Growth, symbiotic, and proteomics studies of soybean Bradyrhizobium in response to adaptive acid tolerance

Bradyrhizobial inoculated soybean often performs poorly on acid-soil because of the acid sensitivity of their associated root nodule bacteria. Acid tolerance in rhizobia has been considered as a key phenotypic characteristic in that it enables the bacteria to perform well under the restrictive conditions of excessive acidity. Since bacteria could develop acid tolerance to a more acid condition by using adaptive acid tolerance response (ATR), it is interesting to investigate whether bradyrhizobia could have this response and what proteins are involved in ATR. Bradyrhizobium sp. DASA01007 was selected for this study based on its ATR ability and symbiosis efficiency with soybean under acid condition. To establish an ATR in bradyrhizobia, late log phase culture of cell grown in mild acid condition was subsequently used as inoculum to more acid conditions. The 2D-gel and proteomic analyses were used to investigate the proteins response during ATR compared with non-adaptive conditions. The 29 identified proteins were grouped into 8 categories based on category orthologous group (COG) and one group of unknown categories. Hypothetical protein, transport and binding proteins, and translation protein were up-regulated at pH 4.5N (non-adaptive condition). While up-regulated proteins found during growth at pH 4.5A (adaptive condition) consisted of proteins in cellular processes, translation, energy metabolism, regulatory functions, interconversions and salvage of nucleosides and nucleotides, and conserved hypothetical proteins group. However, transport and binding proteins were absent in adaptive condition. At pH 5.5A, proteins involved in cellular processes were also detected. Several proteins overproduced in adaptive condition may be involved in ATR of bradyrhizobia. An importance of ATR in root nodule bacteria would support a better chance of survival in low pH soils than those conventionally grown in neutral pH. These results suggest that the use of ATR condition could provide an improvement in the production of inoculants.Keywords: Adaptive acid tolerance, Bradyrhizobium, Soybean, 2D-gel electrophoresi

Co-inoculation effects of Bradyrhizobium japonicum and Azospirillum sp. on competitive nodulation and rhizosphere eubacterial community structures of soybean under rhizobia-established soil conditions

Bradyrhizobial inoculants used for soybean seed inoculation to maximize the benefit of N2-fixation should include bradyrhizobial strain with high N2-fixation rates and ability to compete with the indigenous rhizobial populations. In this study, co-inoculation of plant growth promoting rhizobacteria (PGPR) Azospirillum sp. with either of Bradyrhizobium japonicum CB 1809 or USDA 110 increased shoot and root dry weight of soybean over non-inoculated control under pot condition with no indigenous soybean nodulating bradyrhizobia. Moreover, competition for nodulation and the effects on rhizosphere soil eubacterial community structures by using single or co-inoculation of B. japonicum and Azospirillum sp. under rhizobia-established Myanmar and Thailand soils were investigated. By inoculation of gus-marked USDA 110 singly or its co-inoculation gave 93.21 to 94.75% and 74.21 to 100% in nodule occupancy, and 23.50 to 41.95% and 50.37 to 73.24% promotion in biomass dry weight over non-inoculated control in Myanmar and Thailand soil samples, respectively. Each of all the tested inoculum levels, that is 106, 107 and 108 cfu/ml of Azospirillum sp. enhanced nodulation in combination with USDA 110 with a corresponding increase in 73.8, 62.25 and 95.34%; and 51.52, 62.38 and 79.46% over non-inoculated control, respectively in Myanmar and Thailand soil, respectively. In addition, soybean rhizosphere soil eubacterial community structures were not shifted by bacterial inoculation. Therefore, Azospirillum sp. could be used in co-inoculant production with B. japonicum for soybean.Keywords: Bradyrhizobium, plant growth promoting rhizobacteria (PGPR), soybean, co-inoculation, competition, rhizosphere eubacterial community structureAfrican Journal of Biotechnology Vol. 12(20), pp. 2850-286

Selection and evaluation of Bradyrhizobium inoculum for peanut, Arachis hypogea production in the Lao People’s Democratic Republic

The interaction between leguminous plants and Bradyrhizobium is limited, known as host specificity. Therefore, the selection of an appropriate Bradyrhizobia for use as biofertilizer inoculum for legumes is necessary. The Arachis hypogea L. is the most popular legume produced in the Lao People's Democratic Republic (PDR). Therefore, this research aimed to obtain the appropriate Bradyrhizobia that provides high efficiency in A. hypogea production in the Lao PDR. The 14 isolates were obtained from root nodules of A. hypogea L. trapped with Lao PDR soil samples. Three were the top isolates PMVTL-01, SMVTL-02, and BLXBL-03 showing high efficiency for peanut growth promotion. Strains PMVTL-01 and SMVTL-02 were closely related to the Bradyrhizobium geno sp. SA-3 Rp7b and B. zhanjiangense, respectively, whilst strain BLXBL-03 was closely related to Bradyrhizobium sp. CCBAU51745 and B. manausense BR3351. The competitiveness of these strains with Bradyrhizobium sp. SUTN9-2::GFP was analyzed, and only Bradyrhizobium sp. SMVTL-02 performed a number of occupied nodules higher than SUTN9-2::GFP. In addition, the competitiveness of the selected strain Bradyrhizobium sp. SMVTL-02 in a soil sample from the Lao PDR in the pot level was employed by tagging the SMVTL-02 with the DsRed gene. The results demonstrated that the DsRed-expressing tagged strain showed higher nodule occupancy than indigenous strains. Moreover, the results of the acetylene reduction assay (ARA), nodule number, nodule dry weight, and total plant dry weight from the pot experiment that inoculated with the SMVTL-02::DsRed were presented as having high potential to promote peanut growth as compared to non-inoculation. Thus, Bradyrhizobium sp. SMVTL-02 could be considered a potential biofertilizer inoculum for A. hypogea production in the Lao PDR

Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice

タイにおけるササゲ属アズキ亜属野生種とそれらに着生する根粒菌の収集

Wild species in the subgenus Ceratotropis in the genus Vigna are believed to be a useful gene (s) source for the Ceratotropis cultigens such as mungbean (V. radiata), black gram (V. mungo), moth bean (V. aconitifolia), rice bean (V. umbellata) and azuki bean (V. angularis). A collaborative mission to collect seed samples and root nodules from wild Ceratotropis species in Thailand was conducted from Nov. 28 to Dec. 6, 1996. Exploration covered central and northern Thailand including Chai Nat, Phichit, Phitsanulok, Sukhothai, Tak, Lampang, Chiang Mai, Mae Hong Son, and Chiang Rai provinces. Aa a result, 29 seed samples and 24 nodule samples from wild Vigna umbellata, 3 seed samples and 1 nodule sample from cultivated V. umbellata (rice bean), 2 seed samples and 3 nodule samples from escaped V. umbellata 2 seed samples and 3 nodule samples from V. grandiflora, 14 seed samples and 13 nodule samples from V. hirtella, 3 seed samples and 2 nodule samples from V. minima, 2 seed samples and 3nodule samples from V. trinervia, 1 seed sample and 1 nodule sample from V. mungo var. silvestris, 2 seed samples and 1 nodule sample from mungbean, 1 seed sample and 4 nodule samples from escaped black gram, 1 seed sample and 1 nodule ample from cowpea (V. unguiculata cv-gr. Unguiculata), 1 seed sample and 1 nodule sample from yard long bean (V. unguiculate cv-gr. Sesquipedalis), and 1 seed sample and 1 nodule sample from common bean (Phaseolus vulgaris) were collected. Seed samples were shared between Chai Nat Field Crops Research Center, Thailand and NIAR, Japan, In Japan, rhizobia from nodules and after regeneration seed samples collected will be preserved in the MAFF genebank. Herbarium specimens are deposited in the MAFF genebank herbarium. Color photographs of flower, seed, plant and taxonomic descriptions, distribution, characteristics of wild Ceratotropis species are available in the homepage "The Illustrated Legume Genetic Resources Database" on the WWW via the internet. The URL is as follows http : //www. gene. affrc. go. Jp/image/legume. htm

Survival and effectiveness stability of cowpea rhizobia as affected by soil temperature and moisture

Typescript (photocopy).Vigna unguiculata (L.) Walp and Arachis hypogaea L. are economically important food legumes in the cowpea cross-inoculation group that are nodulated by Rhizobium. Benefits from inoculation depend on survival and effectiveness stability of rhizobia introduced into the soil. The objectives of this research were to evaluate the effect of soil temperature and moisture on rhizobial survival and the effectiveness of surviving rhizobia. To determine survival of rhizobia in soil, five strains of cowpea rhizobia were used. They were incubated under moisture conditions of air dry, moist (-0.33 bar), and saturated and temperature conditions of 20°C, 35°C, and 45°C for nonsterilized soil and 40°C in sterilized soil. Three strains of cowpea rhizobia were used to determine effectiveness stability of surviving rhizobia. Incubations were under dry and moist (-0.33 bar) soil conditions at 40°C in sterilized soil for 45 days and at 45°C in nonsterilized soil for 15 days. To evaluate effectiveness, eighty single colony isolates of each strain were obtained from nodules of plants inoculated with soil samples at time 0 and at 15 days of incubation in dry and moist nonsterilized soils. Forty isolates were obtained from pour plates of soil dilutions of sterilized soil at time 0 and 60 isolates were obtained from soil samples of dry and moist soil after 45 days. Effectiveness of isolates was measured as dry matter produced by siratro plants. The number of rhizobia determined by the most probable number plant infection method, as compared to plate counts under pure culture conditions, using cowpea, siratro or peanut underestimated the population by 10 to 100-fold but it was the only available method for use with nonsterilized soil. Survival of rhizobia, in nonsterilized soil, was affected by temperature and moisture. At 35°C, the populations of T-1, TAL309, and 3281 decreased approximately 100-fold for the dry soil and the saturated soil during 45 days of incubation. However, populations of strain 201 decreased by only 10-fold under the same environmental conditions. When strains 201 and TAL309 were incubated at 45°C the population decreased rapidly and could not be detected at 45 days. In sterilized moist soil, at 40°C the population of all strains increased during the first 2 weeks. For dry soil conditions the populations began decreasing within 5 days but in contrast to the nonsterilized soil, the populations did not decline under saturated soil conditions..

Survival and effectiveness stability of cowpea rhizobia as affected by soil temperature and moisture

Typescript (photocopy).Vigna unguiculata (L.) Walp and Arachis hypogaea L. are economically important food legumes in the cowpea cross-inoculation group that are nodulated by Rhizobium. Benefits from inoculation depend on survival and effectiveness stability of rhizobia introduced into the soil. The objectives of this research were to evaluate the effect of soil temperature and moisture on rhizobial survival and the effectiveness of surviving rhizobia. To determine survival of rhizobia in soil, five strains of cowpea rhizobia were used. They were incubated under moisture conditions of air dry, moist (-0.33 bar), and saturated and temperature conditions of 20°C, 35°C, and 45°C for nonsterilized soil and 40°C in sterilized soil. Three strains of cowpea rhizobia were used to determine effectiveness stability of surviving rhizobia. Incubations were under dry and moist (-0.33 bar) soil conditions at 40°C in sterilized soil for 45 days and at 45°C in nonsterilized soil for 15 days. To evaluate effectiveness, eighty single colony isolates of each strain were obtained from nodules of plants inoculated with soil samples at time 0 and at 15 days of incubation in dry and moist nonsterilized soils. Forty isolates were obtained from pour plates of soil dilutions of sterilized soil at time 0 and 60 isolates were obtained from soil samples of dry and moist soil after 45 days. Effectiveness of isolates was measured as dry matter produced by siratro plants. The number of rhizobia determined by the most probable number plant infection method, as compared to plate counts under pure culture conditions, using cowpea, siratro or peanut underestimated the population by 10 to 100-fold but it was the only available method for use with nonsterilized soil. Survival of rhizobia, in nonsterilized soil, was affected by temperature and moisture. At 35°C, the populations of T-1, TAL309, and 3281 decreased approximately 100-fold for the dry soil and the saturated soil during 45 days of incubation. However, populations of strain 201 decreased by only 10-fold under the same environmental conditions. When strains 201 and TAL309 were incubated at 45°C the population decreased rapidly and could not be detected at 45 days. In sterilized moist soil, at 40°C the population of all strains increased during the first 2 weeks. For dry soil conditions the populations began decreasing within 5 days but in contrast to the nonsterilized soil, the populations did not decline under saturated soil conditions..

Survival of Cowpea Rhizobia in Soil as Affected by Soil Temperature and Moisture

Successful inoculation of peanuts and cowpeas depends on the survival of rhizobia in soils which fluctuate between wide temperature and moisture extremes. Survival of two cowpea rhizobial strains (TAL309 and 3281) and two peanut rhizobial strains (T-1 and 201) was measured in two soils under three moisture conditions (air-dry, moist (−0.33 bar), and saturated soil) and at two temperatures (25 and 35°C) when soil was not sterilized and at 40°C when soil was sterilized. Populations of rhizobia were measured periodically for 45 days. The results in nonsterilized soil indicated that strain 201 survived relatively well under all environmental conditions. The 35°C temperature in conjunction with the air-dry or saturated soil was the most detrimental to survival. At this temperature, the numbers of strains T-1, TAL309, and 3281 decreased about 2 logs in dry soil and 2.5 logs in saturated soil during 45 days of incubation. In sterilized soil, the populations of all strains in moist soil increased during the first 2 weeks, but decreased rapidly when incubated under dry conditions. The populations did not decline under saturated soil conditions. From these results it appears that rhizobial strains to be used for inoculant production should be screened under simulated field conditions for enhanced survival before their selection for commercial inoculant production