Characterization and Diversity of plant growth promoting Endophytic and Rhizosphere Bacteria isolated from theRhizosphere and tissues of Pepper and their effect on plant growth promotion and disease suppression of Phytophthora capsici


 Plant growth promoting rhizo and endophytic bacteria were isolated from different parts of pepper from south eastern Ethiopia. Plant growth promoting bacteria (PGP) are those that may be used to promote plant growth and suppress plant diseases. The objectives of this study were to identify and characterize PGPB indigenous to pepper rhizosphere and endophyte bacteria in Ethiopia, and to determine their capacity to suppress Phytophthora capsici in pepper. From a total of 60 isolates, 20 were selected based on their in vitro antagonism activity of phytopathogens and plant growth promoting traits. From the total 60 strains representing, 38 rhizosphere, and 22 endophytic bacteria were identified based on biochemical assays of semi-automated Vitec 2 compact and ten potential bacteria further identified by molecular methods. Results revealed that only one isolate of rhizosphere and three endophytic bacteria showed more than 50% suppression of test pathogens. The isolates were evaluated for their ability to solubilize phosphate, as well as for ammonia, indole acetic acid, hydrogen cyanide, and biofilm production. The selected isolates produced (0.2–93 µg mL− 1) of indole-3-acetic acid, without supplemented with tryptophan, while supplemented with tryptophan it produces (11.23–159 µg mL− 1). The activities of plant growth-promoting were assessed by measuring their effect on the number of lateral roots, root and shoot length of Arabidopsis plants, and germination percentage of pepper plants. Pepper plants grown from seeds that were treated with these PGPB strains showed significantly higher levels of germination, seedling vigor, and growth, compared to non-treated control plants. Since these PGPB inoculants showed multiple characters useful to the host plants, they may be used as an alternative in the production of new, safe, and effective seed treatments as bio-fungicides. Generally, this work exhibits the potential of bacterial isolates to control Phytophthora infection and promote plant growth.


Introduction
The increment of crop productivity is essential for feeding the increasing population in developing countries and often depends on the use of synthetic fertilizers. However, contentious use of these synthetics microcidal was shown to decrease bacterial diversity in soil 1 and can also have harmful impacts on the environment, such as leaching of phosphorus and nitrogen into groundwater, and increasing the pollution of soil and water 2 . To reduce the continuous use of chemicals and to increase the sustainability of agricultural practices is the use of e cient, nutrient mobilizing microorganism's fertilizers 3,4 .
Plant growth promoting bacteria (PGPB) that form a close relationship with their host plants are important to increase plant productivity and health under various environmental conditions 5 . Endophytes and rhizosphere bacteria colonize plant tissues and roots without any apparent pathogenic symptoms and begin bene cial associations with their plant host by synthesis of phytohormone, production of an enzyme, and nutrient mobilization and translocation, such as phosphate (PO4 − 3 ) solubilization, nitrogen xation, and ammonia (NH3) production 6,7 . The rhizosphere and the internal tissues of most plants are commonly invaded by bacteria and should have various effects on the event and physiology of the host plant. The internal tissue and rhizosphere of plants get bene ts from these bacteria.
Endophytic bacteria that reside within the interior of plant tissues provides different bene ts to plants, which include the promotion of plant growth and induce defense mechanisms to plants, and producing antibiotics against fungal pathogens 8 . Also, it plays a crucial role in the healthiness and development of plants under different environmental stresses. Endophyte bacteria, which inhabit healthy plant tissues without causing any damage to the host, have enormous potential as biological control agents against plant diseases. 9 . Even though it has different ecological niches, rhizo-bacteria and endophytic bacteria use the same mechanisms to promote plant growth and control phytopathogens 10 . For instance, they promote plant growth by producing indole-3-acetic acid (IAA), synthesis of phosphate solubilizing, and production of ammonia 11 . 12 studied bacterial inoculants as bio fertilizers and found that improved growth and increased yield of cereal crops 13 has been reviewed the importance of endophytic, and rhizosphere microorganisms within the plant growth, particularly in unfavorable soil conditions. The impact of some plant-associated microorganisms is connected to their unique metabolic activities, like nitrogen (N2) xation, phosphate solubilization, the ad, production of IAA, 14 . To grasp the interaction between host plants and their rhizosphere and endophytic bacteria, it's important to spot the variety of this bacteria related to the plants.
In the present study isolation of plant growth-promoting (PGP) endophytic and rhizosphere bacteria from pepper cultivated region in the Eastern part of Ethiopia and investigating the effects of these bacteria on inoculated pepper seedlings growth enhancement, and genus wise screening of their diversity.

Isolation of Rhizosphere bacteria from pepper plants
The rhizosphere soil samples were collected from all the elds' surveys by uprooting the healthy chili plant carefully without damaging the basis system. It is then, shakes gently to get rid of the surplus soil and brings it to the laboratory by a sterile polythene bag. The procedure was implemented using the following methods 15 .
Ten grams of soil sample was taken separately and suspended in 90 ml of sterile water and stir well to give 1:10 dilution (10 − 1). One ml from this was transferred to a tube containing nine ml of sterile water to give 1:100 dilutions (10 − 2 ). One ml from this was transferred to a tube containing nine ml of sterile water to give 1:1000 (10 − 3). Similarly, 10 − 4 and 10 − 5 dilution for isolation of bacterial spp. were used. One ml of every dilution was added into Petri plates and 20 ml of separate sterile and cooled macConkey and nutrient agar medium were poured and every treatment was replicated 3 times. The representative bacterial colonies were picked up, puri ed, and preserved at -22 0 C for further use.

Determination of CFU/ml
The pour plate methods were used for the determination of CFU/ml of the bacterial suspension.
The number of CFU/ml was calculated as under 16 No. of bacterial colonies CFU/ml = --------------------Dilution ×amount plated 2.2 Isolation of endophytic bacteria from pepper plants Endophytic bacteria were isolated from the inner tissues of roots, leaves, fruits, and stems of apparently healthy pepper varieties (different cultivars) collected from major Eastern parts of Ethiopia pepper growing regions following the methods of 17 with some modi cations. Samples were surface sterilized with 2% sodium hypo chloride followed by 70% ethanol for 3 min and rinsed ve times in sterile distilled H 2 O. Five grams of surface sterilized plant parts were grinded separately with a waring blender in phosphate buffer saline (PBS) and were centrifuged (60 g) for ve minutes. The supernatant was serially diluted up to 10 − 5 , poured on nutrient agar and macCkonkey agar plates, and incubated at 28 0 C for 48 hrs. The bacterial colonies from each tissue were selected and subcultured on nutrient agar and stored for further studies. Surface sterility was checked for every sample to observe the e ciency of the disinfection procedure.
For this, 0.1 ml of the last wash was poured to agar media and incubated at 28 0 C for a sterility check.
3 Bacterial Identi cation 3.1 Morphological Identi cation 3.1.1 Gram stain A loop full culture was taken and smear prepared on a clean glass slide. After the smear dried, it absolutely was stained as per instruction given in Gram's staining reagents kit. Then, the morphological characters were noted.

Biochemical and physiological characterization
The pro ciency of bacteria to utilize different metabolites was done through performing biochemical tests. The bacterial bio agents were preliminarily identi ed using Gram stain, and biochemical tests (Catalase, Indole production, Oxidase test, urea test, Citrate utilization test, gas glucose production, H 2 S production test, Urea utilization test, Lysine Decarboxylase test, Fermentation of Sugars (Sugar Utilization Test)). Subsequently, selective media like MacConkey agar was used to isolate gram-negative bacteria, and was also identi cation was performed with a semi-automatic Vitek 2 compact (bioMérieux) system using GP ID REF21342 (Identi cation-Gram-positive bacteria) and GN ID REF21341 (Identi cation-Gram-negative bacteria) cards. All the test procedures were followed in keeping with the manufacturer's instructions.

Molecular Identi cation
A volume of 1.5 mL of overnight-grown cultures of each isolate were harvested by centrifugation at 11,000 g for 5 min and removal of the supernatant by gentle pipetting. Genomic DNA of the bacterial isolates was extracted using the ISOLATE II Plant DNA Kit (Bioline, Inc., Taunton, MA, United States) according to the manufacturer's instructions. The isolated DNA was subjected to PCR ampli cation with a universal 16S primer set consisting of forward primer 63f (5'-CAG GCC TAA CAC ATG CAA GTC-3') and reverse primer 1387r (5'-GGG CGG WGT GTA CAA GGC-3') (Pharmacia) 18 . PCR was performed in a 25.5 µL reaction mixture containing: 18 µL pure water, 5 µL of 10X TBE buffer, 0.5 µL of dNTPs (10 µM), 0.5 µL of 10 µM 63F primer, 0.5 µL of 10 µM 11387R primer, and 1µl of DNA template. The PCR thermocycler conditions were 30 s at 95•C for initial denaturation, 30 cycles of 30 s at 95•C, 30 s at 55•C for annealing and 1min at 72•C, followed by 5 min at 72•C for nal extension. Ampli cations were performed in duplicate for each sample, and the presence of the predicted PCR products was con rmed by 1% agarose gel electrophoresis. The PCR products were puri ed using the Wizard R SV PCR Clean-Up System (Promega), and purity was con rmed using a Nano Drop on spectrophotometer, then cloned, and sends to the Molecular cloning laboratories in San Francisco CA, for sequencing by DNA Analyzer (Applied Biosystems).

Plant Growth Promoting Bacteria
PGPB are bacteria that can promote plant growth, including those that are free-living, have symbiotic relationships with rhizosphere plants, and bacterial endophytes that can colonize interior tissues of plants. PGPB could promote plant growth directly by either enhancing resource acquisition or managing plant hormone levels, or indirectly by decreasing the deleterious impacts of different pathogenic microbes on plant growth and development 19 . The production of indole acetic acid was veri ed with some modi cations via the colorimetric method developed by 20 . At 500 rpm for 7 days of 10^8 cfu/ml of an overnight bacterial culture grown on tryptophan broth was centrifuge. The supernatant was reserved and 1ml was mixed with 2ml of Salkowski's reagent (2% 0.5 FeCl3 in 35% perchloric acid (CLO4) solution) and kept within the dark. Then the optical density (OD) was recorded at 530 nm at different L-tryptophan concentrations.
L-Tryptophan utilized was studied by estimating the residual l-tryptophan remaining in the broth by previously estimated spectrophotometric test 21 . Brie y, 1 ml of cell-free aliquots was taken from the broth and evaporated on a boiling water bath to dryness, followed by the addition of 1 ml of nitric acid (16 mol/l) and incubation at 50 0 C for 15 min. The contents were then cooled at room temperature following the addition of 4 ml of sodium hydroxide (5 mol l/l) solution; ethyl alcohol (97%) was used to make the nal volume of 10 ml. After mixing the contents, absorbance was recorded at 360 nm. Quanti cation of ltryptophan was performed using the standard curve. 4.1.2 Qualitative and Quantitative Measurement of Phosphate Solubilization Qualitative tests of bacterial isolates were screened for their TCP solubilizing activity on PKV plates. Isolates had been spot inoculated at the middle of the agar plate aseptically. All of the plates are incubated at 28 0 C for 5 days. A clean region around a growing colony showed solubilization of phosphate and recorded as phosphate solubilization index (PSI). PSI was calculated by dividing the ratio of the full diameter (colony + halo region) by to the colony diameter 22 (2,4,8,10,20,30,40, and 50µg mL − 1 ) were made to get the standard curve of KH 2 PO 4 . Uninoculated sterilized Pikovskaya broth medium was used as control. There were three replicates for each treatment and each treatment received an equal amount of culture broth (inoculum adjusted to ~ x 10 8 CFU mL − 1 ). Change in pH of broth culture in response to phosphorus was recorded for cell-free supernatant measured with a pH meter at each sampling time. The correlation coe cient (r) between soluble P and pH value was calculated using Graph pad prism software (Graph pad prism Inc, California, USA) at 1% level of signi cance.

Plate Method
Qualitative assay of bio lm-producing microorganism was detected using the congo red agar method, as a result of the dark color of colonies inoculated on CRA medium 24 . The CRA medium is prepared by mixing 0.8 g of congo red, and 36 g of sucrose to 37 g/L of Brain heart infusion (BHI) agar. The mucoid nature of the bacterial colonies was studied through the cultivation of all the strains on Congo red agar (CRA) plates 25 . Fresh bacterial cultures were streaked on the CRA plates. Plates were incubated at 28°C for 48 h and seen for the attribute of black colony morphology of the media.

Tube Method (TM)
The tube methods of bio lm formation through the bacterial isolates have been located by the way of adherence to the partitions of culture tubes 26 .
The inoculum was prepared using 5 ml of nutrient broth. After 48 h of incubation at 28°C, turbidity was adjusted to 0.5 (10^8 CFU/ml) McFarland standards for the bio lm experiment, 100 µl of inoculum was transferred into three ml of nutrient broth in10-13 ml of test tubes.
All the test tubes were incubated at 28 0 c for 48h.in nutrient broth. The tubes were washed with three ml of 2X phosphate buffer saline (PBS). The isolates of which bio lms formed on the walls of test tube are stained with 3 ml of 2% crystal violet for 1 hr. Then, crystal violet-stained test tubes were rinsed twice with PBS and allowed to dry, and the occurrence of visible lm lined the walls, and the bottom of test tubes indicates the presence of bio lm 27 . All the tubes had been added with 33 % of 1.5 ml glacial acetic acid and blended gently. Optical density (OD) was measured in the spectrophotometer at 570 nm. The OD values of the samples were measured with the PBS as control. The bio lm formation experiment in tubes was carried out by using both glass and plastic (PVC) tubes. To interpret results, categorization can be done as no bio lm production (0), weak (+ or 1), moderate ( + + or 2), and strong bio lm production (+++ or 3) by the calculation of cutoff value (ODc) shown below 28 : OD ≤ ODc no bio lm production ODc < OD ≤ 2 × ODc weak bio lm production 2 × ODc < OD ≤ 4 × ODc moderate bio lm production 4 × ODc < OD strong bio lm production.

Qualitative and quantitative estimation of ammonia production
The bacterial isolates were inoculated in 10ml of peptone water broth and incubated at 28°C for 5 days. Then, Nessler's reagent (0.5 ml) was added in every tube. The color of the media changed from brown to yellow, which indicates the presence of ammonia production, and its OD was measured at 450 nm using a spectrophotometer (6405 UV-Visible, JENWAY) following 29 . The concentration of ammonia was estimated using the well-known curve for ammonium sulfate for concentrations in the 0.1-1 mol/ml range. 4.2.2 Qualitative screening of hydrogen cyanide production Hydrogen cyanide production was evaluated using the qualitative technique described by 30 . Bacterial isolates are streaked on nutrient broth supplemented with glycine (4.4 g/l), Whatman paper no.1 (soaked in 2% of Na2CO3 in 0.05% picric acid) is put on the peak of the plate which is then sealed with Para lm and incubated at 28°C for 6 days. The color of the lter paper strips change from yellow to light brown, brown, and brick red was recorded as weak (+)moderate (++), or strong (+++) reaction, respectively. If there is no change in color, it is recorded as a negative (−) reaction.

Screening PGPB isolates for plant growth promotion on Arabidopsis thaliana
In this study, Arabidopsis thaliana seed wild type ecotype Col-0 were purchased by the Institute for Scienti c and Technological Research of San Luis Potosi, (IPICYT), Mexico from Ohio University, Arabidopsis Biological Resource Center. The stock is conserved in IPICYT laboratory, and seed is harvesting every year to refresh and get new more seeds. Then, the experiment was performed following IPICYT guide lines. The seeds were surface-sterilized with 20% sodium hypochlorite and centrifuge at 1500 rpm for 7 minutes, rinsed ve timeswith sterile deionized water at 1500 rpm for 5 minutes and vernalized for 2 days at 4°C in thedark. 31 . The vernalized seeds were placed on bipartite Petri dishes containing 0.5% strength Murashige-Skoog (1/2x MS) medium (Sigma-Aldrich, St.Louis, MO) with 0.8% agar and 1.5% sucrose, and the plates were incubated for 2days (16 h light and 8 h dark at 22°C) 31 .Two days before the plant experiments, bacterial strains were cultured on Nutrient broth and grown overnight. Bacterial cells were counted by hemocytometer and diluted to 8× 10 8 CFU/ml. and fteen microliters of a bacterial cell suspension were placed in the direct interaction (Ba-MS), and in the split interaction (Ba/MS) of MS medium. Plates were sealed with plastic lm and incubated in the growth chamber as described above. The fresh shoot weight, root length and lateral root per seedling were measured 15 days after planting. The assay was in ve replication for each treatment.

Diversity and characterization of Rhizosphere and Endophytic bacteria
A total of 60 bacteria were isolated from different parts of pepper plant from the Eastern parts of Ethiopia (table 1) among them 7 were from roots, 6 were from stem, 5 from leaves, 4 from fruits and 38 from rhizosphere of pepper plants. In other words from 60 isolates, 38 were from the rhizosphere (63.33%) and 22 were from endophytic isolates (36.66%). Gram staining test on those isolates con rmed that 95% of those isolates had been gram-negative and the rest of 5% isolates were gram-positive. Sixty morphologically different bacterial endophytes and rhizosphere isolated from the pepper grow on Nutrient agar, and Blood base agar media. The bacterial endophytes and rhizosphere isolated from the root, stem leaves and rhizosphere of pepper were abbreviated as AAURE, AAUSE, AAULE and AAUR respectively. Preliminary screening for the antagonistic potential of the bacterial isolates were done by dual culture plate assay on PDA medium that supported the growth of both the bacterial isolates and the pathogen. Out of the total 60 bacterial isolates 20 (33.3%) were selected based on dual culture plate assay, plant growth promoting effect and other parameters as shown in the table-2 and 3 below. Among the 20 antagonistic bacteria, AAULE51 (un identi ed) isolates were dominant followed by isolates of AAUSR23 (Enterobacter hormaechei), AAUFE29 (Rhizobium sp.), and AAULE41 (Pseudomonas ourscence). The antagonistic activity of mycelial growth inhibition of Phytophthora capsici was recorded from 54.8-94.65% and these were selected for further study.   Table.3. Twenty bacterial isolates selected based on the above criteria We obtained a total of 60 rhizospher and endophyte bacterial strains from the tissue and rhizosphere of pepper plants. Twenty isolates were selected based on their ability to produce IAA, phosphate solublization, bio lm, and ammonia and hydrogen cynide production and show in vitro antagonism against Phytophthora capsici and Fusarium sp. pathogens in a preliminary screening. All 20 isolates were tested to the Gram staining, citrate, catalase, indole and oxidase tests (Table 4).
Phylogenetic trees of three gram positive and seven gram negative bacterial strains constructed from 16S rRNA sequences showed that the selected isolates were mainly members of genus Bacillus, Pseudomonas, Rhizobium, Enterobacter, Serracia, and Pantoea (Fig. 9a &b). The sequences of the isolates AAUSR1, AAUSR2, and AAUSR18 showed 100% similarity. Isolate AAULE41 had 98% homology with Pseudomonas ourscence. Isolate AAUSR29 was identi ed as Rhizobium sp. with Gen Bank accession number MW89263. Isolates AAUSR16 showed 98 % sequence homology with Serracia marcescens (Fig. 9b).  Salkowski's reagent (Fig. 1a, table 1). Isolates of AAUSR7, AAUSR15, AAURE16, AAUSR23, AAULE41, AAUSR43, AAUSE44, and AAUSR48 produced more than 40 µg /ml of IAA when supplemented with L-tryptophan (Fig. 1b). The maximum production of IAA (159 µg/ml) was attained by utilizing 35.5% of ltryptophan, when medium was supplemented with 400 µg/ml concentration of l-tryptophan as a precursor (Fig. 1c) isolates were found to have more than 10 mm phosphate solubilizing index (PSI). Maximum PSI was observed by AAU51 (14.3 ± 0.9 a ) followed by AAU29 (11.7 ± 0.9 b ) (Fig 2a). Quantitative estimation of phosphate solublization is shown in (Fig. 2b). Out of 11 phosphate solublizer on pikovskaya broth, only 5 showed phosphate solublization of 40 µg/ml or more on quantitative phosphate solublizaton. Consequently, as shown in Fig. 2c, it can be seen that the Among the total 60 isolates, 31 and from 20 selected 15 bacterial isolates were produced bio lm both in tube and slide methods (Fig. 3a &b). Bio lm detection from endophytic and rhizosphere bacteria was done by Congo red agar method. It was observed that the strains produced white colored colonies on CRA medium, were negative while positive isolates produced black colored colonies, which was a typical indication for the production of bio lm (Fig. 3a). This study has been reported that the nutrient composition of the CRA medium in particular, supplementation of various sugars were greatly in uenced the colony morphology as well as bio lm formation e ciency of the rhizosphere and endophytic bacteria.

.2 Tube method
In this method, the bio lm formation was seen by the formation of a visible thick lm inside the wall of the tube and bottom of the tube. The isolates were showed thick lm inside the bottom of the glass tube indicating strong bio lm production, while other isolates did not show the formation of bio lm in the glass tubes (Fig. 3b). And its optical density after washed with methanol were measured.16 isolates had strong bio lm production (+++) OD (570nm), 1 isolate showed moderate bio lm production (++) whereas, 12 isolates were showed weak bio lm formation (+).

Ammonia production
All 60 bacterial isolates were showed production of ammonia in peptone water. Ammonia produced by different bacterial isolates is showed in Fig. 6. AAULE51 and AAUSR1 isolates produced maximum ammonia, which was 121.9 µmol/ml (OD 450). The 9 isolates produced more than 50µmol/ml (OD 450) of ammonia.

Qualitative and quantitative screening of HCN production
Sixty isolates recovered from different locations and different parts of pepper were tested for their ability to produce hydrogen cyanide (HCN). Only 8 isolates were able to produce HCN. The 8 HCN producing isolates were isolated 4 from rhizosphere soil, and 4 from endophytic of pepper plants grown in Eastern Ethiopia (AAUSR10, AAURE12, AAURE13, AAUSR15, AAUSR20, AAUSR22, AAUSR41 and AAUSR51. A strong (+++) HCN production was recorded by the isolate AAURE13, AAUSR22, and AAULE41; while a moderate (++) reaction was recorded by AAURE12, AAUSR20, and AAULE51. A weak (+) reaction was recorded by AAUSR1 and AAUSR15 (Table 5 and Fig. 8). Quantitative assay of HCN production by bacterial isolates showed that isolates AAURE13 and AAURE41 were produced a maximum absorbance value of HCN (0.13 and 0.23 respectively), followed by AAUSR22, AAURE51, and AAUSR12 which recorded absorbance values of 0.086, 0.063, and 0.057), respectively. The lowest absorbance were recorded by AAUSR10 (0.032 (Fig. 5)

Plant Growth Stimulation Assay of Selected Bacterial Isolates on Arabidopsis thaliana (Col-0) Plant in a bipartite petridish
To determine the plant promotion effect of selected Bacterial isolates on Arabidopsis which is grown on MS culture media with Arabidopsis plant, two experiments were analyzed such as in the direct interaction (Bacteria-Arabidopsis on MS) and split interaction (Bacteria/Arabidopsis on MS). Physiological parameters at 10 days post inoculation (dpi), such as fresh weight, main root length, and lateral root number of plants, were used to evaluate the effect of bacteria on Arabidopsis growth (Fig. 8a, b, c & d ). All isolates were showed an increment in the Arabidopsis fresh weight. While isolate AAUFE29 in the split system stimulated signi cantly the plant growth which reached double the fresh weight of un-inoculated control plants (Fig. 8a). In the main root length, there was no signi cant difference observed in both split and direct interaction systems in comparison with un-inoculated control (Fig, 8b). In respect to the lateral roots, in all isolates except AAUSR23 signi cant difference was observed (Fig, 8c)  unknown strains. In the present study, sixty rhizo and endophytic bacterial strains were recovered from chilli rhizosphere, root, stem, leaf and fruits, majorly belonged to the genera Entrobacter and Pseudomonas. Concerning the isolated bacteria, 11.67 % were from roots, 63.33 % from the rhizosphere, 8.3 % from leaves and 11 % from stem. Previous studies also report on various biocontrol potential of bacteria belonging to the genera Pseudomonas and Bacillus 36 , Bacillus subtlis and Actinomycetes 37 being the most common bacterial groups showing in pepper plants. Bacterial colonization appeared to be abundant in the rhizosphere pepper plants, which may be a re ection of a primary site for bacterial nutrients. Antibiotic production may be one of the mechanisms used by these bacterial isolates to dominate the host by preventing other potential colonizers 38 . The preference of bacteria for the rhizosphere re ects the presence of high levels of nutrients in the rhizosphere and the capability of the nutrients to support higher bacterial growth and metabolism in rhizosphere soils compared with other tissues 39 . The results showed that some of the plant growths promoting bacteria revealed multiple plant growths promoting traits and disease suppressing ability in in vitro. PGPR has often been found to reveal multiple modes of action, including biological control 40 . Presently, from all 60 bacterial isolates (n = 8, 13.3%) of endophytic bacteria and (n = 12, 20%) of rhizospheric bacteria that were antagonists against oomycetes and Fusarium as well as has plant growth prompting effects. 41,42 previously studied on bacterial antagonists and found that 27.5-62.6% are antagonistic against the common plant pathogenic fungi like Phythophotora capsici, Rhizoctonia solani, Py. ultimum, Fusarium oxysporum, and Botrytis cinerea. This may be because we used only Phytophthora capsici and Fusarium spp as potential organisms, while in the previous study the initial screening included many potential pathogens.
The antagonistic effect of the bacterial strains used as biological control for P. capsici 43 . Initially, from sixty bacterial strains, twenty were screened for antagonism against P. capsici. All the twenty tested bacterial strains showed varied levels of antagonistic potential. In this study results showed that four bacterial isolates, i.e., AAUSR23-Enterobacter hormaechei, AAUFE29-Enterobacter sp. V.H.18, AULE41-Pseudomonas uorescence and AAULE51-un identi ed showed > 52.5% mycelial growth inhibition of Phytophthora capsici. 36 studied the antagonistic effects of bacterial strains and found that, out of fteen tested rhizospheric bacteria, ve bacterial strains showed > 70% of antagonistic potential against P.capsici. In other study from 48 threeisolates were found to control P.capsici both in vivo and in vitro 44 . Bacillus amyloliquefaciens also, have been reported as both plant growth promoters and biocontrol agents against soilborne pathogens including Fusarium wilt of banana and potato dry rot caused by Fusarium sp. 45 . In our study 20 isolates showed both plant growth promotors and biological controls against P. capsici. From this isolate Enterobacter hormaechei, Entrobacter sp.V.H.18, Pseudomonas orecense, and AAULE51 -Unidenti ed that showed highest antagonistic effect against P. capsici and plant growth promotion activity were selected for further study.
The role of plant growth promotion microbes can be grouped into direct and indirect. Direct mechanisms include nitrogen xation, phosphate solubilization, bio lm production, and IAA production while indirect mechanisms include cell wall degrading enzyme production, ammonia production, HCN production and antibiosis 46 .
The direct growth promoting characters of bene cial bacteria are the ability to produce bio lm formation, solubilize phosphate, and production of Indole acetic acid. These traits increase the available nutrients in the soil, which the plant can absorb for growth 47 . Most of the tested bacteria showed multiple PGP traits which help to growth promotion and disease reduction ability of PGPB. The multiple modes of action have been researched to be the main reasons for the plant growth promotion and disease suppressing potential of PGPR 48 . It is essential to screen the potential of rhizo bacterial and endophytic strains for their plant growth promotion characters to obtain the desired bene ts of disease management, and plant growth promotions. In this study, four bacterial strains with better antagonism effect against P. capsici and plant growth promotion traits were selected.
Indole-3-acetic acid (IAA) is a secondary metabolite, and its production in bacterial agents is generally described based on their ability to use tryptophan supplemented in the growth medium, which is the major precursor of IAA biosynthesis via the indole pyruvic acid (IPA) pathway 49 . IAA supports root development, elongation, and proliferation and helps plants to take up water and nutrients from the soil 50 . L-Tryptophan is commonly considered as an IAA precursor because its addition to IAA producing bacterial culture enhances IAA biosynthesis 51 . 52 reported that all tested endophytic bacterial strains had the ability to produce IAA in the absence and presence of tryptophan. In the present study from 17 IAA producing isolates, 12 had the ability to produce IAA in the absence of L-tryptophan. The maximum production of IAA was recorded in the medium supplemented with 400 µg/ml tryptophan for isolate AAUSR17, AAUSR 43, AAUSE 44, 200 µg/ml for isolate AAURE16 and AAUSR48 (Fig. 1b ).
The plant growth is affected by low pH since the concentrations of metals like Al3 + and Mn2 + increase in the soil solution, and able to reach toxic levels. It is known that soil pH and metal cations might disturb many processes arising in the rhizosphere. The impact of different levels of pH (4.28-8.5) on media after 7 days incubation was determined. The maximum amount of IAA was produced when the pH of the culture medium was 6.44 and 6.22 for isolates AAUSR43 and AAUSR7 (Fig. 1b). 51 have reported br12, br2, and br3 for elaborated high levels of IAA production in a medium having pH 8. 53 studied on IAA production by Streptomyces sp. and reported that pH 7.0 was suitable for maximum IAA production. 54 studied on IAA producing bacteria and reported that most of the organisms are gram-negative. 55 reported a few known gram positive bacillus strains to produce IAA. Present study showed that from 17 IAA producing strains 16 were gram-negative and 1 gram positive. Except for isolate AAURE16 (Pantoea cypri pedii) and AAULE41 (Pseudomonas orecense) the IAA production of l-tryptophan increased with increasing l-tryptophan concentration but the utilization of l-tryptophan decreases with increasing l-tryptophan concentration. This might be due to a change in pH. 56 reported that the optimal IAA production and bacterial growth were at neutral pH, therefore acidic and alkaline conditions may affect the production of IAA as well as the growth of an organism. 56 also on IAA production and l-tryptophan utilization and found that the IAA production and utilization of l-tryptophan decreased with increasing l-tryptophan concentration.
Phosphate solubilization bacteria is one of the very crucial trait of plant growth promotion that solubilizes insoluble phosphate and making it available for plants. It has been found that P is important for plant growth, and its de ciency limits plant growth. Although synthetic fertilizers are added to the soils, because of the immobilization of phosphorus, plants can only use a small amount of phosphatic fertilizer. Therefore, the choice of highly e cient phosphate solubilizing bacteria is very important; it will practically increase phosphorous in the rhizosphere and tissue of plants. 57 reported that the isolates which solubilize tri-calcium phosphate (TCP) in a liquid and solid medium capable for produce organic acids making the pH of the medium acidic.
In the present study from 60 isolates, 11 strains showed phosphate solubilization and almost all of the isolates that can solubilize TCP showed drop in the pH of the medium when compared to the control. The decrease in the pH is supported by the production of different organic acids by consumption of sugars 58 . 59 also studied on phosphate solubilization of potential rizosphere fungi and reported that the maximum pH decrease was recorded from the 5th and 10th days of incubations by the most fungal isolates but was later increased or nearly constant in all liquid culture. From the nine isolates, the biggest decrease of pH in the medium was from an original value of 7.0 to pH values 4.00, 4.05, 4.13, and 4.23 for the isolates JUHbF60, JUCaF37, JUHbF95, and JUFbF59, respectively, 10 days after incubation. However, no decrement of pH was found in the last 15-20 days of incubation in PVK liquid medium. In this study the maximum pH drop was recorded in the 15th days of incubation by isolate AAUSR15 (from pH 7.00 to 3.7). Further bacterial isolates of AAUSR15 and AAUSR43, AAUSR23 and AAUFE17 showed a signi cant increment of pH after 5, 10, and 15 days of incubation respectively. An increment of pH could be microorganisms face the lack of nutrients and begin to consume the organic acids as the nutrient sources.
In our study, the concentration of phosphate solubilizing isolates in the PVK medium showed, ranged between 4.25 ± 0.005 µg/ml to 86.75 ± 0.005 µg/ml, 13 µg/ml ± 0.055 µg/ml to 94.25 µg/ml ± 0.055 µg/ml and 11.75 µg/ml ± 0.034 µg/ml to 90.5 µg/ml ± 0.034 µg/ml after 5, 10 and 15 days of incubation respectively. The highest concentration was recorded in isolate AAUFE29 (Enterobacter sp.V.H.18)a t pH of 6.3 after 10 ten days of incubation. 60  With regard to the incubation period, the solubilization capacity of the organism was high till 10 days of incubation. The isolate AAUFE 29 (Enterobacter sp.V.H.18) solubilizes more amount of phosphate during 10 days of incubation, which was readily available and declined later. The decrease in phosphate solubilization after a maximum value might be attributed to the shortage of nutrients in the culture medium and a change in pH. Also, the decrease in soluble P at 15 days of incubation may be either due to decreased solubilizing activity or increased P absorption and re-xation of solubilized phosphorus with metal ions present in the medium. These results are in con rmation with the results observed by 62 , the P concentration in the liquid medium did not have a sigmoid curve type but with some variabilities, which could be due to cell lysis and phosphorus precipitation brought about by organic metabolites. In our study, the decrease in phosphorus solubilizing concentration for the duration of 5 days of incubation in some phosphate solublizing bacteria as shown in (Fig. 3a) can be recognized as a result of the consumption of accessible phosphorus for growth and development of the bacteria.
Additionally, as shown in Fig. 3b, the increased concentration of the P-solublization in the pikovskaya liquid medium by bacterial isolates is in accordance with the increase of the PSI values, with the correlation and P-value value of 0.79 and P ≤ 0.0037 respectively. Also in previous studies where the correlation of Psolubilization and PSI was 0.89 63 .
Bio lms are surface-associated microbial cells, enclosed in a self-produced EPS that predominantly contain proteins, polysaccharide,, and lipids 64 . 65 studied on bio lm producing PGPR and reported that they are more effective under eld conditions than any planktonic PGPR In this study, 31 (51.67 %) from 60 bacterial strains isolated from pepper plants were found to form bio lms (Fig. 4Aa, &b). Among them, 16 (51.8%) strains showed strong bio lm production. 66 reported on bio lm production of rhizobacterial strains isolated from rhizosphere of tomato plants, and found that from 78 tasted strains 21 (26.9%) were found to form bio lms... All these bio lm-producing rhizobacterial strains are nonpathogenic to human and animals based on the hemolytic test using 5% sheep blood.
Another important PGPR mechanism that indirectly in uences plant growth is ammonia production. It accumulates and supply nitrogen to their host plants and promotes plant growth. In this study all the isolates were able to produce ammonia and the highest value of 121.9 µ mol/ml were measured for strains AAULE51 and AAUSR1 ( Fig. 5). 67 reported that ammonia produced by plant growth promoting bacteria has been shown to supply nitrogen to their host plants and thus promote root and shoot elongation and their biomass. Bacterial isolates can improve plant development by the production of ammonia through the hydrolysis of urea into ammonia and carbon dioxide. 67 also reported that the ammonia production by the rhizobacterial isolates was observed in the range of 2.5 µmol/ ml to 7.54 µmol /ml. About 67% of bacterial isolates revealed more than 4 µmol ml − 1 of ammonia production, and strain AB331 produced the highest amount of ammonia (7.54 µmol/ ml). In the present study, the ammonia production by bacterial isolates was observed in the range of 11.72 µmol/ ml to 124 µmol /ml). About 30% (18 isolates) showed ammonia production of more than 12 µmol /ml.
Hydrogen cyanide production are suggested indirect mechanisms of plant growth promotion. HCN is a volatile product which has an antifungal activity. It was believed that initially HCN production play its role in plant growth promotion by suppressing the plant pathogens 68 . However, this idea has recently been changed. It has been supposed that the production of HCN indirectly increases phosphorus availability by chelation and sequestration of metals, and indirectly increases the nutrient availability to the bacteria and host plants 69 . In our study, from the tested bacterial strains eight were positive for HCN production test. Under this study a highest (0.23) HCN production was recorded by the isolate AAULE (Pseudomonas oursence), while a lowest production was recorded by AAUSR10 (Table 5 & Fig. 6).
Plants and bacteria can interact with one another in a variety of different ways. The interaction may be bene cial, harmful. or neutral for the plant, and sometimes the impact of a bacterium may vary as the soil conditions change 70 . In nature, plants are not isolated organisms, so that during their evolution they have adjusted to interact with diverse communities of symbiotic, bene cial, and pathogenic microorganisms 71 . Plant growth-promoting bacteria are naturally existing in soil and tissue that aggressively colonize plant roots and plant tissue and bene t plants by giving growth promotion. Inoculation of crop plants with certain strains of PGPB at an early stage of development improves biomass production through direct effects on root and shoots growth 72 .
Microorganisms that can strongly in uence plant performance, for example, by modulating nutrient uptake and thereby either enhance or decrease nutrient availability. Although many studies have been studied with the bene cial plant-microbe interaction, relatively few have provided a more thorough description of the effects on the plant by the interaction when it involves PGPB. In this study, we used Arabidopsis as a model system to investigate the effect on phenotypic properties, plant growth regulation, and root development by using four bacterial isolates which provide disease suppression. A positive effect was observed with growth promotion for Arabidopsis after treated with bacterial isolates. After inoculation of bacteria, the growth of primary roots, fresh weight of Arabidopsis, and number of lateral roots were showed increment when compared to control experiment (growth without the cultivation of bacteria) after 14 days of inoculation. The fresh weight of AAUSFE29 (Enterobacter sp.V.H.18) on a bi-partite Petri dish reached an approximately 2-fold increase. This suggests that bacterial metabolites might synthesis of bioactive VOCs and privileged accumulation of carbon dioxide. In the present study also, we evaluated the effect of potential bacterial isolates on Arabidopsis thaliana growth when the bacteria was grown in an appropriate bacterial culture medium, such as nutrient agar medium. When both bacterial strtains were grown on MS medium, major growth and development of the Arabidopsis plants were observed in comparison to the control condition. Particularly, the number of lateral roots was higher in the MS medium of bacterial Arabidopsis interaction experimental condition with both bacterial isolates. Microorganisms produce a broad spectrum of volatile organic compounds with various functions in plants such as defense, growth, variety of root building, and in microbe-plant communication 73 .

Conclusion
Out of sixteen bacterial isolates, four e ciently suppressed the mycelial growth of pathogenic Phytophthora capsici in dual culture assays in vitro. Bacterial isolates with strong anti-fungal potential were found positive for HCN, catalase test, Indole acetic acid (IAA), ammonia and phosphate production. This study shows the taxonomic diversity and existence of antagonistic and plant growth endophytic and rhizosphere bacteria with different physiological and biochemical capabilities, which may give a basis for the isolation of new potent biocontrol agents of oomycete plant pathogens. Furthermore, it appears that pepper plants can be an important source of endophytic and rhizosphere bacteria that induce pepper plant growth promotion and defense against pepper pathogens. Future studies will focus on AAULE51 (unidenti ed) strain regarding development as a commercial biocontrol agent and determination of the structure of antibiotic compounds, and also to con rm the signi cant role of native endophyte and rhizobacteria for the control of soil-borne oomycetes and the possible use of this potential isolates in bio-fertilizers and bio-fungicides production. Additional studies regarding eld applications and bio formulations are in progress. Qualitative estimation of ammonia production by selected bacterial isolates in peptone water using Nesslerization reaction. Different letters above bars indicate a statistically signi cant difference (P < 0.05).

Figure 5
Quantitative assay of HCN produced by bacterial isolates at 600 nm wavelength. Different letters above bars indicate a statistically signi cant difference (P < 0.05) Figure 6 Qualitative estimation of ammonia production Figure 7 Qualitative estimation of HCN production