de blocos casualizados, com quatro repetições e os tratamentos arranjados em esquema de parcela subdividida, no qual a parcela consistiu de dois níveis de sombreamento (50% e 75%) e a subparcela de quatro tipos de substrato: solo; solo + areia (1:1); solo + areia + serragem (2:1:1); e solo + vermiculita (1:1). Cada parcela experimental foi constituída por cinco mudas. As mudas foram avaliadas quanto a um conjunto de variáveis morfológicas e fisiológicas, da parte aérea e da raiz. No final do experimento foi também realizado o isolamento e a caracterização fenotípica de bactérias oriundas de nódulos coletados nas mudas. A caracterização foi realizada pela avaliação morfológica das colônias. O sombreamento de 50% é o indicado na produção de mudas de taxi-branco, enquanto que, em relação ao substrato, aquele composto por solo + areia é o que proporciona mudas de melhor qualidade. A espécie apresentou simbiose com grande diversidade de rizóbios, apesar de o número de isolados não ter sido influenciado pelo tipo de substrato e nível de sombreamento. A maioria dos isolados obtidos apresentam tempo de crescimento muito rápido e pH ácido do meio de cultura

Compatibility of the cowpea seed treatment with fungicides and inoculation with Bradyrhizobium strains

O objetivo deste trabalho foi avaliar a compatibilidade do tratamento de sementes com fungicidas e a inoculação com estirpes de Bradyrhizobium em feijão-caupi. Em laboratório, avaliou-se a sobrevivência de células nas sementes da cultivar BRS Guariba, tratadas ou não com fungicidas (fludioxonil, carbendazim, carbendazim + thiram e carboxin + thiram) e inoculadas ou não com Bradyrhizobium (estirpes UFLA3‑84, BR 3267, INPA3‑11B e BR 3262). Em casa de vegetação, conduziu-se experimento em vasos de Leonard, com os mesmos tratamentos. Foram avaliados: massa de matéria seca da parte aérea, além de número e massa de nódulos 25 dias após a emergência das plantas. No campo, dois experimentos foram conduzidos, tendo-se utilizado a estirpe BR 3262, com aplicação de fungicidas nas sementes: um em área de primeiro cultivo e outro em área cultivada anteriormente com culturas anuais. Avaliaram-se, aos 35 dias, o número de nódulos, a massa de nódulos secos e a massa de matéria seca da parte aérea, e, na colheita, a produtividade de grãos. Os fungicidas não tiveram efeito significativo sobre a sobrevivência de Bradyrhizobium, a nodulação das plantas e o rendimento de grãos, que, em média, foi superior a 1.200 kg ha‑1. O tratamento de sementes de feijão‑caupi com fungicidas é compatível com a inoculação das estirpes avaliadas.The objective of this work was to evaluate the compatibility of seed treatment with fungicides and Bradyrhizobium strains inoculation for cowpea. Cell survival on seeds of cultivar BRS Guariba treated or not with fungicides (fludioxonil, carbendazim, carbendazim + thiram, and carboxin + thiram) and inoculated or not with Bradyrhizobium (strains UFLA3‑84, BR 3267, INPA3‑11B, and BR 3262) were evaluated in laboratory. Another experiment was carried out in greenhouse, in Leonard jars, using the same treatments. The following were evaluated: shoot dry matter weight, besides number and dry matter weight of nodules 25 days after plant emergence. Two experiments were performed in the field with the strain BR 3262, with fungicides on the seeds: one in a first cultivation area and the other in an area previously cultivated with annual crops. Nodule number, nodule dry weight, and shoot dry matter weight were evaluated at 35 days, and grain yield at harvest. Fungicides did not have significant effect on Bradyrhizobium survival, plant nodulation, and grain yield, which was, in average, higher than 1,200 kg ha‑1. Cowpea seed treatment with fungicides is compatible with the inoculation of the evaluated strains

Diversidade e eficiência na fixação do nitrogênio de rizóbios isolados de nódulos de Centrolobium paraense

The objective of this work was to isolate and characterize rhizobia from nodules of Centrolobium paraense and to evaluate their symbiotic efficiency. Soil samples collected from four sites of the Roraima Cerrado, Brazil, were used to cultivate C. paraense in order to obtain nodules. Isolates (178) were obtained from 334 nodules after cultivation on medium 79. Twenty‑five isolates belonging to six morphological groups were authenticated using Vigna unguiculata and they were characterized by 16S rRNA. Isolates identified as Bradyrhizobium were further characterized using rpoB gene sequencing. A greenhouse experiment was carried out with C. paraense to test the 18 authenticated isolates. Approximately 90% of the isolates grew slowly in medium 79. The 16S rRNA analysis showed that 14 authenticated isolates belong to the genus Bradyrhizobium, and rpoB indicated they constitute different groups compared to previously described species. Only four of the 11 fast‑growing isolates nodulated V. unguiculata, two of which belong to Rhizobium, and two to Pleomorphomonas, which was not previously reported as a nodulating genus. The Bradyrhizobium isolates ERR 326, ERR 399, and ERR 435 had the highest symbiotic efficiency on C. paraense and showed a contribution similar to the nitrogen treatment. Centrolobium paraense is able to nodulate with different rhizobium species, some of which have not yet been described.O objetivo deste trabalho foi isolar e caracterizar rizóbios de nódulos de Centrolobium paraense e avaliar sua eficiência simbiótica. Amostras de solo, coletadas de quatro locais do Cerrado de Roraima, foram utilizadas para o cultivo de C. paraense, para obtenção dos nódulos. Os isolados (178) foram obtidos dos 334 nódulos coletados após o cultivo em meio 79. Vinte e cinco isolados, pertencentes a seis grupos morfológicos, foram autenticados com uso de Vigna unguiculata e caracterizados pelo 16S rRNA. Os isolados identificados como Bradyrhizobium foram caracterizados depois pelo sequenciamento do gene rpoB. Um experimento em casa de vegetação foi realizado com C. paraense, para testar os 18 isolados autenticados. Aproximadamente 90% dos isolados mostraram crescimento lento em meio 79. A análise do 16S rRNA mostrou que 14 dos isolados autenticados pertencem ao gênero Bradyrhizobium, e o rpoB indicou que eles constituem grupos diferentes em comparação às espécies já descritas. Somente quatro dos 11 isolados com crescimento rápido nodularam V. unguiculata, dois dos quais pertencentes a Rhizobium e dois a Pleomorphomonas, que não foi relatado anteriormente como gênero nodulífero. Os isolados de Bradyrhizobium ERR 326, ERR 399 e ERR 435 apresentaram a maior eficiência em C. paraense e mostraram contribuição similar ao tratamento nitrogenado. Centrolobium paraense é capaz de nodular com diferentes espécies de rizóbios, algumas das quais ainda não foram descritas

Bradyrhizobium neotropicale sp. nov., isolated from effective nodules of Centrolobium paraense

Root nodule bacteria were isolated from Centrolobium paraense Tul. grown in soils from the Amazon region, State of Roraima (Brazil). 16S rRNA gene sequence analysis of seven strains (BR 10247(T), BR 10296, BR 10297, BR 10298, BR 10299, BR 10300 and BR 10301) placed them in the genus Bradyrhizobium with the closest neighbours being the type strains of Bradyrhizobium paxllaeri (98.8 % similarity), Bradyrhizobium icense (98.8 %), Bradyrhizobium lablabi (98.7 %), Bradyrhizobium jicamae (98.6 %), Bradyrhizobium elkanii (98.6 %), Bradyrhizobium pachyrhizi (98.6%) and Bradyrhizobium retamae (98.3 %). This high similarity, however, was not confirmed by the intergenic transcribed spacer (ITS) 16S-23S rRNA region sequence analysis nor by multi-locus sequence analysis. Phylogenetic analyses of five housekeeping genes (dnaK, gin/I, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EKO5(T) (=LMG 24129(T)) to. be the most closely related type strain (95.7% sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [major components being C-16:0 and summed feature 8 (18:1 omega 6c/18:1 omega 7c)] DNA G+C content, slow growth rate and carbon compound utilization patterns, supported the placement of the novel strains in the genus Bradyrhizobium. Results of DNA-DNA relatedness studies and physiological data (especially carbon source utilization) differentiated the strains from the closest recognized species of the genus Bradyrhizobium. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nil/-I) placed the novel species in a new branch within the genus Bradyrhizobium. Based on the current data, these seven strains represent a novel species for which the name Bradyrhizobium neotropicale sp. nov. is proposed. The type strain is BR 10247(T) (=HAMBI 3599(T))

Diversity and nitrogen fixation efficiency of rhizobia isolated from nodules of Centrolobium paraense

The objective of this work was to isolate and characterize rhizobia from nodules of Centrolobium paraense and to evaluate their symbiotic efficiency. Soil samples collected from four sites of the Roraima Cerrado, Brazil, were used to cultivate C. paraense in order to obtain nodules. Isolates (178) were obtained from 334 nodules after cultivation on medium 79. Twenty-five isolates belonging to six morphological groups were authenticated using Vigna unguiculata and they were characterized by 16S rRNA. Isolates identified as Bradyrhizobium were further characterized using rpoB gene sequencing. A greenhouse experiment was carried out with C. paraense to test the 18 authenticated isolates. Approximately 90% of the isolates grew slowly in medium 79. The 16S rRNA analysis showed that 14 authenticated isolates belong to the genus Bradyrhizobium, and rpoB indicated they constitute different groups compared to previously described species. Only four of the 11 fast-growing isolates nodulated V. unguiculata, two of which belong to Rhizobium, and two to Pleomorphomonas, which was not previously reported as a nodulating genus. The Bradyrhizobium isolates ERR 326, ERR 399, and ERR 435 had the highest symbiotic efficiency on C. paraense and showed a contribution similar to the nitrogen treatment. Centrolobium paraense is able to nodulate with different rhizobium species, some of which have not yet been described