Proposed some interactions at molecular level of PGPR coinoculated with Bradyrhizobium diazoefficiens USDA110 and B.japonicum THA6 on soybean symbiosis and its potential of field application

Applied Soil Ecology 85 (2014) 38–49

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Proposed some interactions at molecular level of PGPR coinoculated with Bradyrhizobium diazoefficiens USDA110 and B. japonicum THA6 on soybean symbiosis and its potential of field application Janpen Prakamhang a , Panlada Tittabutr a , Nantakorn Boonkerd a , Kamonluck Teamtisong b , Toshiki Uchiumi c , Mikiko Abe c , Neung Teaumroong a, * a

School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand The Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand c Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 March 2014 Received in revised form 19 August 2014 Accepted 20 August 2014 Available online xxx

The objectives of this research were to select the appropriate plant growth promoting rhizobacteria (PGPR) and evaluate their influence in promoting nodulation and N2-fixing efficiency of soybean (Glycine max) by coinoculation with Bradyrhizobium diazoefficiens USDA110 and B. japonicum THA6 strains. Selected 12 PGPR performed a significant capability of promoting N2-fixation, nodule number, nodule and plant dry weight with both of the commercial bradyrhizobial strains, USDA110 and THA6 (P < 0.05). Furthermore, isolates S141 and S222, which are closely related to Bacillus subtilis and Staphylococcus sp., were selected for coinoculation with USDA110 and THA6. The effective coinoculation doses of PGPR: Bradyrhizobium on soybean were 106:106 colony forming unit (CFU) ml
1 seed
1. The expression levels of soybean and Bradyrhizobium related genes including Glyma17g07330, otsA, phbC, dctA and nifH in nodule discontinuously triggered both up- and down-regulation at different time frames (2–7 WAI). The transmission electron microscopy (TEM) micrograph of coinoculated soybean nodule showed the compact cluster of bacteroids which was densely packed with poly-ß-hydroxybutyrate (PHB) granules. The amounts of PHBs remained in mature nodule of coinoculation treatments whilst single inoculation nodules were senescence. The induction of soybean root subsequently increased the nodulation signaling and then activated the trehalose accumulation and the transport of carbon that represented an increase in PHB accumulation, resulting in the enhancement of the nodulation and N2-fixation in soybean. These results were accordingly related with phenotypic characters in Leonard’s jar experiment in terms of enhancing the nodulation and N2-fixation in soybean. The effect of coinoculation experiment under field condition could increase 9.7–43.6% of seed yield per hectare which was higher than those of uninoculation or single inoculation of PGPR or Bradyrhizobium. Therefore, the efficiency to enhance soybean N2-fixation by coinoculation of S141 and S222 with Bradyrhizobium strategy could be developed for supreme soybean inoculants. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Bradyrhizobium japonicum B. diazoefficiens Coinoculation PGPR

1. Introduction Bradyrhizobium plays a special role in the nitrogen cycle of agroecosystems by infecting the roots of soybean (Glycine max) and forming dinitrogen-fixing nodules. In this way, significant amounts of nitrogen are fixed and transferred to the plant, reducing the need for nitrogen fertilizer. However, there is a number of factors which impede the nodulation on soybean root. Several abiotic and biotic factors can inhibit the formation of the N2-fixing symbiosis

* Corresponding author. Tel.:+ 66 44 223350; fax: +66 44 216345. E-mail address: [email protected] (N. Teaumroong). http://dx.doi.org/10.1016/j.apsoil.2014.08.009 0929-1393/ ã 2014 Elsevier B.V. All rights reserved.

between rhizobia and leguminous plants. The lack of nodules or formation of ineffective ones on soybean roots may be resulting from host micro-symbiont incompatibility as well as the function of both known and unknown bio-molecule such as flavonoides, polysaccharides and hormones (Daayf et al., 2012). Furthermore, the inoculation dose is an important factor in agricultural application of microbial inoculants. Many countries have the dose standards for rhizobial inoculants (Smith and Hume, 1987). In a field experiment, soybean nodule number and mass including grain yield were all related to B. japonicum inoculants with bacterial density ranging from 103 to 106 cells seed
1. However, the nodule number did not increase as much as the density of inoculant cells (Duzan et al., 2004).

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Enhancement of legume nitrogen fixation by coinoculation of rhizobia with some plant growth promoting rhizobacteria (PGPR) is an alternative approach to improve the nitrogen availability in sustainable agricultural production systems. Some PGPR strains are capable of promoting growth of leguminous plants, nodulation, and nitrogen fixation when coinoculated with rhizobia. Examples of these are Azospirillum (Aung et al., 2013), Azotobacter (Wu et al., 2012), Bacillus (Atieno et al., 2012), Serratia (Zahir et al., 2011), and Streptomyces (Tokala et al., 2002). Rhizobia and PGPR share common microhabitats in the root and rhizosphere soil interface so they must interact during the root colonization process. The effect of Rhizobium–PGPR coinoculation has been observed in different symbiotic and plant growth parameters. Compared to single Rhizobium inoculation, coinoculation of Rhizobium spp. and Azospirillum spp. can enhance the number of root hairs, the amount of flavonoids exuded by the roots, and the number of nodules formed (Remans et al., 2008). The beneficial influence of PGPR on nodulation of legumes by Rhizobium has been variously attributed to their ability to produce phytohormones (Drogue et al., 2013) as well as by other unidentified mechanisms (Dudeja et al., 2012). The possibility that metabolites other than phytohormones, such as siderophores and flavonoids which enhance nod gene expression, might enhance nodule formation has also been proposed (Lugtenberg et al., 2013), but these hypotheses have not been well verified. It is unlikely that any one rhizobacterium would be predominant and effective in all environments and hence mixtures of compatible strains might be more significant than single bacterial specie in promoting plant growth. It is widely accepted that plant growth promotion by PGPR does not rely on a unique mechanism, but rather is the net result of multiple mechanisms. According to this view, the general objective of this research is to select the appropriate PGPR and evaluate an increase of soybean (Glycine max) yield via nodule nourishment mediated by coinoculation PGPR with B. diazoefficiens USAD110 and B. japonicum THA6 compared to single inoculation as well as to propose some phenomena involved in Bradyhizobiumsoybean–PGPR interactions. 2. Materials and methods 2.1. Bacterial strains Bradyrhizobium diazoefficiens USDA110 and B. japonicum THA6, which are commercially used in rhizobial inoculants production for soybean in Thailand, were obtained from the Department of Agriculture, Bangkok, Thailand. B. diazoefficiens USDA110 and B. japonicum THA6 were cultured in yeast extract-mannitol (YEM) broth (Somasegaran and Hoben, 1994). The PGPR isolates were originally isolated from soybean rhizosphere soil as described by Piromyou et al. (2011) from 30 soybean fields in 11 provinces of Northern and North–Eastern part of Thailand including Chiang Mai (18 340 33.4700 N/98 520 52.9100 E), Chiang Rai (20 200 36.0200 N/99 540 38.9700 E), Lampang (18 130 44.4900 N/99 350 30.20“E), Lamphun (18 360 21.8300 N/99 20 42.9100 E), Lopburi (15 60 18.9700 N/101 110 49.4500 E), Mae Hong Sorn (19 210 14.0600 N/98 250 54.4500 E), Nakhon Ratchasima (15 1022.9900 N/101 360 27.4800 E), Nakhon Sawan (15 390 45.7500 N/100 340 12.0200 E), Phitsanulok (16 520 28.3100 N/100 210 19.6000 E), Phrae (17 580 25.8000 N/99 590 52.4000 E) and Uttaradit (17 410 13.6300 N/100 80 27.1700 E). The PGPR on N-free LG plates (Lipman, 1904) were incubated for 2 days at 30  C, and bacteria representative of the predominant morphologically distinct colonies present on the plates was selected for further analysis. All 285 bacterial isolates were screened against both B. diazoefficiens USDA110 and B. japonicum THA6 by antimicrobial spot test. Each bradyrhizobial culture was swab over YEM agar plates and after the plates were dried, 20 ml of each strains of PGPR

39

suspension was spotted onto bradyrhizobia agar plate. After 4days cultivation at 30  C, the plates were daily examined for growth inhibition by observing the clear zones. Only PGPR isolates which did not show any clear zone on both USDA110 and THA6 agar plate were selected for further coinoculation assay. All bacterial cultures were maintained by periodic transferred and stored in the refrigerator for further studies. 2.2. Effect of PGPR inoculation dose One ml of the bacterial broth culture (106 Colony Forming Unit (CFU) ml
1 seed
1) was inoculated onto each seed according to the following treatments. For the single inoculation treatment, the seedlings were inoculated separately with 106 CFU ml
1 seed
1 of S141, S222, USDA110, and THA6. For the coinoculation treatment, the USDA110 or THA6 culture at 106 CFU ml
1 seed
1 (as recommended using for soybean cultivation in Thailand) was mixed in a ratio of 1:1 with PGPR isolate S141 or S222 at five inoculum doses; 103, 104, 105, 106 and 107 CFU ml
1 seed
1. In the control treatment, the cell suspensions were replaced by sterilized distilled water. The plants were cultivated in growth chambers using modified Leonard's jar assemblies and harvested at 45 days after inoculation (DAI). The nodule number, nodule dry weight, and plant dry weight were measured. Nitrogenase activity was measured by the Acetylene Reduction Assay (ARA) (Somasegaran and Hoben, 1994). 2.3. Coinoculation effect of bradyrhizobia and PGPR on soybean 2.3.1. Leonard’s jar experiment The soybean (Glycine max (L.) Merrill) cv. Chiang Mai 60 is a recommended commercial line for use under Thai field condition. Seeds of soybean were cultivated in growth chambers using modified Leonard’s jar assemblies (Blauenfeldt et al., 1994). Seeds were surface sterilized and gnotobiotically germinated on wet tissue paper (Somasegaran and Hoben, 1994). The early stationary phase of PGPR and bradyrhizobia cultures were centrifuged (4000 g for 5 min) and washed with sterilized 0.85% (w/v) NaCl to remove the excess media, and the cell pellet was resuspended in 0.85% (w/v) NaCl solution. A preliminary Leonard’s jar experiment was conducted to evaluate the coinoculation effects of B. diazoefficiens USDA110 and B. japonicum THA6 with 45 PGPR isolates on soybean. For the single inoculation, the seedlings were inoculated separately with 1 ml seed
1 of 106 CFU ml
1 of PGPR or B. japonicum, and mixed in a ratio of 1:1 for coinoculation treatment. In the control treatment, the cell suspensions were replaced by 0.85% (w/v) NaCl solution. Plants were grown in growth chamber at 25  C light room under 16/8 h light/dark photoperiod. During the experiment, the plants were watered regularly with N-free nutrient solution (Zhang et al., 1996). The experiment was laid out with five replicates for each treatment. Plants were sampled at 45 DAI and the nodule number, nodule dry weight, and plant dry weight were recorded. The top 12 PGPR that can promote soybean growth were selected for further PGPR characterization and identification studies. Only 2 PGPR isolates which showed the highest capability of growth promoting were selected for further experiments. 2.3.2. Field experiment Single and coinoculation inocula were prepared as described in Leonard’s jar experiment by 9 different treatments as follow: (1) Control; Non inoculation, (2) USDA110; single inoculation with B. diazoefficiens USDA110, (3) THA6; single inoculation with B. japonicum THA6, (4) S141; single inoculation with Bacillus subtilis strain S141, (5) S222; single inoculation with Staphylococcus sp. strain S222, (6) U110 + S141; coinoculation with B. diazoefficiens USDA110 and B. subtilis strain S141, (7) U110 + S222; coinoculation

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with B. diazoefficiens USDA110 and Staphylococcus sp. strain S222, (8) THA + S141; coinoculation with B. japonicum THA6 and B. subtilis strain S141, and (9) THA + S222; coinoculation with B. japonicum THA6 and Staphylococcus sp. strain S222. The experiment was performed in two sites. Site 1 was Organic Farm located in Suranaree University of Technology (SUT) campus at Nakhon Ratchasima province, Thailand (14 520 16.100 N and 102 10 31.9500 E) which has history of legume cultivations but no history of chemical fertilizer application. This experiment was conducted during January–April, 2012 with the average rainfall 31.07 mm, average temperature range 24.4–30.6  C, and the average humidity range 63.1–69.2%. The soil was sandy clay in texture having a pH 6.41, EC 0.031 ms cm
1, 2% organic matter content, available P 11 ppm, and exchangeable K 330 ppm. Site 2 was rice paddy field at Buriram province (14 340 3.6300 N and 102 320 38.9300 E) which has a history of chemical fertilizer application but no history of legume cultivations. This experiment was conducted during August– November, 2012 with the average rainfall 152.03 mm, average temperature range 26.4–29.1  C, and the average humidity range 71.1–83.5%. The soil was clay loam in texture having a pH 5.79, EC 0.420 ms cm
1, 1.92% organic matter content, available P 19.80 ppm and exchangeable K 43.57 ppm. Each field site was laid out under a completely randomized design (CRD) by dividing into nine treatments (as mentioned above), and each treatment consisted of 3 replicates making a total of 27 plots according to 9 different treatments as mentioned above. Four-row plots were used with 50 cm between rows and 10 cm between plants in a row and plots were kept 0.5 m spacing between blocks. Each size of plot of site 1 was 2  5 m (10 m2) and site 2 was 2  2 m (4 m2). Inoculum containing approximately 1 ml bacteria (106 CFU seed
1) was poured over the seed in each row prior to covering the seeds with soil. The regular agricultural practices were done except chemical fertilizer application and pesticide spraying. The plants were harvested at 100 DAI. Seed yield was taken from the two middle rows of each plot. Pods were removed and threshed by hand and seed yield was determined. 2.4. Bacterial plant growth promoting characteristics and identification 2.4.1. Indole-3-acetic acid (IAA) production assay Bradyrhizobia and PGPR isolates were grown in YEM and LB medium, respectively, and the IAA production was colorimetrically determined as described by Costacurta et al. (1998). Pure IAA (Sigma, USA) was used as standard. 2.4.2. ACC (1-aminocyclopropane-1-carboxylate) deaminase activity Assay Bradyrhizobial and PGPR isolates were grown in YEM and LB media, respectively until cells reached the early stationary phase. The cells were collected by centrifugation at 5000  g for 5 min and washed twice with minimal medium (Penrose and Glick, 2003). Cell pellets were suspended in 15 ml of minimal medium supplemented with 1 mM ACC-deaminase, and further incubated at 28  C for 24 h with shaking at 200 rpm to induce ACC-deaminase enzyme production. ACC deaminase activity was measured according to a protocol of Tittabutr et al. (2008). 2.4.3. Siderophore production assay Bacterial isolates were assayed for siderophores production on the Chrome azurol S (CAS) agar medium (Sigma, Ltd.) described by (Schwyn and Neilands, 1987). The CAS agar plates were prepared and divided into equal sectors and spot inoculated with test organisms (10 ml of 106 CFU ml
1) and incubated at 28  2  C and observed daily for up to 7 days. Development of yellow–orange

halo around the growth was considered as positive for siderophore production. 2.4.4. 16S rRNA gene analysis The selected PGPR were identified at species level based on the full length sequence analysis of 16S rRNA gene. The genomic DNA was extracted following the method of Prakamhang et al., 2009. The 16S rRNA universal primers 27F and 1492R were used to amplify approximately 1.5 kb internal region of the 16S rRNA gene (Weisburg et al., 1991). The nucleotide sequence of purified PCR products was analyzed at the Macrogen Service Center (Seoul, Korea). The DNA sequences were generated and the most closely related sequences were obtained from the NCBI database. 2.5. Transmission electron microscopy (TEM) studies of bacteroids in soybean nodule sections The fresh soybean nodules from Leonard’s jar experiment were harvested at 2, 3, 4, 5, 6 weeks after inoculation (WAI) and used for further investigation. The procedures for preparation of nodule were basically performed according to a method reported by Fuentes et al. (2002). The embedded specimens were trimmed and sliced into ultrathin sections with an ultramicrotome (Ultracut RMC Boeckeler1, Boeckeler Instruments Inc., USA). The sections were mounted on grids and stained according to Chansatein et al. (2012). The sections were viewed and digitally photographed using a TEM (JEOL JEM-1230, JEOL, Japan). 2.6. Gene expression analysis 2.6.1. Sample preparation Soybean samples were prepared and conducted in Leonard’s Jar experiment as mentioned above. The fresh and upper most soybean nodules from the main root were harvested weekly from 2 to 7 WAI. Nodule samples were sterilized with 95% ethanol for 10 s and washed 5–6 times with sterilized water, and then immediately frozen in liquid nitrogen and stored at –80  C for further total RNA extraction. The experiment was laid out with CRD with three biological replications. 2.6.2. Total RNA extraction and RT-PCR analysis The frozen nodules were ground in TissueLyser (QIAGEN, USA) to a fine powder. Total RNA were directly isolated from plant samples using RNeasy Plant Mini Kit (QIAGEN, USA) according to the manufacturers protocol. RNAs were treated with DNaseI to prevent contamination of genomic DNA, and then cleaned by using RNeasy MinElute Cleanup Kit (QIAGEN, USA). Transcription levels were determined by reverse-transcription polymerase chain reaction (RT-PCR). Primers for transcript amplification and gene description are provided in Supplementary Table S1. The geometric mean of relative expression ratios for three biological repetitions and the corresponding upper and lower 95% confidence intervals were calculated. All RT-PCR were performed in Techne1 TC512 Thermal Cycler (TECHNE, UK), and the PCR products were visualized using 1% agarose gel electrophoresis and stained with a 1:10,000 dilution of SYBR Green I nucleic acid stain (Sigma– Aldrich, St. Louis, MO), then documented on gel documentation (FireReader, Uvitec Cambridge) and analysis (UVIband, Uvitec Cambridge). 2.7. The statistical analysis Data from each experiment were first submitted to tests of normality and homogeneity of variances for each variable and then to analysis of variance (ANOVA). When confirming a statistically significant value in the F-test (P  0.05), a post hoc test (Duncan’s

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Fig. 1. Nodule number per plant (A) nodule dry weight per plant (B) and plant dry weight (C) of soybean after single and coinoculated between B. diazoefficiens USDA110 or B. japonicum THA6 with 12 selected PGPR isolates at 45 DAI. Treatments are represented by control (non-inoculated); USDA110 (B. diazoefficiens USDA110); THA6 (B. japonicum THA6); + (coinoculation treatment). Significance at P  0.05 is indicated by mean standard error bars (n = 5).

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multiple-range test at P  0.05) was used as a multiple comparison procedure (Duncan, 1955) by SPSS1 software for WINDOWSTM, Version 14.0; SPSS, Chicago, IL). 3. Results 3.1. Screening of PGPR Out of 285 soybean rhizosphere soil bacterial isolates, only 45 PGPR isolates did not show antimicrobial activity or clear zone on both Bradyrhizobia USDA110 and THA6 agar plates. All 45 PGPR isolates were tested with soybean in Leonard’s jar trails, and only 12 PGPR showed significant capability of promoting nodule number, nodule dry weight and plant dry weight when coinoculated with both bradyrhizobia when compared to single inoculation on soybean at 45 DAI (P < 0.05) (Fig. 1). Variations in nodule number at 45 DAI as consequence of different microbial inoculation and coinoculation were presented in Fig. 1A. The highest mean number of nodules per plant was recorded in USDA110 + S222 treatment (49.00 nodules plant
1) followed by THA6 + S222 (46.75 nodules plant
1) when compared with all other treatments and control. Enhancement in number of soybean nodules obtained from coinoculated with USDA110 and S141 or S222 was higher than those of single coinoculation with USDA110 by 63.1 and 76.6%, respectively. Similar trend was also found in case of THA6 and its both coinoculation S141 and S222 by 90.8 and 54.1%, respectively. The data on the nodule dry weight were presented in Fig. 1B. At 45 DAI, significantly higher nodule dry weight of 102.8 mg plant
1 was recorded in USAD110 + S222 treatment followed by USDA110 + S111 (100.43 mg plant
1). Coinoculation of USDA110 with S141 and S222 enhanced nodule dry weight of soybean compared with single inoculation with USDA110 by 148.1 and 102.8%, respectively. Soybean nodule dry weight was significantly improved when coinoculated THA6 with S141 and S222 by 150.1 and 141.2%, respectively. Coinoculation USDA110 + S141 treatment showed significant maximum plant dry weight (1400.5 mg plant
1) (Fig. 1C) followed by USDA110 + S222 treatment (1377.9 mg plant
1). Coinoculation of USDA110 with S141 and S222 enhanced soybean plant dry weight compared with USDA110 single inoculation by 39.3 and 37.1%, respectively. Similar results were also found in case of THA6, in which soybean plant dry weight was increased when coinoculated THA6 with S141 (40.4%) and S222 (50.1%) when compared with single inoculation of THA6. The IAA production and strain identification using 16S rRNA gene sequences of 12 PGPR strains including 2 strains of commercial bradyrhizobia were assessed (Table 1). Isolate SPT122 produced highest amounts of IAA followed by S141 and

S131b (23.00, 19.33 and 13.67 mg ml
1, respectively). However, the IAA production was neither detected in USDA110 nor THA6. All the PGPR used for inoculation were assessed for their ACC-deaminase activity and siderophore but none of them produced ACCdeaminase or siderophore. Among the coinoculation treatments, S141 and S222 performed significantly highest in all plant growth parameters when coinoculated with USDA110 and THA6. Therefore, S141 and S222 were selected for further experiments. 3.2. Determination for dose of Bradyrhizobium and PGPR There were differences among PGPR doses for all soybean parameters including plant dry weight (Fig. 2A), nodule number (Fig. 2B) nodule dry weight (Fig. 2C), and also nitrogen fixing activity (Fig. 2D). The results of the controls and single inoculation of each PGPR were not different from each other on soybean plant dry weight. All inoculant doses (103–107 CFU ml
1 seed
1) of single inoculation of USDA110 and THA6 or coinoculation treatments promoted higher plant dry weight than those obtained from uninoculated control or single inoculation of PGPR treatments. Soybean nodule number, nodule dry weight, and N2-fixing activity of single inoculation of bradyrhizobia and all coinoculation treatments were significantly different (P < 0.05) among the bacterial concentrations tested. In case of USDA110 with S141 and S222, the inoculum dose at 106:106 CFU ml
1 seed
1 increased plant biomass by 115.5 and 65.8%, nodule number by 64.85 and 113.3%, nodule dry weight by 127.0 and 133.9%, and ARA activity by 98.5 and 114.8%, respectively. In case of THA6, the optimum coinoculation dose was also 106 CFU ml
1 seed
1 for S141 and S222. This dose increased plant biomass by 78.6 and 48.7%, nodule number by 38.0 and 23.4%, nodule dry weight by 67.0 and 142.5%, and ARA activity by 69.9 and 128.8%, respectively. Thus the optimum coinoculation dose was at 106 CFU ml
1 seed
1 for USDA110 and THA6 with 106 CFU ml
1 seed
1 for both PGPR isolates S141 and S222. 3.3. The relative genes expression levels in soybean nodule after single and coinoculation in the different time frames The relative expression levels of Glyma17g07330 gene were upregulated at the 2 WAI and almost stopped expressing at the 6 WAI (Fig. 3A). The relative expression level of Glyma17g07330 gene in nodule inoculated with USDA110 was strongly up-regulated from 3 to 5 WAI and then the expression stopped at 6 WAI. In case of THA6, the highest up-regulated expression of this gene was recorded at 4 WAI and the expression level completely stopped at 6 WAI. Interestingly, significantly higher expression level of

Table 1 IAA production and partial 16 S rRNA gene sequence analysis of selected PGPR isolates. Isolates

Accession number

% Identity

IAA production(mg ml
1)a

USDA110 THA6 MC12 MC3 S131a S131b S141 S221 S222 SPT1 SPT122 SPT422 TS22 TS413

AB909430 AB909431 AB909432 AB909433 AB909434 AB909435 AB909436 AB909437 AB909438 AB909439 AB909440 AB909441 AB909442 AB909443

99% Bradyrhizobium diazoefficiens USDA110 99% Bradyrhizobium japonicum USDA91 100% Serratia marcescens d4 100% Bacillus sp. C-24 99% Bacillus cereus DZ4 100% Arthrobacter sp. RSBA1 100% Bacillus subtilis GB03 100% Pseudomonas putida CY06 99% Staphylococcus sp. JMP-C 99% Bacillus megaterium XA7-7-1 99% Staphlococcus sciuri LH-T5 100% Bacillus sp. C-1 98% Bacillus megaterium TOBCMDU-1 96% Unculturable Staphylococcus sp.

0.02g  0.01 0.01g  0.01 2.00fg  1.00 11.33cd  1.53 2.33fg  1.53 13.67c  2.08 19.33b  3.21 5.33ef  2.08 5.00ef  2.00 5.33ef  2.31 23.00a  3.00 8.33de  2.89 7.00e  1.00 1.33g  1.15

a

Means (n = 3) from a same column followed by different letters are significantly different (P  0.05, Duncan’s test), standard deviation.

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Fig. 2. Effects of inoculation dose between B. diazoefficiens USDA110 or B. japonicum THA6 at 106 cells seed
1 with various dose of PGPR isolates S141 and S222 on soybean plant dry weight (A) nodule number per plant (B) and nodule dry weight per plant (C) and N2-fixing activity (nmol C2H4 g
1 nodule dry weight) (D). The numbers at the x-axis symbolize the varied inoculation doses of PGPR are 103 to 107 CFU ml
1 seed
1 and the coinoculation ratio between bradyrhizobia at 106 CFU ml
1 seed
1 and varies inoculation dose of PGPR from 103 to 107 CFU ml
1 seed
1. Treatments are represented by Control (non-inoculated); U110 (B. diazoefficiens USDA110); THA6 (B. japonicum THA6); + (coinoculation treatment). Significance at P  0.05 is indicated by mean standard error bars (n = 5).

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Fig. 3. Relative expression level in 2–7 WAI soybean nodules of Glyma17g07330 (A), otsA (B), dctA (C), phbC (D) and nifH (E) genes. Soybean b-tubulin and 16S rRNA genes were used as an internal control. Significance at P  0.05 is indicated by mean standard error bars (n = 3).

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Glyma17g07330 gene was recorded in the coinoculation of THA6 + S222 treatment at the late of nodulation (4–7 WAI) especially at 7 WAI that was still expressed, while the expression of other treatments was stopped. In case of otsA gene expression, all coinoculation treatments gave the strongly expression since the 2 to the 7 WAI but the single inoculation with bradyrhizobia showed trend of declining at the 6–7 WAI (Fig. 3B). At the 4 WAI, the relative otsA gene expression levels of coinoculation with USDA110 and S141 or S222 and THA6 with S222 were highest while THA6 + S141 treatment was strongly expressed at the 3 WAI. The relative dctA expression levels in all coinoculation treatments were highest at the 6 WAI (Fig. 3C). The relative expression levels in coinoculation with USDA110 and S141 treatment were elevated 3.1 folds while USDA110 + S222 promoted 1.9 folds. The phbC gene of single and coinoculation treatment was expressed at 4 WAI and remained up-regulated until the 7 WAI (Fig. 3D). The relative phbC expression levels in coinoculation with USDA110 + S141, USDA110 + S222, THA6 + S114 and THA6 + S222 were raised 2.5, 1.9, 3.3 and 2.3 folds at the 6 WAI, respectively. The expression levels of nifH gene were up-regulated in all treatments, both single and coinoculation treatments (Fig. 3E). The expression level of nifH gene was strongly induced by the coinoculation with bradyrhizobia and PGPR. Coinoculation of USDA110 with S141 and S222 induced the expression level was higher than those of single inoculation of USDA110 since 2 WAI until the 7 WAI. The upregulation levels of nifH gene were also found higher in THA6 + S141 and S222 than single inoculation of THA6 by 44.53 and 87.82% at 2 WAI, 41.52 and 42.86 at 3 WAI. 3.4. Effect of coinoculation on bacteroid morphology The TEM was used to observe the changes of bacterial morphology when single inoculation with bradyrhizobia was compared to the coinoculations with S141 or S222. There was no obvious difference in size, spacing or morphology of infected cell in nodule at the 2, 3, 4 and 5 WAI in every treatment. The nodules derived from USDA110 single inoculation at 6 WAI performed the widespread of cytoplasmic disruption, including cytoplasmic breakdown and lesions in the symbiosome membranes as indicated by arrow in Fig. 4A and B. Moreover, the coinoculation of USDA110 and S141 had symbiosome containing few bacteroids with appendages within the symbiosome large air space (Fig. 4C and D). However, the coinoculation of USDA110 and S222 showed the compact cluster of bacteroids which was densely packed with poly-ß-hydroxybutyrate (PHB) granules (Fig. 4E and F). The single inoculation with THA6 also exhibited the visibly disintegrated symbiosome membrane similar to USDA110 (Fig. 4G and H). In case of the coinoculation of THA6 with either PGPR strains S141 or S222, the symbiosome composed of either a single or multiple bacterial cells surrounded with the peribacteroid space and a plant derived peribacteroid membrane. Moreover, the PHB granules within the abundant of bacteroids in symbiosome were densely packed (Fig. 4I–L). 3.5. Effects of coinoculation on soybean grain yield under field condition

Fig. 4. Transmission electron micrographs (TEM) of infected cells of 2–7 WAI nodules single inoculation with B. diazoefficiens USDA110 (A and B), B. diazoefficiens USDA110 coinoculated with S141 (C and D), and B. diazoefficiens USDA110 coinoculated with S222 (E and F), single inoculation with B. japonicum THA6 (G and H) B. japonicum THA6 coinoculated with S141 (I and J) and B. japonicum THA6 coinoculated with S222 (K and L). The magnification is the same in panels A, C, E, G, I and L (bars = 1 mm) and in panels B, D, F, H, J and L (bars = 0.2 mm). The widespread of cytoplasmic disruption, including cytoplasmic breakdown and lesions in the symbiosome membranes, is indicated by arrow. CW; cell wall, B; bacteroid, PBM; peribacteroid membrane, PHB; poly-b-hydroxybutyrate granule.

In order to evaluate the effects of coinoculation on nodule number, nodule dry weight, and soybean seed yield under field condition, the experiments were carried out at two different locations, Buriram province and SUT Organic Farm field. At Buriram site, the number of soybean nodule obtained from coinoculations of USDA110 and S141 and S222 treatments was higher than those of single coinoculation with USDA110 by 47.4 and 58.5%, respectively. Similar trend was also found in case of THA6 both coinoculations of S141 and S222 by 66.7 and 59.1%,

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Fig. 5. Effects of coinoculation between B. diazoefficiens USDA110 or B. japonicum THA6 with PGPR isolates S141 and S222 at 106:106 cells seed
1 on nodule number per plant (A and B), nodule dry weight per plant (C and D), and soybean seed yield (E and F) performed at Buriram site (panel A, C, and E) and SUT Organic farm site (panel B, D, and F), respectively. Data represent the means of nine experiments, each with three replicates. Values represent mean  SD (n = 3). Within treatment, means labeled with different letters are statistically different at P < 0.05.

respectively (Fig. 5A). At SUT organic farm site, the nodule numbers obtained from coinoculations of USDA110 and S141 and S222 treatments were significantly increased and higher than those of single coinoculation with USDA110 by 63.1 and 72.7%, respectively. Parallel trend was also found in case of THA6 coinoculation with S141 and S222 by 47.7 and 37.2%, respectively (Fig. 5B). The coinoculations of USDA110 with S141 and S222 increased the nodule dry weight per plant when compared to the single coinoculation with USDA110 by 48.3 and 40.1%, respectively. In case of THA6, the coinoculations with S141 and S222 increased the nodule dry weight by 40.8 and 37.5%, respectively (Fig. 5C). Moreover, at SUT Organic farm site, the

nodule dry weights per plant of coinoculations of USDA110 with S141 and S222 were higher than those of USDA110 single coinoculation by 48.3 and 40.1%, respectively. For THA6, the coinoculations with S141 and S222 induced the nodule dry weight by 40.8 and 37.5%, respectively (Fig. 5D). The highest soybean seed yield was noticed in USDA110 + S141 (2811.70 kg ha
1) followed by THA6 + S222 (2732.45 kg ha
1) at Buriram site (Fig. 5E). The seed yields from coinoculations with USDA110 and S141 and S222 treatments were increased by 28.4 and 23%, respectively. In case of THA6, the coinoculation with S141 and S222 induced the seed yield by 25.2 and 27.3%, respectively. Furthermore, all coinoculation treatments were on par with each other. At SUT

J. Prakamhang et al. / Applied Soil Ecology 85 (2014) 38–49

Organic farm site, the highest seed yield was noticed in THA6 + S141 (2868.87 kg ha
1) followed by USDA110 + S141 (2748.23 kg ha
1) as shown in Fig. 5F. The seed yields in coinoculation with USDA110 and S141 and S222 treatments were increased by 9.8 and 1.4%, respectively. In case of THA6, the coinoculation with S141 and S222 induced the seed yield by 43.6 and 27.5%, respectively. 4. Discussion In this study, the PGPR isolates were preliminary isolated from soybean rhizosphere soil using LG N-free medium in order to obtain the most abundant root-adhering bacteria (Piromyou et al., 2011). Enhancement of nitrogen fixation is the ultimate goal of any Rhizobium-legume symbiosis. Therefore, all the PGPR selection experiments were carried out in N-free conditions. These might result in more chances for PGPR to persist and provide some nitrogen to plants via nitrogen fixation. We hypothesized that increase in nodule number, nodule dry weight, and plant dry weight of soybean due to coinoculation with Bradyrhizobium and PGPR could be attributed to a greater nitrogen fixation. The most commonly concerned modes of action of PGPR were phytohormone production which enhances plant development and growth, plant ethylene levels reduction through ACC-deaminase activity, iron uptake enhancement by siderophore production, phosphate mobilization for dissolving insoluble forms of phosphate, and a variety of mechanisms like flavonoids leading to changes in the pattern of root exudation (Vacheron et al., 2013). This research investigated the main modes of action of PGPR isolate including IAA production, ACC-deaminase activity and siderophore production. However, the ACC-deaminase activity and siderophore production could not be detected in all the selected PGPR. These results indicated that plant growth promotion may be derived from other factors rather than ACC-deaminase activity and siderophore production. The most effective PGPR isolates in this study were S141 and S222 which performed significantly highest in all plant parameters. Interestingly, isolates SPT122 and S131b showed the highest IAA production but did not show the highest capability of promoting soybean biomass when coinoculated with both bradyrhizobia (Table 1). It can be postulated that IAA production by PGPR does not always stimulate the soybean-rhizobia symbiosis. The component in the root and/or root exudate may alter the amount of IAA produced and/or genes expression. These results are consistent with the report of Kochar et al. (2013) that IAA secreted by PGPR interferes with the many plant developmental processes because the secreted IAA by PGPR altered endogenous plant IAA. In case of otsA, phbC, nifH, dctA, and MYB transcription factor (Glyma17g07330) genes, they might have an important function in various stages of nodule development, such as control of the nodules development and transport of nutrients between plant and bacteria. We suggested that a significant number of those of regulatory genes were specifically expressed in developing and/or mature nodules. Furthermore, at 2–7 WAI the single nodules can be observed and harvested for analysis. Our observations support the finding that a large number of putative genes were differentially expressed in mature nodules (Libault et al., 2009). The MYB transcription factor functions are necessary in different plant biological processes and may possibly be involved in legume nodulation (Du et al., 2012). The MYB transcription factor of soybean encoded by Glyma17g07330 gene is affected by rhizobial inoculation. Moreover, Glyma17g07330 has been established to be nodulation-specific (Libault et al., 2009). These results indicated that the MYB transcription factors have a key role in regulation of legume-specific nodulation. However, this might be responsible for legume-specific nodulation after coinoculation with bradyrhizobia and PGPR. During nodulation, the

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Glyma17g07330 gene was expressed during the first WAI but was strongly disrupted at 7 WAI (Fig. 3A). This indicated that Glyma17g07330 gene was expressed specifically in developing nodules but not in the late mature nodules which might be involved in bacterial infection or in the early steps of nodule development. However, functional analyses of soybean MYBs found that they regulated numerous processes including responses by various abiotic stresses (Liao et al., 2008). Thus, abiotic stresses may also be considered as another factor involved in our results. However, the Glyma17g07330 functions in legume-specific nodulation remain to be characterized. The otsA gene is regulated for trehalose 6-phosphate synthase which controls trehalose synthesis. Trehalose accumulation in B. japonicum influenced nodulation of G. max cv. Lambert and was related to the soybean nodule development (Sugawara et al., 2010). We hypothesized that the expression of the otsA gene may be modulated by bradyrhizobia and/or PGPR. Gene expression analysis reported here indicated that the trehalose biosynthesis gene otsA was induced by coinoculation with PGPR, resulting in an increase of trehalose accumulation levels. Various studies have reported that the osmotic homeostasis stress induced the trehalose biosynthetic genes in B. japonicum (Sugawara et al., 2010) or when the Bradyrhizobium entered a symbiotic relationship with the soybean plant. However, there are no reports considering the effect of coinoculation with PGPR. Our results demonstrated that expression of the otsA gene may be modulated by the accumulation of trehalose in bradyrhizobia and then influence its symbiosis with soybean. Taken together with soybean yield results, these indicated that the increase of trehalose accumulation caused by PGPR coinoculation enhanced N2 fixation and soybean yield. These results are similar to those found in studies of Rhizobium etliPhaseolus vulgaris symbiotic interaction (Suárez et al., 2008) and R. tropici CIAT899 in P. vulgaris (Bargaz et al., 2013). The higher gene expression levels in coinoculation experiment than those of single inoculated with bradyrhizobia at the late nodulation (7 WAI) may be caused by the effect of their plant-rhizobial interaction. In young soybean nodules, the bulk of trehalose is located in the cytosol and in only a small proportion in the bacteroids, but the older plants change to the opposite. The increasing retention of trehalose in bacteroids with increasing nodule age indicated that its function changes during nodule development, or trehalose may play a role during some stage of the life cycle of the bacterium outside of the nodule (Streeter and Gomez, 2006). The dicarboxylate transport protein DctA encoded by dctA gene was responsible for transporting of carbon from the host plant to the bacteroids in order to sustain bacteroid metabolism (Batista et al., 2009). The dctA encoded a structural protein necessary for C4-dicarboxylate transport and there has been little direct information concerning the effects of coinoculation to dctA expression. From our study, the expression of dctA gene was observed in all developmental stages from early to late symbiotic bacteroid differentiation (6–7 WAI). These findings suggested that the coinoculation seems to influence the expression of dctA gene, especially during the bacteroid differentiation into the fully differentiated bacteroids. Moreover, the dctA gene could be consistent with an increased need for transport of C4-dicarboxylic acids by the nitrogen-fixing bacteroids (Boesten et al., 1998). The phbC gene encoded the PHB polymerase and influenced PHB accumulation in bacteroids of soybean. Although most of the bacteroid carbon provided by the plant was channeled into energy for nitrogen reduction, some carbon was diverted by the bacteroids into the production of PHB (Resendis-Antonio et al., 2011). The PHB accumulation during symbiosis might increase rhizobium fitness, proliferation of survival or imitation inside senescing nodules or later in the soil, resulting in their own reproductive success. This could make a conflict of interest between host and symbiont

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especially the influence of coinoculation bradyrhizobia and PGPR on soybean. The expression of phbC gene was observed at 4–7 WAI probably because PHB accumulation took place after the peak of nitrogenase activity had passed. However, the ability to accumulate PHB during symbiosis fluctuated relative to nitrogenase activity by competing for the same energy and reductant sources. Thus, PHB synthesis in bacteroids and nitrogen-fixation might compete for the photosynthate (Trainer and Charles, 2006). Interestingly, the data presented in this study suggested that coinoculation with bradyrhizobia and PGPR forced accumulation of PHB during the symbiosis and did not appear to have a negative effect on plant yield. Moreover, the phbC was expressed under different bacterial inoculations because the growth and metabolism of different species of bacteria may be influenced by the increase or decrease in PHB accumulation. However, studies of the effect of coinoculation on PHB synthesis and transporters of soybean nodules are needed to analyze in more detail. The nifH gene encoded the structure of denitrogenase reductase (NifH) which is used to fix atmospheric nitrogen. The coinoculation of PGPR may influence the expression of bradyrhizobia nifH gene expression. A significant increase in nifH expression level in soybean root nodule was observed after coinoculation of Bradyrhizobium and PGPR. This suggested that Bradyrhizobium was supported by PGPR in order to enhance nitrogen fixation and nodulation. Interestingly, the nifH gene was highly expressed in mature nodules (at 4 WAI) while the dctA, phbC, and otsA genes were expressed after the nitrogen fixing had passed. Soybean may overcome the problem of nitrogen limitation by stimulating physiological adaptations. These adaptations consist of the increase of nodule size and nodule number per plant, and therefore the increase of nitrogenase activity (Libault, 2014). Moreover, an increase in the allocation of plant photosynthates to the bacteroides and the balances of varied ions also occurred. The essential role of PHB during the nodulation process is also demonstrated that phbC mutant defective in PHB biosynthesis does not fix N2 (Wang et al., 2007). Therefore, the controlled exchange of carbon and nitrogen sources caused balance between PHB accumulation and N2 fixation in nodules. The TEM micrograph of 6-week-old of either USDA110 or THA6 nodules showed different responses to the indicators of senescence of symbiosis whilst the coinoculation treatments demonstrated densely packed PHB granules within the bacteroids. The bacteroids accumulated large amount of trehalose, glutamate, myo-inositol and intermediates of the primary carbon metabolism (Vauclare et al., 2013). The accumulation of the storage compound (PHB), which diverts carbon supply from nitrogen fixation, is very frequently observed in reversibly differentiated bacteroid. Inhibition of PHB accumulation could be a direct or indirect consequence of the terminal differentiation. Moreover, terminally differentiated bacteroids with their weakened membranes and locked-within nodule cells might be more effectively digested during nodule senescence than bacteroids-differentiating nodule. Thus, recycling components of nodules might provide more nutrients for the plant (Kereszt et al., 2011). Relatively few studies have investigated the genetics and biochemistry of PGPR that control nodule senescence. Nodule senescence can be triggered by a signal or signals from the tight metabolic controlled by the host plant such as reactive oxygen species (ROS) (Silveira et al., 2011), antioxidants (ascorbate and glutathione), hormones and proteinases (Puppo et al., 2005) as well as certain cysteine proteases (Vorster et al., 2013). Yang et al. (2009) proposed the term ‘induced systemic tolerance’ (IST) for PGPR-induced physical and chemical changes in plants that resulted in enhanced tolerance to abiotic stress. Moreover, the PGPR strain induced a higher increase in these antioxidant enzymes in response to severe salinity (Kohler et al., 2009). Taken together, PGPR may delay senescence of nodule by reducing

stresses via adequate balance in the oxidative metabolism or manipulation of nodule hormone contents and production within legume nodules. However, senescence-related genes expression needs to be further elucidated. However, the coinoculation of both bradyrhizobia with S222 was not obviously different from single inoculation of bradyrhizobia in the field experiment at Buriram site. This might be because of the effect of indigenous rhizobia or rhizospheric bacteria on PGPR such as indigenous microbial community structure (Piromyou et al., 2011), competitive colonization, and rhizosphere eubacterial community structures of soybean (Aung et al., 2013). Moreover, the soybean yield trend on the SUT Organic farm site was obviously increased, especially when coinoculated with THA6 + S141. This might also depend on the soil property such as legume planting history or the chemical fertilizer application history. Moreover, both soil nitrogen and symbiotic nitrogen are required for the optimum soybean production. These results suggested that the availability of adequate amount of plant nutrient in soil could support the soybean production. Moreover, the organic matter available can promote the dispersal and activity of applied PGPR. 5. Conclusion The coinoculation with bradyrhizobia and PGPR leads to an increased number of the most active nodules and plant yield as well as a greater nitrogen fixation. The maximal levels of nifH gene expression are found at 4 WAI, and it is interesting that at the 7 WAI, the expression levels of bacteroid dctA, phbC and otsA genes are increased in the presence of PGPR. Furthermore, expression of bacteroid nifH gene is somewhat higher in presence of PGPR at the 7 WAI. Altogether, these data can figure out a role of the PGPR in delaying nodule senescence, a conclusion supported by the transmission electron microscopy analysis of nodules. Inoculation modes of PGPR and rhizobia may result in variable effects on legume growth and nodule morphology, and this may depend on the phase of the process modified by PGPR: infection, nodulation, and/or nitrogen fixation. Acknowledgements This work received financial supports from the program Strategic Scholarships for Frontier Research Network for the Ph. D. Program Thai Doctoral degree from the Office of the Higher Education Commission, Thailand; and Suranaree University of Technology, Thailand. Thanks are to Miss Nual-anong Narkkong (Central Instrumentation Unit, Faculty of Science, Mahasarakham University, Thailand) for her help on TEM technique. Thanks are also extended to Dr. Issra Pramoolsook for advice and comments on the language of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsoil. 2014.08.009. References Atieno, M., Herrmann, L., Okalebo, R., Lesueur, D., 2012. Efficiency of different formulations of Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different strains of Bradyrhizobium japonicum. World J. Microb. Biotechnol. 28, 2541–2550. Aung, T.T., Tittabutr, P., Boonkerd, N., Herridge, D., Teaumroong, N., 2013. Coinoculation effects of Bradyrhizobium japonicum and Azospirillum sp: on competitive nodulation and rhizosphere eubacterial community structures of soybean under rhizobia-established soil conditions. Afr. J. Biotechnol. 12, 2850– 2862.

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