Plant Growth Promotion and Biocontrol Potentiality of Endophytes Isolated from Root Nodules of Sulla flexuosa L. Plants

Legumes, native to the Mediterranean, harbor reservoirs of endophytes that help plants adapt to various environmental stresses. Te current study was carried out to evaluate the plant growth characteristics and antifungal activity of root nodule endophytes as biocontrol agents and plant growth promoters. Eleven bacterial endophytes isolated from root nodules of Sulla fexuosa L. grown in Northwest Morocco were assessed for their plant growth-promoting (PGP), and antifungal properties. Four endophytic bacteria were selected for their efciency in solubilizing inorganic phosphate. Te selected strains were positive for more than 2 PGP traits, including indole acetic acid, ACC deaminase, siderophore, and ammonia production. Te screening for lytic enzyme production revealed that all strains were capable of producing chitinase, cellulase, catalase, and protease, while the secretion of amylase and urease was not detected. Te HFB11 was the only strain incapable of producing pectinase. In vitro experiments revealed the strains’ potential to withstand salt and drought stresses by being able to grow in high concentrations of NaCl and PEG. Based on 16S rRNA gene sequencing, the strains were identifed as Enterobacter and Serratia . Te antagonistic activity of the strains against Botrytis cinerea , Aspergillus ochraceus , and Fusarium oxysporum was detected and they were shown to inhibit the fungal growth with various percentages. Te highest percentage of inhibition was observed for HFB3 against B. cinerea with 50% inhibition followed by HFB8 which was able to inhibit 47% of F. oxysporum ’s growth. In contrast, a weak inhibition was observed against A. ochraceus . All these fndings indicate that the chosen endophytes, halotolerant Serratia inhibens HFB8 and Enterobacter hormaechei HFB11, might be used as candidates for efective biocontrol and growth promotion of legumes.


Introduction
Te Mediterranean Basin supports a diversifed ecosystem of pasture legumes; however, recent research indicates that the Mediterranean area is particularly sensitive to climate change, with signifcant decreases in overall precipitation and increases in mean annual temperatures, soil mineral defciencies, and/or toxicities [1].Additionally, toxigenic soil fungal pathogens such as Fusarium sp., Aspergillus sp., and Botrytis sp., which are the most virulent fungi prevalent in the Mediterranean ecosystems due to their potential to survive on infested residues, can cause enormous diseases and losses in production cost in agroecosystems [2].Nevertheless, a variety of legume species have adapted to growing in stressful environments, including Hedysarum spp.(Sulla fexuosa L.), a naturally occurring plant in Northern Morocco noted for its high fodder value and capacity to prevent soil erosion due to its deep and robust root structure [3].Te benefts of efcient symbiosis between legumes and nitrogen-fxing bacteria in agricultural output are widely known.Tey can fx substantial amounts of atmospheric nitrogen, allowing them to live in nitrogendepleted soils without N fertilizers, while also maintaining higher nitrogen content in the soil.In addition to rhizobia, a variety of endophytic bacteria belonging to diferent bacterial genera such as Bacillus, Pseudomonas, Enterobacter, Burkholderia, Serratia, and Pantoea have been widely reported to originate from diferent legumes such as alfalfa, soybean, chickpea, and peanut [4][5][6][7][8][9].Microbial endophytes are a special group of soil microorganisms that successfully colonize the root systems of their host plants and could efectively support plant growth [10].Unlike rhizobacteria, endophytes form symbiotic relationships with many plants, fourish within the internal tissues of plants, and use specialized mechanisms to infltrate the host [11].Although the host mostly reaches the rhizosphere soil surrounding the roots, it is, however, important to note that this infltration is not beyond the control of the host plant [12].Numerous reports have highlighted the advantageous efects of endophytic bacteria in promoting plant growth directly and/or indirectly, regardless of the disadvantageous conditions.Endophytic bacteria are benefcial to the host plants due to several factors including, their ability to increase the intake of nutrients such as phosphorus or nitrogen, their production of phytohormones implicated in root growth, such as auxin and cytokinin, their involvement in development and biomass, in addition to inducing resistance to abiotic stresses [13][14][15][16].Tey can also protect plants from pathogens by producing bioactive secondary metabolites, notably lytic enzymes, or by competing for nutrients via siderophores, which chelates the iron present in the soil, rendering it unavailable to pathogenic microorganisms [17].Te search for sustainable alternatives to enhance plant defense mechanisms is tempting.Te use of endophytic bacteria as biocontrol agents ofers an attractive alternative solution for tackling pathogen-related crop losses.Tere is a growing interest in using endophytic bacteria as bioinoculant agents to promote plant health and crop productivity, as they can be employed as biofertilizers, seed treatments, or foliar sprays [18].Tis study, therefore, was carried out to isolate and identify root endophytic bacteria from Sulla fexuosa along with determining their multiple plant growth-promoting traits, tolerance to drought and salt stress, and antagonistic activity.

Sample Collection.
Root nodule samples of Sulla fexuosa plants wildly grown in Boukhalef province of Tangier, Morocco (35 °728240′N, 5 °873565′W), were collected aseptically in sterile cotton swabs and contained in screw cap plastic tubes containing silica gel as described by Vincent [19], all sample tubes were stored at 4 °C before being brought to the laboratory and processed for isolation.Te soil sample analysis at the National Center of Scientifc and Technical Research (CNRST) in Rabat, Morocco, revealed a clay texture with a low level of N, P 2 O 5, and OM and an alkaline pH of 7.9 (Table 1).

Bacterial Strain Isolation.
Healthy nodule samples were collected from the roots of Sulla fexuosa.Te endophytic bacterial strains were isolated from surface-sterilized nodules as described by Vincent [19].To guarantee purity, single colonies were picked and streaked repeatedly on Yeast extract mannitol (YEM) medium containing Congo red.Te isolates were stored at − 20 °C in 25% (v/v) sterile glycerol.Successful surface sterilization of nodules was assessed by inoculating the water of the fnal rinse into YEM medium agar for 7 days at 30 °C.

In Vitro Plant Growth Promoting
Activities.Te isolated strains were tested for PGP attributes in vitro, including their capacity to solubilize tricalcium phosphate, produce hydrogen cyanide (HCN), indole-3-acetic acid (IAA), siderophore, ACC deaminase, and ammonia.

Inorganic Phosphate Solubilization.
Te endophyte bacterial colonies were streaked into Pikovskaya medium (PVK) [20] to test their ability to dissolve Tri-Calcium Phosphate (TCP).Te plates were incubated for 7 days at 28 ± 2 °C, and colonies with a clear halo were considered Phosphate Solubilizing Bacteria (PSB).Te diameters created by the bacterial colonies along with the clear zone were measured and used to determine the Phosphate Solubilization Index (PSI) using the following equation [21]: Te PSBs were inoculated in 50 mL of PVK broth, with uninoculated broth serving as control.Te solubilized phosphorus was calculated using the Ames method [22] and the pH was measured using a pH meter (pH-221 Biobase).

Hydrogen Cyanide (HCN)
Production.HCN secretion was qualitatively analyzed according to the method described by Bakker and Schippers [23].Te tested strains were streaked on a YEM medium containing 4.4 g•L − 1 of glycine.A piece of Whatman flter paper No. 2 was soaked in a 0.5% solution of picric acid and placed on top of the plates.When exposed to HCN gas, the color of the paper changed from orange to brown.

Indole Acetic Acid (IAA) Production. Gordon and
Weber's coulometric approach to investigate tryptophandependent IAA production was adopted [24].Bacterial isolates were grown in Sucrose Minimal Salts (SMS) medium containing 0.05% of tryptophan.After two days of incubation at 28 °C, the culture was centrifuged for three minutes at 13000 rpm. 1 mL supernatant was supplemented with 2 mL of Salkowski reagent.Absorbance of the test 2 International Journal of Agronomy mixture was taken at 535 nm after 20 minutes of incubation at room temperature.
2.3.4.Siderophores Production.Te Schwyn and Neiland assay was followed to determine the synthesis of siderophores by the selected endophytes [25].After incubating the CAS solution and culture bacteria in the dark for 30 min, the siderophore production was measured at 630 nm and computed.Te formula proposed by Pal and Gokarn [26] was adopted to calculate the percentage of siderophores in units: where, Ar � OD of reference (CAS reagent); As � OD of the sample.

ACC Deaminase Production.
Te presence of ACC deaminase was determined by the ability of the selected bacterial strains to use ACC as the sole source of nitrogen.
According to the Jacobson et al. method [27], we compared the growth rates of bacterial strains cultured in the presence of two diferent nitrogen sources: ACC and ammonium sulfate (NH 4 ) 2 SO 4 , as well as a mineral source, magnesium sulfate (MgSO 4 •7H 2 O).After 48 hours, the optical density at 600 nm was measured.Isolates with an OD greater than that of the MgSO 4 •7H 2 O solution were considered positive for ACC production.
2.3.6.Ammonia Production.Te production of ammonia was tested by inoculating 10 6 UFC/mL of one-day-old culture into 10 mL of peptone broth and incubating at 28 °C for 72 hours.After adding 0.5 mL of Nessler's reagent to each tube, a color change from yellow to brown indicated the production of NH 3 [28].

Extracellular Enzyme Production.
Te capacity of the bacterial isolates to degrade cellulose was tested by streaking the inoculant on cellulose, and Congo-Red agar media, as described by Gupta et al. [29].Amylase activity was determined using a soluble starch-yeast extract medium [30,31].Te clear zone in skimmed milk agar was used to determine the protease activity [32].As for the determination of urease activity, the analysis was performed as described by Maheshwari et al. [33].Te pectinase production was determined as described by Cattelan et al. [34], while the appearance of a clear halo around the colonies on the pectin medium was employed as an indication of the presence of pectinolytic activity.Agar medium supplemented with colloidal chitin was used to screen for chitinaseproducing strains, through the detection of clear zones around bacterial colonies on a cream background [35].Te catalase activity was examined as described by Dacre and Sharpe [36].
Total DNA was extracted using the phenol/chloroform method [38] and adjusted to a fnal concentration of 100 ng•µL − 1 using a Nanodrop spectrophotometer (Termo Scientifc ™ NanoDrop 2000).Te obtained sequences were assembled using the sequence alignment editing program Bioedit (7.0.5.3), checked manually, and compared with those from GenBank using the BLAST algorithm (https:// blast.ncbi.nlm.nih.gov/blast.cgi/).Using Clustal W software, the sequence data were visually compared and aligned.Te neighbor-joining method was used to generate phylogenetic trees while the bootstrap analysis used 1000 resamplings.Te Molecular Evolutionary Genetics Analysis (MEGA) was used for all phylogenetic analyses.

Antagonism Activity.
Te antifungal efects of endophytic isolates on the growth of Aspergillus ochraceus and Fusarium oxysporum, isolated and identifed by El Aaraj et al. [39], as well as Botrytis cinerea, isolated from cultured strawberries in Larache, Morocco, were evaluated.Te antifungal activity was tested by a dual culture assay [40].A 5 mm disk of the tested fungal strain was placed in the center of plates containing Potato dextrose agar (PDA) medium, the bacterial cultures were streaked on both sides within a 30 mm distance from the fungal disc.Control experiments (without bacteria) were performed for each fungal strain.Te plates were incubated at 25 °C for 7 days and examined for mycelial growth inhibition.Te inhibitory activity was defned using the following formula [41]:

In Vitro Growth-Promoting Potential of Endophytes.
Eleven endophytic bacteria were isolated from nodules of Hedysarum sp.Four isolates were selected based on their ability to solubilize TCP, which was confrmed by the presence of a clear zone surrounding the colonies on PVK agar medium.Tese isolates were further evaluated for their growth-promoting potential and biocontrol capacities.Te screening results for the PGP characteristics are shown in Table 2. Te selected isolates demonstrated important phosphate-solubilizing ability, with phosphate index solubilization ranging from 2.43 to 3.23.Te quantitative estimation of TCP solubilization by the tested strains in PVK liquid medium was recorded.Two strains, HFB11 and HFB3 recorded the highest concentration needed for P solubilization (79.74 mg•L − 1 ), followed by HFB7 (68.94 mg•L − 1 ), while HFB8 reported the lowest value with a concentration of 52.58 mg•L − 1 .Te results illustrated in Table 2 regarding the available P and pH values were found to be inversely linked (negative correlation).All of the selected endophytes were capable of producing high levels of IAA and siderophores in addition to their phosphate-solubilizing ability, however, no traces of HCN were detected with any of the tested strains.Te amount of IAA produced in the presence of L-tryptophan as a precursor was very low, ranging from 0.27 mg•L − 1 (HFB3) to 1.35 mg•L − 1 (HFB8).As for siderophores production, the percentage ranged from 2.94% (HFB3) to 52.58% (HFB8).Moreover, HFB7, HFB8, and HFB11 were found to produce ACC deaminase upon adding ACC as the sole nitrogen source for bacteria.In terms of ammonia production, HFB3, HFB7, and HFB8 were the three strains that demonstrated a discoloration in peptone water from yellow to brown, therefore indicating the presence of a positive reaction.

Hydrolytic Enzymes.
Regarding enzymatic activities, the screening revealed that all tested strains had the ability to produce chitinase, cellulase, catalase, and protease, but none could produce urease and amylase, while only one strain, HFB11, tested negative for pectinolytic activity (Table 2).

Molecular Identifcation Using 16S rRNA Gene Sequencing of Selected Endophytic Strains.
Te genetic characterization of the selected endophytic bacterial strains based on the nearly complete encoding of the 16S rRNA gene revealed a close relationship to Enterobacteriaceae family, inferring two genera, Enterobacter sp., and Serratia sp., with similarity values ranging from 95% to 100% (Figure 1).According to the phylogenetic tree inferred from the 16S rRNA gene sequences, the four strains isolated from Sulla fexuosa L. HFB3, HFB11, HFB8, and HFB7, were linked to Enterobacter bugandensis 247BMC T , Enterobacter hormaechei subsp.xiangfangensis 10-17 T , Serratia inhibens S40 T , and Serratia liquefaciens ATCC 27592 T respectively.Te sequences of these strains were deposed in NCBI GenBank under the following accession numbers HFB3 (OP316890), HFB11 (OP316893), HFB8 (OP316892) and HFB7 (OP316891).

Halotolerance Assay.
Te capacity of the chosen endophytic bacteria to grow in various concentrations of NaCl and PEG 6000 was investigated (Table 3).A drop in OD in conjunction with the increasing levels of NaCl and PEG was noticed, indicating the presence of various levels of stress tolerance.All tested isolates exhibited a halotolerant profle, with the exception of Enterobacter bugandensis HFB3 (Table 3).Te ability of the strains to adapt to abiotic stresses revealed that three isolates, Serratia grimesii HFB7, Serratia inhibens HFB8, and Enterobacter hormaechei HFB11, were halotolerant with Enterobacter hormaechei HFB11 having the highest drought tolerance.

Antagonistic Activity.
Te in vitro screening revealed that the four strains can sufciently disrupt the radial mycelial growth of F. oxysporum (16.67%-40%) and B. cinerea (35.56%-50%), while the sensitivity of A. ochraceus towards the selected bacteria appeared to be minimal in comparison, with inhibition percentages reported to be less than 20% (Table 4).Compared to other strains, Serratia inhibens HFB8 presented optimum antifungal efcacy against phytopathogens, efectively suppressing F. oxysporum and B. cinerea's growth at a rate of 47% and 45%, respectively (Table 4).

Discussion
In this study, four endophytic bacterial strains were selected from a collection of eleven strains isolated from root nodules of Sulla fexuosa L., a Mediterranean forage plant with substantial ecological and economical signifcance.It is demonstrated that Sulla fexuosa harbors endophytic strains presenting numerous plant growth-promoting and antimicrobial abilities.Te present study examined the inorganic phosphate solubilization, phytohormone IAA, siderophore, ACC deaminase, and ammonia production, in addition to extracellular enzyme secretion in order to select isolates displaying the most promising growth promotion and biocontrol properties of leguminous plants [42,43].Te 16S rRNA sequencing confrmed the identity of these endophytic isolates.Te four endophytic bacterial strains belonged to Enterobacter and Serratia genera, which are common species reported to be isolated from root nodules of various leguminous plants [7,40].Interactions between plants and endophytes have been reported to improve plant nutrition and to have a protective efect on the host plant 4 International Journal of Agronomy  International Journal of Agronomy against several biotic and abiotic stresses, they may boost host plant form by enhancing resistance to herbivores, heat, salt, diseases, and drought, as well as raising roots and leaves biomasses.Tus, endophytes can be considered as excellent host cohabitants [11,41].Phosphorus is considered a macronutrient that is required for multiple enzymatic activities widely implicated in numerous plant physiological processes.Te data obtained in this study regarding endophytic bacteria revealed that the selected strains could boost phosphorus availability for plants through the solubilization of precipitated phosphates.Tey can equally improve soil phosphorus availability through diferent mechanisms, such as acidifcation, ion exchange, chelation, and organic acid synthesis, or by secreting acid phosphatase, which mineralizes organic phosphate [44].Te ability to generate IAA has a signifcant impact on plant growth and development, including root formation and proliferation, which improves water and nutrient uptake [45].Endophytic bacteria that produce IAA, as is the case in this study, may directly boost plant development by increasing root surface area and length by promoting plant cell elongation or infuencing cell division, allowing plants more access to soil nutrients.On the other hand, bacterial strains generating ACC deaminase have been shown to help plants cope with environmental stresses by promoting root and shoot growth and decreasing the inhibitory efects of ethylene secretion.Te preliminary

HFB7 (OP316891)
Serratia liquefaciens ATCC 27592 T (NR 121703.6 International Journal of Agronomy analysis revealed that all endophytic bacterial strains were positive for ACC deaminase activity (Excluding Enterobacter hormaechei HFB11).Although siderophores may not be directly implicated in plant growth, these microbial-derived compounds can provide a source of iron and sustenance which can be used for growth stimulation [46].Furthermore, siderophore-producing bacteria can inhibit the growth of competitive species by restricting the availability of Fe in the environment [47,48].Te selected endophytes exhibited a capacity to produce a signifcant amount of siderophores that could be involved in boosting and protecting plants under stressed conditions.Numerous studies have reported the efcacy of plant growth-promoting endophytic bacteria to alleviate drought and salt stresses in plants [45,46].According to Kang et al. [49], inoculating alfalfa plants with drought-resistant Enterobacter ludwigii AFFR02 and Bacillus megaterium MJ1212 minimized the detrimental infuences of drought stress, and increased plant growth and biomass content.Te results obtained for the halotolerance assay indicated that the selected endophytic bacteria were able to grow in a NaCL concentration as high as 10% which is considered to be a higher tolerance rate than the one previously reported by Patel and Parekh [50]  In regards to the strain's antagonistic efect, the endophytes Serratia HFB8 and Enterobacter HFB11 demonstrated strong antifungal activity in the current investigation, inhibiting over 40% of Fusarium sp. and Botrytis sp.'s mycelial growth.Tese fndings contribute to the knowledge surrounding the well documented potential of strains such as Bacillus, Pseudomonas, Streptomyces, Enterobacter, and Serratia species as potential biocontrol agents against various plant pathogenic fungi [52].Te current study showed that bacterial strains with high biocontrol potential, Serratia inhibens HFB8 and Enterobacter hormaechei HFB11 are mutually capable of producing siderophores and cell walldegrading enzymes, namely chitinases, cellulases, proteases, and catalases.Te ability of Serratia sp., in particular, to produce its own chitinase has attracted the most attention in terms of its potential for biocontrol.In fact, it has been recognized by several studies as a biocontrol agent against F. oxysporum, B. cinerea, and Rhizoctonia solani [49,50], which is in agreement with our fndings.Further studies have been conducted that focused on the efect of B. subtilis on the reduction of the infection rate of F. solani, a phytopathogen widely responsible for root rot and boosting plant growth under salt stress, through an in vitro antagonistic screening of endophytic strains against fungal isolates from chickpea roots [53].Hydrolytic enzymes could hydrolyze a variety of polymeric materials, including cellulose, hemicelluloses, chitin, proteins, and nucleic acid.
Te secretion of these enzymes may also improve the inhibitory activity against plant pathogens by dissolving their cell walls.Bacterial endophytes producing lytic enzymes are used as biocontrol agents to combat fungal and bacterial infections, as well as plant-parasitic nematodes [52,54].Overall, the antifungal capabilities of the studied potent endophytes could be accredited to a combination of factors such as competition for nutrients and space, synthesis of secondary metabolites, and production of lytic enzymes.
Te current study demonstrated that Serratia inhibens HFB8 and Enterobacter hormaechei HFB11 both possess direct PGP activities such as inorganic phosphate solubilization, IAA secretion, siderophores production, and ACC deaminase production, as well as indirect PGP activities such as protease, cellulose, chitinase, catalase, pectinase, and antifungal activity.Te utilization of advantageous soil bacteria to control plant diseases, a type of biological control, falls within the scope of green strategies.Similarly to our fndings, Serratia and Enterobacter spp.are the most frequently reported for their biocontrol activity against phytopathogens [53,55].Tese PGPR Endophytic isolates have shown important properties that signifcantly promote plant growth, and therefore have proven their efcacy for potential application to alleviate abiotic and biotic stresses in plants, their application can supplementarily provide an ecofriendly and valuable alternative to chemical fertilizers.Since they are indigenous and competent in the rhizosphere, these PGP endophytes (HFB8 and HFB11) provide benefts to the host plant through interaction and metabolism with a high level of antagonistic potential against a variety of fungal phytopathogens.Tus, it could be proposed as a friendly microbiological agent without harming the environment.

Conclusion
Te present study shows that root nodules of the Mediterranean native legume Sulla fexuosa harbors endophytic bacteria presenting at least two plant growth-promoting traits along with antimicrobial activity.Te endophytes Serratia inhibens HFB8 and Enterobacter hormaechei HFB3 displayed a variety of plant growth-promoting properties which makes them strong contenders for bioinoculant production, thereby improving the health of the soil in an eco-friendly manner, all while increasing the population of Sulla plants, and efectively preventing them from becoming endangered species.However, further research into their PGP and biocontrol nature in the presence of plants is required to fully explore their biotechnological potential.

1 ) 1 )Figure 1 :
Figure 1: Phylogenetic tree of selected endophytes (denoted in bold) and their phylogenetically related species based on 16S rRNA sequences.Scale bar represents 0.01 substitutions per nucleotide position.Te tree is rooted on Actinobacillus lignieresii NCTC 4189 T .

Table 1 :
Physical and chemical soil analysis.

Table 2 :
Plant-growth promoting and enzymatic activities of the selected endophytes.

Table 3 :
Growth of endophytes under nonstressed (control), salinity and drought stressed conditions of diferent NaCl and PEG 6000 concentrations at OD 600 nm.

Table 4 :
Percentage of inhibitions of the endophytic bacteria studied on diferent phytopathogenic fungi.Te data presented are the mean of 3 replicates.Means in the same column followed by the same letter are not signifcantly diferent p < 0.05 (Fisher's least signifcant diference (LSD) test).