Screening of endophytic plant growth-promoting bacteria isolated from two Nigerian rice varieties

Probiotic bacteria, inhabiting the endosphere of plants, presents a major opportunity to develop cheap and eco-friendly alternatives to synthetic agrochemicals. Using standard microbiological procedures, culturable bacteria were isolated from the endosphere (root, stem and leaf) of two Nigerian rice varieties (Ofada and ITA 150). Assays including indole acetic acid (IAA) production, phosphate solubilization, seed germination assay, hydrogen cyanide (HCN) and ammonia production, and antifungal assay were conducted to identify the phytobeneficial bacterial strains. A total of 193 bacteria were isolated from all samples. The roots had the highest bacterial count of 8 45 and 61 x 10 cfu/g in Ofada and ITA 150 respectively when compared with the stem and leaf samples. The bacterial genera isolated were Bacillus, Pseudomonas, Enterobacter, Klebsiella, Staphylococcus, Citrobacter and Escherichia. Eight isolates produced IAA as well as ammonia. Phosphate solubilization was observed in five isolates while four isolates produced HCN. The germination rate of rice seeds coated with bacterial isolates ranged from 81.48% to 100%. Eight bacterial isolates inhibited the growth of Fusarium oxysporum and Rhizoctonia solani, but Citrobacter freundii and Enterobacter cloacae showed the highest antagonism. Thus, eight bacteria isolates showed multiple beneficial abilities and may be considered as promising microbial inoculants for rice plant growth and productivity.


Introduction
management and biotic stresses such as pest and The world's population has been projected to disease outbreaks, are limiting factors of optimal rice hit 9 billion by 2050 (Ronald, 2011), and most of this production in Nigeria.The use of chemical fertilizers growth is expected to happen in sub-Saharan Africa, and pesticides to remedy the problem of low soil particularly in Nigeria.The challenge of feeding the fertility and disease outbreak is currently limited due world's poorest people in an environmentally to rising costs and environmental concerns.In order sustainable manner will no doubt be immense.Rice to make rice cultivation more sustainable and less consumption is expected to increase as well.
dependent on chemical nitrogen and phosphorus Increasing rice production worldwide will help to feed fertilizers and pesticides, the use of endophytic plant the world.Despite the 6.74 million tonnes of rice growth-promoting bacteria (PGPB) that could protect produced in Nigeria in 2014 (FAOSTAT, 2015), Nigeria and promote rice growth would be an alternative for has remained one of the largest importers of rice, rice production.(Chinnusamy et al., 2006;Cong et al., second to Indonesia.Its importation bill of rice has 2009; Mäder et al., 2012).increased from $1,000,000 in the 1970s to as high as Endophytic bacteria are generally referred to as microorganisms that colonize healthy plant tissue $800 million in 2005 (WARDA, 2007) and in 2014, without causing obvious symptoms or produce Nigeria spent N1 billion daily importing rice (Osagie, obvious injuries to the host (Bacon andHinton, 2006). 2014).
They are also involved in the supply of biologically Among other factors, soil fertility fixed nitrogen to their host plant (Boddey et al., supplemented with cycloheximide (100 µg/mL) to 2005).The beneficial effects of endophytic bacteria inhibit fungal growth.Inoculated Petri plates were on plants include; the production of phytohormones, incubated at 28°C for 48 hours.After the incubation siderophores, ammonia, HCN, phosphate time, the colony forming units (CFU) for each plate solubilization (Compant et al., 2005;Rajkumar et al., was estimated. 2006), and the production of lytic enzymes Isolates differing in morphological (Nagarajkumar et al., 2004).Bacterial endophytes appearance were selected and were streaked onto colonize the ecological niche similar to that of new plates until pure cultures were obtained.Pure phytopathogens, which makes them suitable as cultures of bacterial isolates were maintained on NA biocontrol agents (Berg et al., 2005).
slants and were stored at 4°C.The use of endophytic bacteria as biofertilizer in the enhancement of rice growth and yield has been Cultural characterization of bacterial isolates reported by Rodrigues et al. (2008) in which the The bacterial isolates were characterized inoculation of Azospirillum amazonense increased dry based on their morphological and biochemical matter accumulation of rice grain (7 to 11.6%), the characteristics (Cappucino and Sherman, 2002) and number of panicles (3 to 18.6%) and nitrogen examined according to the Bergey's Manual of accumulation at grain maturation (3.5 to 18.5%).

Azospirillum brasilense inoculum was reported to
Biochemical tests including catalase, citrate increase yield of rice up to 76% (Thakuria et al., utilization, capsule staining, Voges-Proskaeur, Methyl 2004).Some of these bacteria have already been red and sugar fermentation tests were carried out on applied as biofertilizers and they are available to the isolates.farmers in the developed world (Beatty and Good, 2011).
Assays for phytobeneficial abilities of isolates The use of endophytic bacteria as Indole Acetic Acid Production biofertilizers and biopesticides to improve rice Detection of IAA production was done as production in Nigeria remains largely untapped described by Sharma et al. (2012).Pure bacterial despite the potential benefits of using rhizobacteria in isolates were inoculated in nutrient broth enriched with crop production and protection.According to Abiala et tryptophan (200 µg/mL), followed by incubation at al. (2015), the development of indigenous biofertlizer 28°C for 48 h in a shaker incubator.10 ml of the and biocontrol strains that suit local environments resultant culture was centrifuged at 4°C for 10 min at may help enhance competitiveness with in situ 15, 000 revolutions per minutes (rpm).Two millilitres of microorganisms and effectiveness at suppressing the supernatant was mixed with two drops of local pathogen strains.Also, Howell (2003) suggested orthophosphoric acid and 4 ml of the Salkowski's that biocontrol agents should be isolated from the soil reagent (50 ml, 35 % of perchloric acid, 1 ml 0.5M FeCl 3 locality where they are expected to function in disease solution).This mixture was kept in a dark room for 20 control.Thus, the present study was undertaken to min, afterwards the light absorption of the mixture was isolate, identify and screen bacteria present in the estimated at 540 nm using a spectrophotometer.The endosphere of two Nigerian rice cultivars (Ofada and light absorption estimates were compared to a standard ITA 150) for their multiple phytobeneficial effects.
curve to determine quantity of IAA produced by each isolate in µg/ml This experiment was carried out in MATERIALS AND METHODS triplicates for each bacterial isolate.

Collection of samples and bacteria isolation
Leaf, stem and root samples of two Nigerian Phosphate solubilization rice cultivars (OFADA and ITA 150) were randomly The bacterial isolates were spot-inoculated collected from the FADAMA farm of the Federal on the Pikovskaya media containing tricalcium University of Agriculture, Abeokuta in triplicates.The phosphate in a plate and incubated at 28°C for 72 h samples collected were separately washed with tap (Sharma et al., 2007).Plates were then observed for water, followed by surface sterilization using 70% the presence of halo zones (clear zones) around the ethanol for 30 seconds, 2% Sodium hypochlorite spotted colonies.The halo zone surrounding the (NaOCl) for 5 minutes, 3% Hydrogen peroxide for 30 colonies were measured, and the phosphate seconds and then rinsed five times with distilled solubilization efficiency (PSE), expressed as a water.Ten grams of these samples were cut to 2-3 cm percentage according to Sharma et al. (2007).pieces and macerated using sterilized mortar and pestle with 12.5 mM potassium phosphate buffer (pH Seed Germination Bioassay 7.1), followed by a 10-fold serial dilution where Seedling bioassay was conducted based on -6 -8 aliquots (100 µL) of the 10 and 10 dilutions were the method described by Dey et al. (2004).Bacterial plated using the pour plate method on Nutrient Agar inocula were prepared by incubating bacterial Olayemi and Odedara/ Nig.J. Biotech.33 (2017) 1-10 cultures for 24h and were diluted with sterile distilled colour was the indicator of HCN production.
6 water to give a concentration of approximately 10 6 cells/mL (10 CFU/mL) adjusted with a spectrophotometer Antagonism assay against phytopathogenic fungi (Jenway, UK).Rice seeds (Ofada variety) were The antifungal activity of the bacterial surface sterilized with 0.5% NaOCl for 2 min, followed isolates was assayed according to Noori and Saud by 30 seconds in 70% ethanol and two rinses in (2012).Potato Dextrose Agar (PDA) was prepared, distilled water, followed by air drying.Seeds were autoclaved and poured in sterile petridishes.A 5 mm coated with each bacterial isolate by immersion in a mycelia mat of Fusarium oxysporum and Rhizoctonia 6 suspension of bacteria containing 10 CFU/mL for 30 solani was placed in the center of each plate.Each mins and were then dried in a laminar flow cabinet for bacterial isolate was streaked 3 cm away from the 1 to 2 h.Nine seeds inoculated with each bacterium fungi on both sides of the fungus and the plates were placed in 9-cm-diameter petri dishes lined with incubated for 5-7 days at 28°C.The zone of inhibition sterilized moistened filter paper and were incubated was observed around the fungus used and the for 7 days at 28°C.Germinated seeds were counted at inhibition index was estimated using the formular: day 7. Germination rate, average plumule and radical Inhibition index (%) = (R1-R2) X 100 lengths as well as vigour index were calculated using R1 the method described by the International Seed Where R1=Radial growth of F. oxysporum in control Testing Agency (1999).
plate and R2=radial growth of F. oxysporum interacting with antagonistic bacteria.The control was a plate with Ammonia Production the phytopathogenic fungi only.All experiments were The ability of each bacterial isolate to done in triplicates.produce ammonia was assayed according to Cappucino and Sherman (1992).Bacterial isolates Statistical analysis were tested for the production of ammonia as follows: Data obtained were analyzed using the 24 h old bacterial cultures were inoculated in 10 ml Statistical Package for Social Sciences (SPSS) version nutrient broth and incubated at 30°C for 48 h in a 17.0 for Windows (SPSS, Chicago IL, USA).Data rotator shaker.After incubation, 0.5 ml of Nessler's obtained were analyzed using descriptive statistics reagent was added to each tube.The development of (mean and standard deviation) and Analysis of a yellow to brown colour indicated a positive reaction Variance (one-way) followed by post hoc pairwise for ammonia production.
comparisons using the Student-Newman-Keuls multiple-range test.

Hydrogen Cyanide (HCN) Production
Production of HCN by the isolates was Results detected according to the method of Ngoma et al.
Enumeration of bacterial isolates (2013).Nutrient broth was amended with 4.4 g/L A total of 193 bacterial strains were isolated glycine, followed by autoclaving and pouring in Petri from the endosphere of the two rice cultivars dishes.Each bacterial isolate was streaked on the 8 examined at 10 CFU/g (Plate 1).The endophytic modified agar.A Whatman filter paper soaked in 2% bacterial population of the roots in both rice varieties Sodium carbonate in 0.5% picric acid solution was (61.00±2.44,45.00±2.44)CFU/g were significantly placed directly on each of the streaked agar plate.
higher than those of the stems (29.33±2.44,Plates were then sealed with parafilm and incubated 32.00±2.44)CFU/g and leaves (15.67±2.44,at 28°C for four days.Development of orange to red colony colour ranging from cream to green.Motility Morphological and Biochemical Characteristics of was observed in most isolates examined and the cell Bacterial isolates shape was mostly rod (Table 1).All the isolates were The colonial morphology of isolates was positive for Catalase and were Glucose fermenters.significantly diverse.All the isolates produced round Majority of the isolates were Gram-negative in shaped colonies, the elevation was either flat or reaction (Table 2).raised, had smooth or undulate margin with the  3).

IAA production ability
This assay was carried out on each of the 193 Antagonism assay against phytopathogenic fungus bacterial isolates, out of which eight isolates were positive for IAA production.A standard curve was Eight isolates inhibited Fusarium oxysporum prepared using different concentrations of pure IAA and Rhizoctonia solani.The highest inhibition index and this curve was used to compute for IAA produced for F. oxysporum was observed in isolate OP7 (80% by the bacterial isolates assayed.All of the eight inhibition index), followed by isolate OP6 with 77.22% isolates examined produced significant quantities of while isolate OP4 had the lowest inhibition index of IAA ranging from 1 µg/mL produced by isolate OP8 to 49.44%.For R. solani, isolate OP8 had the greatest 32 µg/mL produced by isolate OP 2. Isolates OP8 and antifungal effect of 86.36% inhibition index, while OP4 produced the largest quantities of IAA at 32 isolate OP7 showed the least inhibition index of µg/mL and 31 µg/mL respectively while isolates OP1 48.18% (Table 3).and OP2 produced the least quantities of IAA at 12 µg/mL and 1 µg/mL respectively (Table 3).
Seed Germination Bioassay All isolates were examined for their effect on

Phosphate solubilization
Ofada rice seed germination in vitro.These isolates Five isolates of the 193 endophytic bacteria had varying degrees of influence on the germination were able to solubilize tricalcium phosphate with rate of the Ofada rice variety used.While the efficiencies ranging from 200% to 128%, while three untreated seeds of Ofada variety (Control) had the isolates did not solubilize the inorganic phosphate.
lowest germination rate of 70.37%, seeds coated with Isolates OP4 and OP7 produced the highest isolates OP1, OP5 and OP8 had the least germination phosphate solubilization efficiency of 170% as rate of 81.48%, while seeds treated with isolate OP3 compared to isolate OP6 with 98% solubilization had the highest germination rate of 100%.The vigour efficiency.Isolates OP1, OP2 and OP3 did not produce index for each isolate was also computed from data any halo zone on the Pikovskaya medium, and thus obtained on the plumule and radicle lengths as well as showed no phosphate solubilizing ability (Table 3).
the germination rate.It was observed that isolate OP3 had the highest vigor index of 682, while isolate OP4 HCN and Ammonia production had the least vigor index of 241.09.All 8 isolates Only four isolates produced HCN of the 193 assayed had significantly higher vigor index values strains in vitro.However, the degree of HCN than the control (Table 4).production ranged from high intensity to weak intensity.Eight bacterial isolates were positive for Table 3: Determination of characteristics associated with plant growth promotion of bacterial strains isolated from rice rhizosphere.KEY:-= not detected.+ = low.++ = medium.+++ = high.Values are mean ± standard error of mean.Values followed by different letters within a column indicates significant differences according to the Student-Newman-Keuls multiple-range test (á = 0.05) Table 4: Beneficial effects of identified bacterial isolates on Ofada seed germination and vigor index Values are mean ± standard error of mean.Values followed by different letters within a column indicates significant differences according to the Student-Newman-Keuls multiple-range test (a = 0.05) Olayemi and Odedara/ Nig.J. Biotech.33 (2017) 1-10

Discussion
(PSE) recorded ranged from 98% to 128%.The range The use of microorganisms and the of PSE recorded is similar to a report by Kaushal and exploitation of beneficial plant-microbe interactions Kaushal (2011).The ability of bacteria to solubilize offer promising and environmentally friendly insoluble phosphates has been attributed to their strategies for conventional and organic agriculture capacity to reduce pH by the excretion of organic worldwide (Berg, 2009).Various endophytic bacteria acids during the assimilation of ammonia that inhabit plant tissues can aid in sustainable (Gyaneshwar et al., 1999).agriculture.
The production of hydrogen cyanide (HCN) In this study, the endophytic bacterial and ammonia, secondary metabolites by certain population was observed to be highest in the roots, plant-associated bacteria is another beneficial trait of followed by the stems and lowest in the leaves of both a PGPB.HCN produced can inhibit the growth and rice cultivars examined.This can be attributed to the metabolism of potential plant pathogen and thus greater concentration of nutrients (exudates) present protect the plant from harm as well as strengthen the in the root region as compared to the phyllosphere.

Based on the morphology and biochemical
This study investigated the effectiveness of characteristics of the isolated bacterial species, 8 bacterial isolates whether they could increase the distinct bacteria morphotypes were tentatively seed germination rate as well as other plant growth identified as Bacillus subtilis, Pseudomonas parameters of the seedlings in vitro (plumule and aeruginosa, Klebsiella sp, Enterobacter sp, Citrobacter radicle lengths).Eight isolates significantly increased sp, Staphylococcus aureus, Staphylococcus the growth parameters of the inoculated rice seeds as saprophyticus and Escherichia coli.The species compared to the uninoculated control.This could be identified in this study are in correlation with other attributed to the suppression of seed pathogens studies on rice endopshere microbiome (Mbai et al., (Abiala et al., 2015) as well as the synthesis of 2013, Mwajita et al., 2013) who both implicated a hormones such as IAA by the isolates in this study (Ng wide variety of bacteria. et al., 2012).Indole acetic acid (IAA) can trigger the The plant growth-promoting abilities of the activity of specific enzymes that promote early isolated endophytic bacteria were assayed with germination and increased plumule and radicle length varying results observed.Endophytes have been and seed inoculation with IAA producing rhizobacteria shown to promote plant growth by producing IAA has been shown to enhance early seedling (Mendes et al., 2007).Indole acetic acid (IAA) is a key establishment (Khalid et al., 2004).phytohormone known for its ability to elongate and The unique ability of certain plant associated enlarge plant root cells in order to permit greater bacteria to control and inhibit the growth of plant nutrient and water absorption.In this study, eight out pathogens is of utmost importance in selecting for of 193 endophytic bacterial isolates produced IAA in potentially plant beneficial bacteria.In this study, concentrations.The range of IAA produced in this eight bacteria were able to inhibit the growth of two study is similar to a report by Ji et al. (2014) who soil-borne fungal pathogens Rhizoctonia solani and observed an IAA range of 3.1-24.6µg/mL while lower Fusarium oxysporum.Enterobacter sp (86.36%) range of IAA produced was reported by Rangjaroen et showed the highest antagonism to Rhizoctonia solani.al. (2015).
Fusarium oxysporum was also highly inhibited by P h o s p h o r u s , a n e s s e n t i a l p l a n t Citrobacter sp (80%).The differential rates of fungal macronutrient, although present in significant inhibition by the bacteria species suggests that the quantities in soils is not readily available for plant mode of action and types of antifungal metabolites uptake due to its insoluble forms being dominant.Of produced vary from one bacterium to another the 193 strains, only 5 isolates solubilized inorganic (Williams and Asher, 1996).The reduction of fungal phosphate by producing halo zones on the Pikovskaya growth by certain plant beneficial bacteria and the in medium.The phosphate solubilization efficiency vitro inhibition zones may be probably due to antifungal substances (such as Zwittermycine-A, Cappuccino, J.G. and Sherman, N. (2002).kanosmine, lipopeptides) and/or lytic enzymes Microbiology: A laboratory manual.New York: released by these isolates (Noumavo et al., 2015).
Pearson Education Incorporated.The results obtained from the various assays indicates that endophytic bacterial isolates OP4, OP7 Compant, S., Reiter, B., Sessitsch, A., Nowak, J., and OP8 corresponding to Bacillus subtilis, Clément, C. and Barka, E.A. (2005).Endophytic Enterobacter sp and Citrobacter sp showed the best colonization of Vitis vinifera L. by plant growthabilities to promote plant growth in vitro as seen by promoting bacterium Burkholderia sp.strain PsJN. the results of the various assays conducted.

Figure 1 :
Figure 1: Comparison of the total heterotrophic bacterial count of the different samples in two different rice varieties (ITA 150 and Ofada).

Table 1 :
Morphological characteristics of bacterial isolates KEY: RSL-Root, Stem and Leaf, RS-Root and Stem

Table 2 :
Biochemical characteristics of bacterial isolates from rice varieties KEY: GR= Gram stain, CA= Catalase, CP= Capsule stain, CO= Coagulase, MO= Motility, IN= Indole, CI= Citrate, UR= Urease, MR= Methyl-red, VP= Voges proskeur, G= Glucose, L= Lactose, M= Mannitol, A= Acid production, + = POSITIVE, -= NEGATIVE Assays for phytobeneficial abilities of isolates ammonia production (Table . In this study, only four bacteria isolated from rice landraces (Rangjaroen et out of the 193 bacterial strains were able to produce al., 2015), the cultivated rice cultivar Khao Dawk Mali HCN as Citrobacter sp and Klebsiella sp produced the 105 (Prakamhang et al., 2009) and wild rice cultivars highest colour intensities and thus the highest HCN while eight endophytic bacteria produced NH3, as Oryza rufipogos, O. nivara and O. granulata observed by the change in colouration of the broth