Genetic diversity of plant growth promoting rhizobacteria and their effects on the growth of maize plants under greenhouse conditions

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Genetic diversity of plant growth promoting rhizobacteria and their effects on the growth of maize plants under greenhouse conditions Sameh H. Youseif National Gene Bank and Genetic Resources, Agricultural Research Center, 12619 Giza, Egypt

A R T I C LE I N FO

A B S T R A C T

Keywords: Isolation PGPR Zea mays Diversity IAA N2- fixation P- solubilization Plant inoculation Plant biomass production

Engineering of plant rhizosphere with beneficial plant growth promoting (PGP) bacteria offers a great promise for sustainable crop productivity. In this context, 49 rhizospheric/endophytic bacterial isolates were purified using N-free medium, screened in vitro for PGP characteristics and evaluated for their beneficial effects on the early growth of maize (Zea mays L.). The biodiversity of isolated bacteria was analyzed by amplified ribosomal DNA restriction analysis (ARDRA) using four restriction enzymes. Out of the 49 isolates, 7 produced high levels (32.1–82.8 μg mL−1) of indole-3-acetic acid (IAA); 11 had potential phosphate solubilizing abilities (101–163 μg mL−1), while significant acetylene reduction activities (100–1800 nmole C2H4 mg−1 protein h−1) were observed in 12 isolates. Five ribogroups (A-E) were identified using ARDRA. The 16S rRNA-sequence analysis of bacterial representatives from different ribogroups revealed that, 89% of isolates belonged to phylum Proteobacteria, while 11% of them were assigned into phylum Bacteroidetes. Phylum Proteobacteria included Achromobacter, Agrobacterium, Bordetella, Cupriavidus, Ochrobactrum, Pseudoxanthomonas and Stenotrophomonas genera. While, phylum Bacteroidetes was represented by Chryseobacterium and Flavobacterium genera. Under greenhouse conditions, all the selected isolates significantly increased shoot and root fresh and dry biomass of maize plants, compared to the non-inoculated control. Inoculation with Chryseobacterium sp. NGB-29 and Flavobacterium sp. NGB-31 showed the highest beneficial effects on plant growth parameters that were tentatively associated with the high efficiency of these isolates to fix nitrogen and produce high amounts of IAA. The study indicates the potential use of these isolates for production of maize biofertilizers under field conditions.

1. Introduction Maize is a major food source for the world and is a high-yield commodity crop. It is a vital source of food security in many developing countries in Africa (FAO, 1997). Furthermore, it serves as a main forage for the production of biogas. In world production, maize is ranked as the third major cereal crop after wheat and rice. In 2016, the total cultivated area of maize in Egypt was valued about 0.75 million hectares and produced 6 million metric tons (USDA, 2017). However, the local production of maize is not sufficient to satisfy the domestic consumption, in the same year, Egypt imported about 9 million tons of maize grains to fulfill the national demand (Wally and Beillard, 2017). Tough a variety of biotic and abiotic stresses affect maize, soil fertility remains one of the main constraints facing the crop productivity, especially in arid and semi-arid countries. Because, nitrogen is considered to be the most critical factor in crop production, the rate of nitrogen application in Egypt is one of the highest rates in the world (FAO, 2005). Maize is a high fertilizer-demanding crop; under Egyptian

soil conditions it requires 330–360 kg N ha−1 to get an optimum yield (FAO, 2005; Hafez and Abdelaal, 2015). Nevertheless, based on worldwide survey, the N fertilizer recovery efficiency has been found to be around 33% for maize (Krupnik et al., 2004). This means that, large portion of N fertilizer is lost from soil to the environment which not only increases the cost of production but also causes severe environmental problems (Tilman et al., 2002). Introduction of plant growthpromoting rhizobacteria (PGPR) as biofertilizers is a more ecologicalfriendly and economical approach to manage this problem and can be used as a sustainable option for improving nutrient availability, plant growth, and crop yields (Vessey, 2003). PGPR are plant-associated bacteria that aggressively colonize the rhizosphere/plant roots, imparting beneficial effects to plants (Panwar et al., 2014). PGPR enhance plant growth both directly and indirectly (Glick, 2012). Generally, PGPR promote plant growth directly by either facilitating nutrient availability and acquisition via N2 fixation (Ahemad and Kibret, 2014) and P- solubilization (Zaidi et al., 2009) or modulating plant growth by providing or regulating various plant

Peer review under responsibility of Faculty of Agriculture, Ain-Shams University. E-mail address: [email protected]. https://doi.org/10.1016/j.aoas.2018.04.002 Received 25 February 2018; Received in revised form 18 April 2018; Accepted 25 April 2018 0570-1783/ 2018Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Youseif, S.H., Annals of Agricultural Sciences (2018), https://doi.org/10.1016/j.aoas.2018.04.002

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fixation was expressed as the quantity of accumulated ethylene (nmoles C2H4 mg−1 protein h−1).

hormones including IAA, abscisic acid, gibberellic acid and cytokinin (Panwar et al., 2014). While, PGPR induce plant growth indirectly by decreasing the inhibitory effects of various pathogens on plant growth via production of antibiotics (Labuschagne et al., 2010), siderophores (Glick, 2012), and induced systemic resistance (Ramamoorthy et al., 2001). PGPR also improve plant defense mechanisms under stressed conditions by producing the enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, facilitating plant growth by decreasing ethylene levels, inducing salt tolerance and reducing drought stress in plants (Zahir et al., 2009). Previous reviews have described the diversity of PGPR in multiple plant species, especially those with agronomical significance (Barriuso et al., 2008; Ahemad and Kibret, 2014). Azotobacter, Azospirillum, Bacillus, Enterobacter, Klebsiella, Paenibacillus, Pseudomonas and Serratia (Ahemad and Kibret, 2014) are genera that comprise common PGPR with known benefits on different crop plants. Research on PGPR has been increasing and many experimental studies have been carried out on different crops including maize (Zahid et al., 2015), wheat (Majeed et al., 2015) and rice (Sen and Chandrasekhar, 2014). These studies have shown the potential of PGPR to increase the growth and yield of such crops with minor inputs of agrochemicals. With the increasing awareness about the economic and environmental concerns of the use of chemical fertilizers, it is important to identify and characterize plant associated rhizobacteria which can be used as potential plant growth promoters to enhance plants fitness and crops productivity. Therefore, the present study aimed at isolation of PGP bacteria from rhizosphere and root- endosphere of some agriculturally important crops (maize, peanut, rice and wheat) grown in Egypt. These bacteria were characterized and screened in vitro for their PGP activities and representative isolates were identified using 16S rRNA sequence analysis. The PGP potential effect of high efficient rhizobacteria on the growth promotion of maize plants at early growth stage was investigated under greenhouse conditions.

2.2.2. Phosphate solubilization All isolates were screened for solubilization of tricalcium phosphate quantitatively in a liquid medium as described by King (1932). Briefly, bacterial isolates were inoculated in a 25 mL Pikovskaya’s broth medium and incubated for 96 h at 28 °C. Bacterial cultures were centrifuged at 15,000 rpm for 30 min. The supernatant (1 mL) was mixed with 10 mL of chloromolibidic acid and the volume was made up to 45 mL with distilled water. Cholorostannous acid (0.25 mL) was added and the volume was made up to 50 mL with distilled water. The absorbance of the developing blue color was read at 600 nm. The amount of solubilized phosphate was detected from the standard curve of a pure substance of KH2PO4 (Sigma-Aldrich®, USA). 2.2.3. IAA production For detection and quantification of IAA production, bacterial colonies were inoculated into LB medium containing 0.5 mg L-tryptophan/mL. The culture was incubated at 28 °C with continuous shaking at 125 rpm for 48 h (Rahman et al., 2010). Approximately 2 mL of culture was centrifuged at 15,000 rpm for 1 min, and a 1 mL aliquot of the supernatant was mixed with 2 mL of Salkowski’s reagent (150 mL concentrated H2SO4, 250 mL distilled water, 7.5 mL 0.5 M FeCl3·6H2O) and was incubated for 20 min in darkness at room temperature as described by Gordon and Weber (1951). IAA production was observed as the development of a pink-red color, and the absorbance was measured at 530 nm using a spectrophotometer (Thermo Scientific, USA). The concentration of IAA was determined using a standard curve prepared from pure IAA (Sigma-Aldrich®, USA) solutions. 2.2.4. Siderophore production Bacterial isolates were assayed for siderophores production on the Chrome azurol S agar medium (Acros Organics®, Belgium) as described by Schwyn and Neilands (1987). Briefly, inoculum (10 µl of 108 CFU/ mL) of bacterial isolates were spotted onto the CAS agar plates and incubated at 28 ± 2 °C for 72 h. Siderophore production was assessed on the basis of change in color of the medium from blue to orange. Quantitative estimation of siderophores was performed by CAS-liquid assay in which 0.5 mL of culture supernatant was mixed with 0.5 mL of CAS reagent (Payne, 1994). Absorbance was measured at 630 nm against a reference consisting of 0.5 mL of uninoculated broth and 0.5 mL of CAS reagent. Siderophore content in the aliquot was calculated by using the following formula:

2. Materials and methods 2.1. Samples collection and bacterial isolation Plant roots and rhizospheric soil samples from different crops including, maize (Z. mays), peanut (Arachis hypogaea), rice (Oryza sativa) and wheat (Triticum aestivum) grown in different governorates in Egypt were collected for this study. Soil samples were collected from 15 to 20 cm depth along with plant roots. Rhizospheric bacteria were isolated from 10 g soil tightly adhering to plant roots by serial dilution plating on Burk’s N-free agar plates (Wilson and Knight, 1952) as described by Somasegaran and Hoben (1994). For endophytic bacteria, fresh roots were surface-sterilized with 70% ethanol for 5 min, followed by 1% of sodium hypochlorite for 2 min and then washed five times with sterilized distilled water (Kuan et al., 2016). The roots were streaked on Luria-Bertani (LB) agar and nutrient agar plates to check the sterilization efficiency and aseptically smashed with mortar and pestle to isolate the endophytic bacteria on Burk’s N-free medium.

%siderophore units = Ar−As/Ar × 100 where Ar = absorbance of the reference at 630 nm (CAS reagent) and As = absorbance of sample at 630 nm. 2.2.5. NH3 production Bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10 mL peptone water in each tube and incubated for 48–72 h at 28 °C. Five hundred microliters of Nessler’s reagent (Fisher®, USA) were added in each tube. Development of brown to yellow color was a positive test for ammonia production (Cappuccino and Sherman, 1992).

2.2. In-vitro screening of PGP activities 2.2.1. Nitrogen fixation by acetylene reduction assay (ARA) Nitrogen-fixing ability of isolated diazotrophic bacteria was tested using ARA as previously described by Hardy et al. (1968). Pure bacterial colonies were inoculated in 7 mL airtight tubes containing 3 mL semi-solid (0.15% agar w/v) Jensen nitrogen-free medium (Jensen, 1951) and were grown at 28 °C for 72 h. Then after, the tubes were injected by 10% (v/v) acetylene and incubated at 28 °C for 24 h. Ethylene production was measured using Gas Chromatography with flame ionization (GC, DANI 1000, Italy). After completion of the ARA, the cells were predigested by adding 10% SDS and protein concentration was determined as described by Lowry et al. (1951). The rate of N2

2.3. Bacterial identification 2.3.1. PCR amplification of 16S rRNA Genomic DNA of bacterial cells was isolated and purified using GeneJet™ Genomic DNA purification Kit (Thermo Scientific®, USA). The procedures were carried out according to manufacture instructions. Primers fD1 and rP2 (Weisburg et al., 1991) were used to amplify a near-full length, approximately 1500 bp fragment of 16S rDNA from those isolates (37) that showed high PGP activities in vitro. PCR was 2

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performed using the standard reaction mixture (25 µl) containing: 1 × PCR buffer, 1.5 mM MgCl2, 5% dimethyl sulfoxide, 200 mM of each dNTPs, 15 pmol of each primer, 1 U of Taq polymerase enzyme (Promega® Corporation, Madison, USA) and 50 ng of DNA template. PCR reaction conditions were: an initial denaturation at 95 °C for 3 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 45 s, and extension at 72 °C for 2 min. PCR products were separated on 1% agarose gel and stained by using ethidium bromide for visualization of DNA fragments.

Table 1 Physical and chemical properties of sandy soil used in the plant inoculation assay.

2.3.2. Amplified ribosomal DNA restriction analysis (ARDRA) The 16S rDNA PCR products were digested with restriction enzymes AluI, HhaI, MspI and RsaI (Thermo Scientific®, USA) as recommended by the manufacturer, and restriction patterns were resolved on a 2% agarose gel electrophoresis system. Gel images were analyzed using Phoretix 1D software (TotalLab, UK). Dendrogram showing the genetic relationships among test isolates was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering method (Sneath and Sokal, 1973). 2.3.3. 16S rRNA phylogenetic analysis The 1500 bp 16S rDNA fragments were purified using QIAquick PCR purification kit (QIAGEN, Germany) according to the manufacturer’s instructions and sequenced using primers fD1 and rP2 (Weisburg et al., 1991) at Macrogen Inc., South Korea. Sequence reads were edited and assembled using the DNASTAR software (Lasergene, Madison, WI). Sequence similarity searches were performed at the National Center for Biotechnology Information (NCBI) server using BLASTN (http://www. ncbi.nlm.nih.gov/blast). The sequences were aligned using Clustal W version 1.8 (Altschul et al., 1997) and subjected to phylogenetic analysis. 16S rRNA phylogenetic tree was constructed using the NeighborJoining (NJ) method (Saitou and Nei, 1987) in MEGA software version 7 (Kumar et al., 2016) and 1000 bootstrap replication to assess branching confidence. The percentage of average nucleotide identity (ANI) within different clustered was calculated using the same program. The evolutionary distances were computed using the Kimura 2parameter model (Kimura, 1980).

Property

Value

Particle size distribution (%) Sand Silt Clay Texture grade CaCo3 (%) Saturation percent S.P (%) pH E.C. (dS m−1 at 25 °C)

90.1 3.9 6.0 Sandy 1.61 21.50 7.82 0.32

Soluble cations (meq/L) Ca2+ Mg2+ Na+ K+

0.54 0.33 1.62 0.65

Soluble anions (meq/L) CO32− HCO3– Cl− SO42−

0.00 0.88 0.59 1.67

Total N (%) Total Soluble-N (mg kg−1) Available-P (mg kg−1) Available-K (mg kg−1) Organic matter (%)

0.021 16.30 6.71 52.10 0.23

DTPA extractable (ppm) Fe Mn Zn Cu

1.62 0.31 0.42 0.18

(Snedecor and Cochran, 1980). 3. Results 3.1. Isolation of diazotrophic PGP- bacteria

2.4. Plant inoculation

Forty-nine diazotrophic bacterial isolates in total; 39 isolates from rhizosphere of maize, peanut, rice and wheat plants; and 10 endophytes from root interior of wheat plants were isolated and purified using Burk’s N-free medium (Table 2). All bacterial cells were Gram negative rod shape.

A pot experiment was conducted in the greenhouse of National Gene Bank, Agricultural Research Center, Giza to evaluate the effects of rhizobaterial inoculation on different growth parameters of maize plants. Thirty-seven isolates were selected based on multiple PGP properties like N2 fixation activity, IAA production and P- solubilization. Seeds of maize (SC 130) were surface sterilized for 5 min in 90% (v/v) ethanol, then rinsed three times with sterile distilled water, followed by 5 min in 10% (v/v) sodium hypochlorite, and finally were washed six times with sterile distilled water. Each pot (13 cm diameter) was filled with 2 kg of autoclaved sandy soil. The soil was analyzed according to Page et al. (1982). The main physical and chemical properties of the soil used are presented in Table 1. Each pot was planted with three surface-sterilized seeds and irrigated with sterilized tab water. All treatments received the recommended dose of super phosphate (15.5% P2O5) and potassium sulfate (48.5% K2O) at the rate of 0.4/pot and 0.2 g/pot, respectively. Pots were arranged in a complete randomized block design with three replicates. For inoculation treatments, seeds were soaked for 2 h in the bacterial suspension of 108–109 cells mL−1 and placed at the same depth (approximately 2.5 cm below the soil surface) in all inoculated pots. The uninoculated control treatment consisted of water-treated seeds (without bacterial inoculation) was included. Plants were grown under controlled conditions: at 35 °C for 12 h (light) and 20 °C for 12 h (dark) and 70% relative humidity. After 35 days, plants were uprooted and shoot, root length as well as shoot, root fresh and dry weights were recorded. Data were subjected to analysis of variance using MSTAT analysis software

3.2. Characterization for PGP traits In vitro PGP traits of all isolated rhzio/endophytic bacteria are described in Table 2. The ARA showed that, bacterial isolates had the nitrogenase activity ranging from 10 to 1800 nmol C2H4 mg−1 protein h−1. Out of the tested isolates, 8 rhizospheric/endophytic isolates showed ARA activities higher than 100 nmol C2H4 mL−1 h−1. The maximum ARA activity (1800 nmol C2H4 mg−1 protein h−1) was recorded by the root endophytic bacterium NGB-31. The results also showed that, bacterial isolates had phosphate solubilization potentiality ranging between 8.0 and 163.0 µg mL−1 (Table 2). Eleven bacterial isolates solubilized more than 100 mg phosphate L−1 from the insoluble tricalcium phosphate. The highest phosphate solubilization was measured for the rhizospheric bacterial isolate NGB-33 (163 µg mL−1) followed by NGB-36 (141 µg mL−1) and NGB-15 (139 µg mL−1). However, among the tested endophytic bacteria, the maximum solubilized phosphate (133 µg mL−1) was achieved by isolate NGB-23. Majority of bacterial isolates produced IAA with or without L-tryptophan induction. However, the amount of IAA was low in the absence of L-tryptophan precursor. In the presence of L-tryptophan, the isolated bacteria produced IAA in concentrations between 0.1 and 82.8 μg mL−1. The 3

4

Maize rhizoshere Maize rhizoshere Maize rhizoshere Maize rhizoshere Maize rhizoshere Maize rhizoshere Wheat rhizoshere Wheat rhizoshere Wheat rhizoshere Rice rhizosphere Rice rhizoshere Rice rhizosphere Rice rhizosphere Rice rhizosphere Rice rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Wheat root Wheat root Wheat root Wheat root Wheat root Wheat root Wheat root Wheat root Wheat root Wheat root Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Maize rhizosphere Wheat rhizosphere Wheat rhizospher Wheat rhizospher Wheat rhizospher Wheat rhizospher Wheat rhizospher Rice rhizosphere Rice rhizosphere Peanut rhizospher Peanut rhizosphere

NGB-1 NGB-2 NGB-3 NGB-4 NGB-5 NGB-6 NGB-7 NGB-8 NGB-9 NGB-10 NGB-11 NGB-12 NGB-13 NGB-14 NGB-15 NGB-16 NGB-18 NGB-19 NGB-20 NGB-21 NGB-22 NGB-23 NGB-24 NGB-25 NGB-26 NGB-27 NGB-28 NGB-29 NGB-30 NGB-31 NGB-32 NGB-33 NGB-34 NGB-35 NGB-36 NGB-37 NGB-38 NGB-39 NGB-40 NGB-41 NGB-42 NGB-43 NGB-44 NGB-45 NGB-46 NGB-47 NGB-48 NGB-49 NGB-50

Gharbia Gharbia Gharbia Gharbia Gharbia Gharbia Gharbia Gharbia Gharbia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Ismailia Qalubia Qalubia Qalubia Qalubia Qalubia Qalubia Qalubia Qalubia Qalubia Qalubia Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Menya Behirah Behirah

Location (governorate)

61 100 55 41 45 62 170 30 39 140 51 45 31 35 91 110 100 40 61 60 11 70 100 140 51 30 60 300 190 1800 100 300 51 41 39 11 15 70 52 65 10 21 15 12 10 19 20 14 11

N2-ase activity

80 ± 2.0 76 ± 1.5 77 ± 2.9 73 ± 3.4 70 ± 5.0 63 ± 1.8 63 ± 3.0 61 ± 3.9 56 ± 2.9 107 ± 5.2 136 ± 3.9 71 ± 4.8 79 ± 1.0 76 ± 2.9 139 ± 5.9 67 ± 3.7 86 ± 2.1 84 ± 4.1 128 ± 2.2 83 ± 2.8 17 ± 3.6 133 ± 7.4 80 ± 4.5 98 ± 4.0 117 ± 1.9 78 ± 1.5 90 ± 2.1 94 ± 3.1 70 ± 3.5 123 ± 2.9 62 ± 1.3 163 ± 2.3 84 ± 1.6 137 ± 4.2 141 ± 2.6 15 ± 2.5 16 ± 3.1 101 ± 3.8 64 ± 1.6 66 ± 2.9 18 ± 1.5 15 ± 3.8 8 ± 1.5 12 ± 2.1 17 ± 4.2 14 ± 2.5 13 ± 0.7 17 ± 2.1 13 ± 2.9

P- solubilization (µg mL−1)

2.59 ± 0.50 2.62 ± 0.58 1.35 ± 0.63 2.40 ± 0.51 1.50 ± 0.53 4.41 ± 1.95 1.29 ± 0.43 4.68 ± 1.24 1.64 ± 0.43 3.30 ± 0.31 9.63 ± 2.15 5.45 ± 1.55 1.98 ± 0.88 2.15 ± 0.30 3.24 ± 1.28 3.04 ± 1.06 2.89 ± 0.56 1.08 ± 0.30 5.37 ± 2.05 1.27 ± 0.46 0.00 4.86 ± 1.14 1.53 ± 0.96 1.75 ± 0.24 1.48 ± 0.66 2.88 ± 1.00 4.14 ± 1.91 25.41 ± 3.12 1.88 ± 0.86 40.27 ± 7.69 4.26 ± 1.77 3.05 ± 1.09 3.26 ± 1.26 6.34 ± 1.25 1.74 ± 0.24 0.00 0.00 1.93 ± 0.50 1.19 ± 0.32 2.67 ± 1.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Without tryptophan

IAA production (µg mL−1)

34.9 ± 3.99 45.4 ± 4.67 17.6 ± 4.85 18.3 ± 3.87 11.2 ± 1.22 18.1 ± 4.00 20.2 ± 3.25 16.9 ± 1.16 19.4 ± 2.05 36.8 ± 5.95 36.6 ± 2.54 16.8 ± 2.25 10.6 ± 1.80 14.6 ± 1.66 32.1 ± 2.10 11.5 ± 1.70 22.7 ± 1.95 17.6 ± 4.35 14.5 ± 2.75 13.6 ± 1.36 2.5 ± 0.66 22.8 ± 1.12 29.8 ± 1.96 22.6 ± 1.92 10.8 ± 1.95 12.4 ± 0.70 17.7 ± 1.88 80.0 ± 3.95 11.6 ± 1.21 82.8 ± 2.05 25.8 ± 1.15 24.4 ± 3.31 15.9 ± 1.16 17.3 ± 2.30 13.5 ± 2.45 3.7 ± 1.14 2.3 ± 1.02 15.4 ± 1.32 11.6 ± 1.51 15.2 ± 2.74 2.9 ± 0.80 2.7 ± 0.54 0.1 ± 0.04 1.0 ± 0.15 0.2 ± 0.01 1.9 ± 0.25 0.2 ± 0.06 0.3 ± 0.02 1.9 ± 0.15

With tryptophan 31.9 ± 0.4 24.7 ± 1.0 15.2 ± 1.4 21.3 ± 1.6 13.8 ± 1.4 14.9 ± 0.5 11.2 ± 0.4 12.8 ± 1.3 11.1 ± 0.6 29.3 ± 1.7 34.1 ± 1.0 16.2 ± 1.2 12.1 ± 1.5 25.5 ± 1.1 28.6 ± 1.0 12.6 ± 1.8 30.7 ± 0.9 21.2 ± 1.3 33.8 ± 1.2 16.9 ± 0.5 10.6 ± 0.3 34.2 ± 1.4 15.2 ± 0.2 27.8 ± 1.0 29.9 ± 1.1 15.0 ± 0.8 16.3 ± 1.4 20.1 ± 0.9 11.3 ± 1.1 28.7 ± 1.5 26.6 ± 1.0 22.5 ± 0.6 17.4 ± 1.5 34.4 ± 1.3 28.3 ± 0.4 8.3 ± 0.2 11.0 ± 0.6 13.5 ± 0.9 32.1 ± 1.8 10.0 ± 0.5 11.7 ± 1.1 0.00 0.00 8.6 ± 0.2 0.00 9.5 ± 0.6 0.00 0.00 0.00

Siderophore production (%)

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + – + – + – – +

Ammonia production

(N2-ase) Nitrogenase, (P) phosphate, (IAA) indole acetic acid, (+) positive; (−) negative test. IAA was tested with (500 µg /µl) and without the addition of precursor. Data are average values of three replicates except for N2-ase activity, ( ± ) standard deviation (SD). Nitrogenase enzyme activity was expressed in nmole C2H4 mg−1 protein h−1.

Source

Isolate No.

Table 2 List of PGPR used in this study and their PGP characteristics.

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Fig. 1. UPGMA dendrogram showing the genetic similarities among different PGPR isolates obtained from the 16S rRNA-RFLP analysis generated by the AluI, HhaI, MspI and RsaI restriction endonucleases.

30% of siderophore units (Table 2). In the same context, 45/49 isolates were able to produce ammonia. It is remarkable that, all root endophytes were ammonia and siderophore producers.

highest amount of IAA was produced by endophytic bacterial isolates NGB-31 (82.8 μg mL−1) and NGB-29 (80.0 μg mL−1). On the other hand, in absence of L-tryptophan, only 37/49 test isolates had the ability to produce IAA with a range of 1.08 to 40.27 μg mL−1. Once again, the maximum amount of IAA in the absence of precursor was recorded for isolates NGB-31 (40.27 μg mL−1) and NGB-29 (25.41 μg mL−1). Although most of tested isolates 43/49 produced siderophores by CAS -blue agar assay exhibiting the color change from blue to orange, however only 7 isolates were able to produce above

3.3. ARDRA of 16S rRNA genes Thirty-seven bacterial isolates were selected for molecular identification based on their potential PGP properties. 16S rRNA gene fragments (ca. 1500 bp) were successfully PCR amplified using fD1 and rP2 5

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Table 3 Sequence analysis of 16S rRNA from rhizo/endophytic PGPR representatives. Strain ID.

RFLP-Ribogroup

Sequence (bp)

Accession No.

Homology to the reference strains

NGB-2 NGB-10 NGB-11 NGB-15 NGB-18 NGB-20 NGB-21 NGB-23 NGB-24 NGB-25 NGB-29 NGB-31 NGB-32 NGB-33 NGB-34 NGB-35 NGB-36 NGB-39

D E D B B B E B B B C C B A A D A A

963 830 948 973 1030 808 1210 1179 1115 881 952 1040 1016 1051 1159 1085 1088 848

LC322225 LC322226 LC322227 LC322228 LC322229 LC322230 LC322231 LC322232 LC322233 LC322234 LC322235 LC322236 LC322237 LC322238 LC322239 LC322240 LC322241 LC322242

Agrobacterium tumefaciens strain R6-364 Achromobacter insolitus strain DSM 23807 Agrobacterium tumefaciens PRE2 Stenotrophomonas sp. strain KG-16-3 Stenotrophomonas maltophilia strain E136 Stenotrophomonas sp. strain ERR 859 Achromobacter sp. strain CH1 Stenotrophomonas maltophilia strain DZSG-6 Stenotrophomonas maltophilia, strain LMG 10989 Stenotrophomonas maltophilia strain EN14ZR5 Chryseobacterium sp. NBRC 101329 Flavobacterium sp. strain JS2 Pseudoxanthomonas mexicana strain NBRC 101034 Cupriavidus sp. strain HBU08093 Bordetella petrii strain AU13195 Ochrobactrum intermedium strain D303 Bordetella petrii strain AU13195 Cupriavidus sp. strain HMT35

Identity (%) JQ659820 CP019325 HQ678680 KX785164 KM894178 MF442343 HQ619222 KF973235 AJ131907 KY486808 AB681454 JF922307 NR113973 HQ906950 EU082174 KJ499780 EU082174 KU358933

98 97 99 98 99 98 99 99 99 98 97 99 98 98 99 98 99 99

endophytic strains S. panacilnani R6-799 and S. maltophilia EN14ZR5 that were isolated from Jatropha (Madhaiyan et al., 2015) and Z. mays plants, respectively. Although, isolate NGB-20 was linked to the outskirt of subgroup B1, but it was defined as Stenotrophomonas sp. because it had 98% sequence homology with root endophyte Stenotrophomonas sp. strain ERR 859 (Table 3). Remarkably, isolates in ribogroup C were recognized as members of phylum: Bacteroidetes, class: Flavobacteria and were separated in two genomic subgroups (C1 and C2). Isolate NGB-29 of subgroup C1 was clustered on a well-supported branch (BT: 100%) along with type strains of the genus Chrysobacterium (ANI: 96%). While, isolate NGB-31 of subgroup C2 formed another tight cluster (BT: 100%) along with reference strains of the genus Flvobacterium (ANI: 96%). Isolates in ribogroup D were assigned as members of class Alphaproteobacteria and were classified into D1 and D2 phylogenetic subgroups. Isolate NGB-35 of subgroup D1 was closely related to type strains of Ochrobactrum intermedium supported by 100% BT and 98.6% ANI. While, isolates NGB-2 and 11 in subgroup D2 were recognized as Agrobacterium sp. and were grouped tightly (BT: 100%, ANI: 98.2%) with reference strains of A. tumefaciens. On the other hand, isolates NGB-10 and 21, the representatives of ribogroup E, were clustered with the defined Acromobacter species (A. insolitus, A. piechaudii, A. aegrifaciens, A. ruhlandii, A. xylosoxidans and A. denitrificans), supported by BT: 99% and ANI: 97.6%.

primers. Five different ribogroups (A, B, C, D and E) were clearly distinguished after restriction fragment length polymorphism (RFLP) analysis using endonucleases AluI, HhaI, MspI and RsaI (Fig. 1). Ribogroup A included 6 rhizospheric bacteria (NGB-33, 34, 36, 39, 40 and 41), that were isolated from Menya governorate. Ribogroup B contained 10 rhizo/endophytic bacteria; rhizospheric isolates NGB-15, 18 and 20 that were isolated from Ismaillia governorate; and endophytic isolates NGB 23, 24, 25, 26, 27, 28 and 32 that were obtained from Qalubia governorate. Ribogroup C comprised 3 bacterial endophytes (NGB-29, 30 and 31) from Qalubia governorate, while ribogroup D was a cluster of 13 rhizospheric bacteria (NGB-1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 19 and 35). Five rhizospheric bacteria (NGB-10, 12, 13, 16 and 21) isolated from Ismaillia governorate were assigned in the ribogroup E. 3.4. Phylogenetic analysis of the 16S rRNA gene Eighteen diazotrophic PGPR were selected to represent different clusters (A-E) revealed by RFLP analysis (Fig. 1). Partial sequences of 16S rRNA (808–1210 bp, Table 3) were obtained and deposited at GenBank®. According to the 16S rRNA genetic similarity, bacterial isolates showed unexpected genetic variations and were closely related to 9 genera distributed in two phyla; Proteobacteria (Alpha, Beta and Gamma-subgroups) and Bacteriodetes (Table 3). The representative isolates clustered together with the corresponding most related type strains. The percentage of average nucleotide identity calculated from 16S rRNA sequences was plotted in Fig. 2 within the putative ribogroups (A–E). According to NJ-phylogenetic tree based on 16S rRNA sequences (Fig. 2), representative isolates in ribogroup A were clearly classified in two different genomic subgroups (A1 and A2) within class Betaproteobacteria. Isolates NGB-34 and 36 in subgroup A1 formed a sister monophyletic lineage to reference strains Bordetella petrii N64 and B. petrii DSM 12804T supported by 100% bootstrapping value (BT) and 98.4% ANI. While, isolates NGB-33 and 39 in subgroup A2 showed high relatedness (BT: 100%, ANI: 97.3%) with references strains Cupriavidus taiwanensis STM6162 and C. taiwanensis PAS15 that were isolated from Mimosa sp. (Chen et al., 2005; Klonowska et al., 2012) as well as C. taiwanensis LMG 19424T. Isolates in ribogroup B were identified as members of class Gammaproteobacteria and were also distributed in two genomic subgroups (B1 and B2). Isolate NGB-32 in subgroup B1 was assigned as Pseudoxanthomonas sp. and was linked tightly (BT: 100%, ANI: 97.5%) to reference strains of P. mixicana. Isolates NGB-15, 18, 23, 24 and 25 in subgroup B2 were designed as Stenotrophomonas sp. and had phylogenetic affiliations to type strains S. rhizophila DSM 14405T and S. maltophilia LMG 10879T. They were also distantly related to

3.5. Plant growth promotion in maize Based on PGP performance, the influence of selected 37 rhizobacteria with multiple PGP activities on maize plant growth was screened in a pot experiment (Table 4, Fig. 3). All the tested bacteria had a significant increase (ANOVA, p < 0.05) in the shoot length compared to the uninoculated control. Maize plants inoculated with Agrobacterium sp. strain NGB-11, showed the maximum shoot lengths (49.3 cm/plant) compared to the uninoculated control (32.3 cm/plant). In the same context, most of the tested bacteria (26/37) produced significant increases (p < 0.05) in root length ranging from 20.0 to 24.0 cm/plant compared to the uninoculated control (17.4 cm/plant). Flavobacterium sp. strain NGB-31 recorded the highest root length (24 cm/plant). Shoot fresh and dry weight as a direct measure of PGP activities, were clearly affected by different bacterial inoculations. All strains resulted in significant increases (p < 0.05) in shoot fresh weight (1.25–3.01 g/plant) and dry weight (0.55–1.15 g/plant) compared to the uninoculated control. Maize plants inoculated by Flavobacterium sp. strain NGB-31 and Chryseobacterium sp. strain 29 gave the highest shoot fresh weight (3.01 and 2.64 g/plant) and dry weight (1.15 and 1.08 g/plant) with significant increases (p < 0.05) over the 6

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Fig. 2. NJ phylogenetic tree based on the 16S rRNA sequences showing the relationships between the represenative isolates of diazotrophic PGPR (rhizospheric and endophytic bacteria) using the Kimura 2-parameter model. Bootstrap values (using 1000 replicates) are indicated at the branching points.

due to the presence of root exudates and rhizodeposits (Compant et al., 2010). In this complex ecosystem, now it is believed that, bacteria can positively improve plant growth while plants can “engineer” their microbiome in order to have beneficial bacterial colonizers, including those living within the plant tissues “plant endophytes” (Marasco et al., 2012; Rashid et al., 2012). Bacterial endophytes may have an advantage over bacteria inhabiting the rhizosphere, since living within plant’s tissues represents an opportunity to interact in a very efficient way with their plant hosts (Santoyo et al., 2016). In this study, 49 rhizospheric soil and roots of some agriculturally important crops widely cultivated in Egypt including maize, peanut, rice and wheat, were screened for the presence of putative nitrogen-fixing and/or PGP bacteria. The diversity of isolated rhizobacteria was investigated using

uninoculated control which recorded 0.98 and 0.32 g/plant, respectively. In the same way, the fresh and dry weight of plant roots were also positively affected by different rhizobacterial inoculation compared to the uninoculated control. Similar to plant shoots, the maximum root fresh weight (1.64 and 1.61 g/plant) and dry weight (0.92 and 0.94 g/plant) were recorded by Chryseobacterium sp. strain 29 and Flavobacterium sp. strain NGB-31, respectively with significant increases (p < 0.05) over the uninoculated control, which gave 0.57 g/plant and 0.40 g/plant, respectively. 4. Discussion Rhizosphere soil is a hot spot of microbial interaction and activity 7

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16S-RFLP analysis which has been used successfully to study microbial diversity in a wider range of ecological niche, including soil rhizosphere and plant associated endophytes (Santoro et al., 2016). The 16S rRNAphylogenetic analysis indicated that, phylum Proteobacteria was the most abundant taxonomic group (89%), followed by Bacteroidetes (11%). Phylum Proteobacteria was represented by 7 genera distributed in classes Alpha-, Betaand Gammaproteobacteria. Among Alphaproteobacteria, several representative isolates belonging to the genera Agrobacterium sp. and Ochrobactrum sp. were identified in this study, with close relationships to A. tumefaciens and O. intermedium, respectively. The genus Agrobacterium is a group of Gram negative soil bacteria found associated with many plant species. Despite the general perception that most of the agrobacteria cause plant diseases, many nonpathogenic A. tumefaciens strains with PGP potentiality and N2fixing efficiency have been also isolated from many members of Graminaceae (Zinniel et al., 2002) as well as from the root nodules of various legumes (Youseif et al., 2014). In agreement with our results, characterization of Agrobacterium strains exhibiting ACC deaminase activity and IAA formation have been previously reported (Ahemad and Kibret, 2014). Similarly, members of the alphaproteobacterial genus Ochrobactrum occur in diverse habitats and play important roles as intracellular symbionts or pathogens in plants, animals and humans (Bathe et al., 2006). Numerous reports have described strains of O. intermedium that could enhance growth of mung bean (Faisal and Hasnain, 2006), peanut (Paulucci et al., 2015) and sugarcane (Hassan et al., 2014) via assisting nutrient uptake, producing IAA and siderophores, exhibiting ACC deaminase activity or preventing plant diseases. In accordance with our results, the diazotrophy of Ochrobactrum has been previously reported through complete symbiotic relationship with some legumes (Trujillo et al., 2005) or by establishing biological nitrogen fixation relationship with other plants (Meng et al., 2014). In the present investigation, Betaproteobacteria were represented by three members of Burkholderiales; Achromobacter sp., Bordetella and Cupriavidus with high homology to B. petrii and C. taiwanensis. There are few reports describing the presence of Achromobacter sp. in the environment, including soils (Mendes et al., 2013) and plants (Jha and Kumar, 2009). Although it is regarded as a potential human pathogen associated with several clinical diseases, the PGP characteristics of many Achromobacter strains have been occasionally described in the literature (Jha and Kumar, 2009; Dawwam et al., 2013). For instance, based on in vitro analysis and greenhouse experiments, they indicated that Achromobacter sp. strain EMCC1936 was capable of producing IAA,

Table 4 Effect of PGPR isolates on different growth parameters of maize plants under greenhouse conditions. Treatment

NGB-1 NGB-2 NGB-3 NGB-4 NGB-5 NGB-6 NGB-7 NGB-8 NGB-9 NGB-10 NGB-11 NGB-12 NGB-13 NGB-14 NGB-15 NGB-16 NGB-18 NGB-19 NGB-20 NGB-21 NGB-23 NGB-24 NGB-25 NGB-26 NGB-27 NGB-28 NGB-29 NGB-30 NGB-31 NGB-32 NGB-33 NGB-34 NGB-35 NGB-36 NGB-39 NGB-40 NGB-41 Control LSD at 0.05

Shoot height cm/ plant

44.7 44.9 46.7 42.0 42.5 41.7 40.3 43.4 42.7 44.3 49.3 42.3 40.4 43.3 48.7 39.3 42.0 42.7 42.9 43.1 40.1 43.3 45.0 44.7 40.3 41.3 48.9 39.9 47.7 41.9 45.0 42.3 43.4 44.7 46.2 44.7 46.3 32.3 3.67

Root length cm/plant

18.3 22.1 20.6 21.0 18.7 22.0 19.3 18.0 18.3 23.3 22.8 20.0 19.0 19.3 22.3 18.0 20.1 20.7 20.3 22.7 18.6 20.2 22.0 21.6 20.3 22.6 23.1 18.6 24.0 22.7 22.9 21.0 22.0 21.4 22.0 20.6 19.6 17.4 2.56

Fresh weight (gm/ plant)

Dry weight (gm/ plant)

Shoot

Root

Shoot

Root

2.37 2.25 1.86 1.92 1.74 1.82 2.07 1.91 1.80 2.40 2.18 1.91 1.34 1.73 2.17 1.65 2.20 1.73 1.72 1.49 1.61 2.24 2.09 1.74 1.65 2.07 2.64 1.57 3.01 2.10 2.40 1.25 1.54 1.57 1.77 1.59 1.60 0.98 0.21

1.14 1.09 1.05 1.25 1.07 0.97 1.21 0.99 0.96 1.44 1.04 0.91 0.77 0.90 1.02 1.06 1.16 1.01 0.89 0.89 0.91 1.33 1.19 0.99 0.90 1.02 1.64 1.01 1.61 0.97 1.47 0.86 0.85 1.01 0.82 1.08 0.89 0.57 0.16

0.84 0.82 0.64 0.71 0.83 0.70 0.64 0.74 0.72 0.95 0.71 0.62 0.55 0.66 0.91 0.80 0.94 0.79 0.65 0.58 0.58 0.89 0.91 0.79 0.79 0.76 1.08 0.68 1.15 0.73 0.93 0.62 0.61 0.68 0.71 0.62 0.65 0.32 0.12

0.72 0.67 0.66 0.73 0.73 0.67 0.68 0.66 0.62 0.85 0.66 0.58 0.50 0.59 0.68 0.73 0.76 0.68 0.59 0.53 0.60 0.85 0.77 0.64 0.63 0.67 0.92 0.68 0.94 0.59 0.88 0.58 0.57 0.68 0.54 0.61 0.54 0.40 0.13

Data per plant are means of 3 replicates (3 plants per each replicate).

Fig. 3. Representation of maize plant health and root system as affected by different PGPR inoculation. Plants were harvested 35 days after sowing. Bar = 10 cm. 8

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found as more efficient phosphate solubilizers compared to endophytic isolates. We identified many genera such as Agrobacterium, Bordetella, Cupriavidus, Flavobacterium, Ochrobactrum and Stenotrophomonas that had high P- solubilization activity ranging between 123 and 136 μg mL−1. The high P- solubilization activity recognized under this investigation, were relatively higher than published by other workers. For example, in a large survey, the maximum P- solubilization activity among 100 PGPR isolated from maize microbiome was 35.6 μg mL−1 corresponding to Bacillus subtili stain HJR3 (Zahid et al., 2015). Similarly, Majeed et al. (2015) reported a range of P- solubilization varied from 2.0 to 19.0 μg mL−1 among 9 bacterial strains isolated from wheat rhizosphere and root-endosphere that belonging to genera Acetobactor, Bacillus and Stenotrophomonas. Production of phytohormones is one of the main mechanisms responsible for the direct PGP effect of rhizobacteria on plants. IAA is generally considered the most important phytohormone that coordinates different developmental processes in plants; a low concentration of IAA promotes primary root elongation, whereas a high IAA concentration stimulates lateral and adventitious root formation (Duca et al., 2014). In nature, IAA is commonly synthesized by plants and PGPR from amino acid tryptophan, a common precursor in root exudates, through transamination and decarboxylation biochemical reactions (Apine and Jadhav, 2011). In this study, Chryseobacterium sp. strain NGB-29 and Flavobacterium sp. strain NGB-31 were identified as the high efficient IAA producers, in both L-tryptophan supplemented (80.0 and 82.8 μg mL−1) or non-supplemented culture medium (25.4 and 40.3 μg mL−1), indicating the potentiality of these strains to have both tryptophan dependent and independent pathways to create the auxin. The amount of IAA produced in the presence of L-tryptophan by these strains was dramatically higher than those presented in other reports. Among other, Marques et al. (2010) reported levels of 3.0–5.0 μg mL−1 for rhizospheric Chryseobacterium palustre and Chryseobacterium humi, whereas Soltani et al. (2010) reported levels of 0.27–12.03 μg mL−1 by Flavobacterium sp. strains that were isolated from wheat microbiome. The variation of IAA production by rhizobacterial species and strains belonging to the same genera has been previously reported (Zahid et al., 2015). This variation is attributed to various biosynthetic pathways, genetic regulation and environmental factors involved in IAA biosynthesis (Duca et al., 2014). Another important PGPR trait, is the production of siderophores. It is believed that, siderophores stimulate plant growth promotion: directly by supplying iron for plants, or indirectly by inhibiting the establishment of phytopathogens through the sequestration of Fe from the environment (Ahemad and Kibret, 2014). Most of tested isolates (43/ 49) showed positive siderophores production ability using CAS -blue agar assay. Potential PGPR strains showing high siderophore producing activity can be further studied for biocontrol capability of plant pathogens. For instance, a collection of 11,520 bacterial isolates recovered from maize microbiome was screened for controlling of fungal species, Fusarium verticillioides (Figueroa-López et al., 2016). Greenhouse experiments revealed the high antagonistic effect of Bacillus cereus sensu lato strain B25. Moreover, in vitro assays indicate that B25 produced siderophores and exhibited protease, glucanase and chitinase activities, suggesting these activities could be possible control mechanisms against F. verticillioides. Inoculation with bacterial strains possess multiple PGP traits, can ultimately benefit plants in terms of stimulating growth. In the plant inoculation assay, most of the tested isolates significantly improved plant growth compared to the uninoculated control. Inoculation with Chryseobacterium sp. strain NGB-29 and Flavobacterium sp. strain NGB31 had the greatest potential to increase shoot and root fresh and dry weights. Plant growth promotion in response to PGPR applied alone or with N or P fertilizers has been frequently reported for different crops under different ecological and environmental conditions (Naqqash et al., 2016; Abdel-Rahman et al., 2017). In consistent with our results (Zahid et al., 2015), reported the positive growth promoting attributes

gibberellin, solubilizing rock phosphate and significantly increased vegetative growth and yield parameters of tomato plants (AbdelRahman et al., 2017). The genus Bordetella is also a well-known opportunistic pathogen; causes respiratory tract infections in mammals, but some species with PGP activities have been isolated from plant rhizosphere (Verma et al., 2015). The occurrence of Bordetella sp. as potent endophytic bacteria has been frequently found in plant roots (Chowdhury et al., 2007), stems (Beneduzi et al., 2013) and fruits (Nithya and Babu, 2017). On the other hand, members of the genus Cupriavidus have been isolated from diverse ecological environments such as soil, water, legume nodules and human clinical sources. Along with Burkholderia, Cupriavidus is the best known Betaproteobacteria “Beta-rhizobia” that can nodulate different legume species (Platero et al., 2016). In addition to the beneficial effect of promoting legume growth by means of N2 fixation, many Cupriavidus species possess PGP characteristics, such as secretion of IAA and siderophores and solubilization of phosphate (Yu et al., 2011; Marcano et al., 2016). Bacterial isolates belonging to Gammaproteobacteria showed high similarities to Stenotrophomonas sp. and Pseudoxanthomonas mexicana. The genus Stenotrophomonas is of increasing biotechnological interest due to its ubiquitous occurrence as rhizospheric and endophytic PGPR (Yu et al., 2011; Marcano et al., 2016). Meanwhile, its capability for bio-controlling of various fungal and soil-borne plant pathogens (Li et al., 2016) and/or improving plant productivity under stress conditions (Singh and Jha, 2017). Our results agree with those obtained by Majeed et al. (2015), who found diazotrophic Stenotrophomonas sp. strains colonizing the rhizosphere of wheat plant as well as in the root interiors that showed multiple PGP traits. In the same trend, consistent with our data, the PGP activities of N2-fixing Pseudoxanthomonas sp. isolated from ryegrass forage crop have been reported by Castellano-hinojosa et al. (2016). They stated for the first time the potentiality of Pseudoxanthomonas sp strain C18 to promote plant growth via production of ACC deaminase and siderophore as well as solubilization of phosphate (Castellano-hinojosa et al., 2016). Remarkably, we identified one representative isolate corresponding to Chryseobacterium and Flavobacterium genera within the phylum Bacteroidetes. Although the members of phylum Bacteroidetes were regarded as a small taxon, but they are commonly existed in association plants in various environments (Zhao et al., 2017). Strains of Flavobacterium and Chryseobacterium were found in the rhizospheres or internally in the tissues of many plants (Ottesen et al., 2013; Raweekul et al., 2016). Our results are in consistent with other publications reported the plant growth capabilities of several strains within Flavobacterium and Chryseobacterium (Marques et al., 2010; Soltani et al., 2010). Nitrogen is the major limiting factor for plant growth and productivity. The application of N2-fixing rhizobacteria as biofertilizers has emerged as a sustainable way for increasing the growth and yields of crop plants (Ahemad and Kibret, 2014). It has been reported that, bacterial occurrence and growth in the form of typical subsurface pellicle in N-free semisolid medium, not generally accompanied by acetylene reduction activity (Tripathi et al., 2002). In this way, large proportions of bacteria isolated from surface-sterilized of various plants using N-free media have been shown to be non-diazotrophs (Chowdhury et al., 2007; Beneduzi et al., 2013). These non-diazotrophs could be nitrogen scavengers, growing on the nitrogen fixed and released by diazotrophs during the enrichments. Our results showed that, only 24% of isolates exhibited a considerable acetylene reduction with values ranging from 100 to 1800 nmol C2H4 mg−1 protein h−1. Phosphorus is a key nutrient required for higher and sustained maize growth and yield especially under semiarid conditions (Amanullah and Khan, 2015). The ability of PGPR strains to solubilize insoluble P and convert it to plant available form is an important characteristic under conditions where P is a limiting factor for crop production. In present study, all tested isolates were able to solubilize phosphate in a range of 8–163 μg mL−1. The rhizospheric isolates were 9

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of maize plants towards inoculation with B. subtilis strain HJR3. Similarly, Kuan et al. (2016) found that, inoculation of maize with N2-fxing PGP strains belonging to genera Bacillus, Klebsiella and Acinetobacter significantly increased the total N content and dry biomass of plants as well as delayed plant senescence.

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