Combined interaction between the diazotrophic Niallia circulans strain YRNF1 and arbuscular mycorrhizal fungi in promoting growth of eggplant and mitigating root rot stress caused by Rhizoctonia solani

Summary. Rhizoctonia root rot of eggplant, caused by Rhizoctonia solani , is an economically important disease. Niallia circulans YRNF1 and arbuscular mycorrhizal fungi (AMF) were assessed for their biocontrol and biofertilizing effects against R. solani , as potential replacements for synthetic fungicides and fertilizers. The diazotrophic N. circulans YRNF1, isolated from soil, reduced in vitro growth of R. solani by 42%. GC-MS analysis of culture filtrate of N. circulans YRNF1 detected bioactive compounds, including butyric acid (85%) and ethylene glycol (8%). In greenhouse experiments, combined application of N. circulans YRNF1 and AMF reduced the severity of egg-plant root rot by 26%. This combined treatment triggered the transcriptional expression of five resistance genes ( JERF3, PAL1, C3H , CHI2 , and HQT ) in the treated egg-plants. Biochemical analyses of the infected eggplant roots treated with the combined bio-inoculants showed enhancement of the phenol content (+188%), and increased antioxidant enzyme activity, mainly of POD (+104%) and PPO (+72%). Combined application of N. circulans YRNF1 and AMF also promoted eggplant growth and improved the total NPK concentrations in treated plant leaves. Inoculation of eggplant with N. circulans YRNF1 in the presence of AMR increased the mycorrhization level. This is the first report of N. circulans and AMF as potential agents for biological con-trol of Rhizoctonia root rot and growth promotion of eggplant.


INTRODUCTION
Solanum melongena L. (eggplant) is the most important member of Solanaceae (Kaniyassery et al., 2022).China is the greatest eggplant producer and Egypt is the 3rd largest producer, recording annual production of 1,396,725 tons (FAOSTAT, 2024).Rhizoctonia root rot of eggplant is caused by Rhizoctonia solani Kühn (Almammory and Matloob, 2019).The soil-borne pathogen causes diseases of several hosts, including root rots, damping-off, leaf spots, and leaf blights, which result in a significant reductions in crop yields (Rashad et al., 2018).
Effective control of R. solani is difficult, due to its wide host range (Cook et al., 2002).The most widely used control method is application of synthetic fungicides, but these can have deleterious effects on human and animal health, may induce fungicide resistance in pathogens (Hollomon, 2015), and lead to environmental pollution (Baite et al., 2021).Biological control of plant diseases has received increasing attention as a possible effective, eco-friendly and safe control strategy against various plant diseases (Al- Askar et al., 2014).
Nitrogen-fixing bacteria convert atmospheric nitrogen to ammonia, which can be absorbed by plants.These bacteria can promote plant growth, facilitate nutrient uptake and phosphorous solubilization, and produce siderophores and phytohormones (Shameem et al., 2023).In addition, Bacillus circulans CB7 Jordan, 1890 (now known as Niallia circulans), a non-symbiotic nitrogenfixing bacterium, with plant growth-promoting properties due to auxin production, P-solubilization and siderophore production, has also shown antagonistic effects against Dematophora necatrix (Mehta et al., 2015).
Triggering immunity-related genes in plants is an important mode of action of biocontrol agents.Jasmonic acid and ethylene-response factor 3 gene (JERF3), the responsive gene that manages several defence-response genes through the jasmonate/ethylene signalling path-way, is elicited by abiotic and biotic plant stresses (Rashad et al., 2022b).
Polyphenols have potential as natural antioxidants, which can protect the living organisms from deleterious effects of the reactive oxygen species (ROS) (Elshafie et al., 2023).These compounds have antioxidant activity, as well as antihypertensive, antimicrobial, and antiviral activity (Losada-Barreiro et al., 2022).Excessive production of ROS causes oxidative stress in humans, leading to the development of several ailments (Forman, and Zhang, 2021).Antioxidant polyphenols have several modes of action, including reductive ability to neutralize ROS, chelation of metal ions that elicit the oxidative stress, inhibition of enzymes involved in the formation of ROS, and activation of the antioxidant enzymes (Dias et al., 2021).Upon infection of plants by the microbial pathogens, their cell walls accumulate large amounts of lignin (Rashad et al., 2020a).Increased lignification is a main barrier against the pathogen spread, and reduces the infiltration of toxins and fungal enzymes into plant cell walls.Lignin compounds also cause fungal pathogens to lose abilities to infect host plants, and prevent pathogen movement and multiplication (Ma et al., 2017).Flavonoids are plant responsive metabolites that contribute to host resistance in response to various abiotic and biotic stresses.These compounds act as physical or chemical barriers to prevent the microbial invasion, and are toxic defences against the microbial pathogens and/ or insects.They interfere with pathogen cellular processes and structures (Ramaroson et al., 2022).The phenylalanine ammonia lyase 1 gene (PAL1) encodes phenylalanine ammonia lyase, which is involved in biosynthesis of polyphenolic compounds that have roles in host plant systemic resistance (Rashad et al., 2020a).During the early stages of lignin biosynthesis, the 4-coumarate 3-hydroxylase gene (C3H) catalyzes conversion of 4-coumarate to caffeate (Shrestha et al., 2022).The Chalcone isomerase 2 gene (CHI2) encodes for chalcone flavonone isomerase that catalyses the first two steps of flavonoid biosynthesis (Chao et al., 2021).Niggeweg et al. (2004) reported that the Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase gene (HQT) catalyzes biosynthesis of chlorogenic acid from caffeoyl-CoA and quinic acid.
The present study had the following objectives: 1) to investigate in vitro suppressive potential of the diazotrophic bacterium N. circulans YRNF1 against R. solani; 2) to assess biocontrol effects of a combined treatment with N. circulans YRNF1 and AMF on Rhizoctonia root rot of eggplant, 3) elucidate the host defensive mechanisms elicited by these combined bioagents, based on the transcription of some responsive genes, phenolic com-pound content, and antioxidant activity; and 4) assess effects of combined bioagents on eggplant development.

Fungi and eggplant cultivar
The pathogenic fungus R. solani (AG-2-2 IIIB) was provided from Mansoura University, Egypt.To prepare the inoculum, a 500 ml glass flask containing 100 g of sterilized oat grains mixed with sand (1:2 v:v) was aseptically inoculated with five discs (6 mm diam.)cut from a 5-d-old culture of R. solani, incubated at 28±2°C, and was shaken daily for two weeks to ensure uniform growth of the fungus (Youssef et al., 2016) (in an equal ratio).AMF were propagated by inoculation of sterilized sandy-clay soil with 10 g of grain of the AMF inoculum (approx.50 AM spores and root pieces g -1 soil).The inoculum was applied as a grain bed before planting grains of maize (potential host) in a sterilized plastic pot under the greenhouse conditions (27/22°C, 75% relative humidity, and 16 h daily light period).No fertilizers were added and irrigation was regularly applied to 50% field capacity.After 3 months, the AMF colonized roots were cut using a sterile scalpel into small segments, and were mixed with the AMF spores in the maize rhizosphere soil to be used as AMF inoculum (approx.45 AM spores and mycorrhizal root pieces g -1 soil) (Nafady et al., 2019).Seeds of the eggplant cultivar 'Rondona' were used, which were provided by the ARC, Giza, Egypt.

Collection of soil samples
Twenty-one soil samples were collected from several cultivated fields in Alexandria and El-Beheira governorates, Egypt, and were immediately transported to the laboratory.These samples were stored for subsequent studies at 4°C.

Isolation of the diazotrophic bacteria
Approximately 10 g of each soil sample were added aseptically to 90 mL of sterilized water in an Erlenmey-er flask, vigorously mixed for 45 min at 150 rpm, and then serial dilutions were prepared (10 -1 to 10 -4 ).Diazotrophic bacteria were isolated from the soil according to Döbereiner (1988) with slight modifications.Plates of nitrogen free (NF) agar medium, containing glucose (20 g L -1 ); K 2 HPO 4 (0.2 g L -1 ), NaCl (0.2 g L -1 ), MgSO 4 .7H 2 O (0.2 g L -1 ), K 2 SO 4 (0.1 g L -1 ), CaCO 3 (5 g L -1 ), and agar (20 g L -1 ), were individually inoculated with 0.1 mL of each serial dilution of soil suspension; Three replicated plates were used for each dilution.The plates were then incubated for 5 d at 28±2°C.Growing bacterial colonies were then singly transferred onto fresh NF plates and incubated at the same conditions.The resulting cultures were stored in 15% glycerol at -80°C until used (Kifle and Laing, 2016).

Screening of the diazotrophic bacteria for antifungal potential against R. solani
The ability of the isolated diazotrophic bacteria to antagonize R. solani was evaluated using the in vitro dual plate method (Jasim et al., 2016) on potato dextrose agar plates (PDA, Difco).In each plate, 10 μL of each bacterial isolate (10 6 cells mL -1 ) were streaked aseptically as a longitudinal line 2 cm away from the border of the plate.A disc (6 mm diam.)cut from a freshly grown R. solani culture was placed 20 mm from the opposite side of the plate.Plates inoculated only with pathogen discs served as experimental controls.Each treatment was applied in six replicates, and the assay was repeated twice.After incubating the plates for 4 d at 25°C, and upon complete coverage of control plates with fungal growth, inhibition of fungal growth in the dual plates was measured using a calibrated ruler, and compared with the fungal diameter in the corresponding control plate.Suppression of the fungal growth (S %) was determined using the equation of Ferreira et al. (1991): where C = radial growth in the control plate, and T = radial growth in the treated plate.

Biochemical analyses of bacterial culture filtrate using gas chromatography-mass spectrometry (GC-MS)
Secondary metabolites in cell free culture filtrate of N. circulans YRNF1 were identified qualitatively using GC-MS.The culture filtrate of N. circulans YRNF1 was obtained by inoculating 200 µL of the bacterial suspension in sterile distilled water (6 × 10 6 cfu mL -1 ) into a 500 mL capacity Erlenmeyer flask containing 250 mL of nutrient broth (NB) [1% (w/v) each of beef extract and peptone, and 5% (w/v) NaCl].The culture was incubated under shaking at 120 rpm at 28°C (Nisa et al., 2019).After 3 d, the NB was centrifuged for 10 min.at 4°C at 11,200 g (Thermo Fisher Scientific).The collected cellfree supernatant was filter sterilized using 0.22 µm filters (Millex-GS, Millipore).The cell-free culture filtrate was lyophilized to complete dryness, re-suspended in methoxyamine hydrochloride (50 µL) dissolved in pyridine, followed by incubation for 90 min.For derivatization of the sample, 50 µL of silylation reagent [bis(trimethylsilyl) trifluoroacetamide + trimethylchloro-silane, 99:1 v:v] were added to the sample.The GC-MS system (GCMS-QP2010 Plus, Shimadzu), equipped with a gas chromatograph (7890B) and a mass spectrometer detector (5977A), was used to analyze the sample, at the Central Laboratories Network, NRC, Giza, Egypt.This system involved an HP-5MS column (30 m × 0.25 mm × 0.25 μm).Hydrogen was used as the carrier gas at the flow rate of 2.0 mL min -1 .In this assay, 1 µL was used as an injection volume according to several processing conditions, including 50°C for 5 min, which rose at 10°C min -1 to 100°C, and then at 20°C min -1 to 320°C.Mass spectra were obtained by electron ionization (EI) at 70 eV, involving a spectral range of m/z 50-700, and a solvent delay of 4 min, where 230°C was the mass temperature used and the Quad was at 150°C.Biochemical characterization of the different bacterial filtrate constituents was obtained by comparing the spectrum fragmentation pattern with that stored in the data of Wiley and the National Institute of Standards and Technology (NIST) Mass Spectral Library.

Detection of the nitrogenase gene (nifH)
Presence of the nitrogenase (nifH) gene was detected in the isolated diazotrophic bacteria according to Tan et al. (2009), as follows: DNA extraction.A 2-d culture of each bacterial isolate grown on NF medium was centrifuged for 2 min at 1792 g.The resulting pellet was re-suspended in sterilized water (100 mL) and re-centrifuged for an additional 2 min at 1792 g, followed by heating in a water bath (90°C) for 10 min.After re-centrifugation, the supernatant was added to a sterile tube (0.5 mL capacity) and used as a DNA template.The resulting crude DNA concentration was estimated at using a UV spectrophotometer at OD 260 and OD 280 (PERSEE).
Molecular identification of the selected bacterium using 16S rRNA gene DNA of the promising nitrogen-fixing bacterial isolate was subjected to amplification of the 16S-rRNA region, using the primer 16S-27F: 5'-AGAGTTTGATC-MTGGCTCAG-3' and 16S-1492R: 5'-CGGTTACCTT- GTTACGACTT-3' (dos Santos et al., 2019).The PCR mixture was subjected to the Exosap-IT (GE Healthcare) PCR clean up protocol.The 16S rRNA gene nucleotide sequence was determined through Sanger sequencing using the DNA Analyzer of Applied Biosystems 3730 × l.Using the Big Dye Terminator from ABI; the two primers 27b F and 1492uR were used for setting up the PCR reactions (dos Santos et al., 2019).The Vector NTI software (Invitrogen) was used to align the sequences from the forward and reverse primers, while the contigs were subjected to BLAST to search for nucleotide similarity (Zhang et al., 2000).The maximum likelihood method through MEGA X software (10.2.4) was used to generate a phylogenetic tree of the selected isolate.

Greenhouse experiment
Eggplant seeds were surface sterilized (using 0.05% sodium hypochlorite) and planted (one per pot) into 20 cm diam.plastic pots containing sterilized clay soil.The soil physical composition was: silt (35 ± 0.13 g kg -1 ), sand (110.2 ± 0.21 g kg -1 ), and clay (453 ± 0.18 g kg -1 ).The soil chemical properties were: pH = 7.58, EC, 1.32 dS m -1 , available P (26 mg kg -1 ), total N (2.84 g kg -1 ), available K (310 mg kg -1 ), organic matter (1.72 g kg -1 ), total organic carbon (0.99 g kg -1 ), and total CaCO 3 (5.9%).For AMF colonization, an AMF inoculum was added to each pot under the seeds (10 g seed -1 of the AMF inoculum).Infestation of the soil was carried out by mixing the upper layer with the R. solani inoculum (3% w/w), and the pots were watered daily for 10 d before planting.For preparation of bacterial inoculum, the diazotrophic bacterium N. circulans strain YRNF1 was grown in NF broth on a rotary shaker for 3 d.The resulting bacterial suspension (adjusted at 10 8 cell mL -1 ) was mixed with 1% Arabic gum.For application of N. circulans YRNF1, eggplant seeds were soaked for 30 min before planting in freshly prepared bacterial inoculum.Three hours before planting, the seeds in control pots were treated with the fludioxonil (50.0%) fungicide at 3.5 mL kg -1 as a positive fungicide control.The negative control involved a set of pots that was untreated.Nine treatments were applied in this experiment.These were: No fertilization was applied in this experiment.Five replicates were used, and the pots were arranged in a randomized complete block design.They were maintained at 70% relative humidity, in a 27°C day 17°C night temperature regime in a greenhouse, and were irrigated with tap water when necessary.This experiment was repeated twice

Assessment of the disease severity
Five plants from each treatment were carefully uprooted, and the adhering soil was removed with tap water.The disease severity was evaluated according to Wen et al. (2005).This scale included six severity categories: 0 = no necrosis; 1 = small root necroses (2.5 mm length); 2 = necrosis (2.5-5 mm); 3 = necrosis ≥ 5 mm; 4 = crown and shoots covered with necrotic lesions; or 5 = seedlings damped-off.The disease severity (DS%) was estimated according to Taheri and Tarighi (2010): where n1 was the number of plants that had severity category 1; n2 was number of plants that had level 2; etc., and N was the total number of evaluated plants.

Quantifying the expression of the defense-related genes using qPCR
Transcription of some responsive genes in eggplant roots was quantified at 14 d post planting (dpp).The studied genes included JERF3, PAL1, C3H, CHI2, and HQT.α-Tubulin and β-actin were used as reference genes, based on their stability in the mycorrhizal plants (Fuentes et al., 2016).The primer sequences used are shown in Table 1.The RNeasy Kit (Qiagen) was used for extraction of total RNA, in accordance with the manufacturer's instructions.A SureCycler 8800 (Agilent, USA) was used to synthesize the cDNA.The total volume (20 μL) of the reaction mixture was composed of 3.5 μL RNase-free H 2 O; 3 μL 5×-buffer; 3 μL RNA (30 ng), 3 μL of dNTPs (10 mM), 7 μL of dT primer (5 pmol μL -1 ), and 0.5 μL of the RT enzyme.
The reaction was carried out using the RotorGen 6000 (Qiagen) real-time system The qPCR was run for 1 h at 43°C, then 10 min at 71°C.The qPCR mixture consisted of 3 μL cDNA, 1.6 μL sterile water, 1.5 μL of each Table 1.Primer sequences of the defense-related genes used in this study (Rashad et al., 2020b).

Primer name
Abbreviation (5'-3') Jasmonate and ethylene-responsive factor 3 primer, and 12.4 μL 2× SYBR® Green Mix.The qPCR reaction was run with one cycle at 95°C (for 3 min), 45 cycles each of 95°C for 15 s and 56°C for 35 s, then 75°C for 250 s.The comparative CT method (2 −ΔΔCT ) was used to estimate the transcriptional expression of the tested genes, in accordance with Schmittgen and Livak (2008).
Triplicates of each treatment (biological and technical) were used, and the assay was conducted twice.

Determination of the biochemical plant defense responses
At 30 dpp, samples from the eggplant roots were collected from each treatment.Estimation of the total phenolics, and activities of peroxidase (POD) and polyphenol oxidase (PPO) were assessed (five replicates).The Folin-Ciocalteu test was used to determine the total phenolic content (Singleton et al., 1999).Approximately 1 g of roots was ground in 5 mL of 80% methanol, at 5°C overnight.This homogenate was then centrifuged at 1344 g for 10 min.The supernatant (100 μL) was then mixed with 20% Na 2 CO 3 (50 μL), 1750 μL of dH 2 O, and 250 μL of Folin-Ciocalteu reagent (Sigma-Aldrich), and the resulting mixture was left at 40°C for 35 min.Caffeic acid was used as a reference.Phenolic contents were estimated using spectrophotometry at 760 nm.For POD activity determination, approx. 1 g of the root was ground in 3 mL of 0.1 M Na 2 HPO 4 buffer, and the homogenate was centrifuged for 10 min at 1792 g.POD enzyme activity was estimated spectrophotometrically at 470 nm, which was represented as ∆A 470 min -1 g -1 f wt (Gong et al., 2001).Estimation of the PPO activity was carried out according to Singh and Ravindranath (1994).Five g of root tissue were homogenized and kept for 35 min in acetone at 4°C.PPO enzyme potential was evaluated spectrophotometrically at 420 nm using catechin as the substrate, and was recorded as ∆A 420 min -1 g -1 f wt.

Estimation of levels of plant mycorrhization
Mycorrhizal colonization in eggplant roots was estimated 45 dpp according to Trouvelot et al. (1986).Plant roots were fragmented (10 mm diam.) using a scalpel and were boiled in 10% potassium hydroxide solution.The treated segments were stained using 0.05% trypanblue, as described by Phillips and Hayman (1970).For each root treatment, approx.50 stained fragments were checked under a light microscope.Three mycorrhization parameters were assessed, including frequency of mycorrhizal colonization, colonization intensity, and frequency of formation of arbuscules (Trouvelot et al., 1986).Esti-mation of mycorrhization was repeated twice, each time on 50 stained fragments.

Evaluation of the eggplant growth parameters
At 45 dpp, approx.five replicate eggplant were carefully uprooted.Adhering soil particles were removed and plant growth was estimated using the following parameters: shoot height, root length, shoot and root dry weights, and leaf area.For the evaluation of the dry weights, the plants were dried at 80°C in an oven (3 d).This assay was conducted twice.

Influence of bioagents on macronutrient contents of eggplant leaves
For quantitative analysis of the nutrients content in eggplant leaves, approx.ten leaves from each treatment were collected, and then air dried.The leaves were fragmented using a grinding machine, and then used for estimation of the total contents of nitrogen (N), phosphorus (P), and potassium (K).Using Kjeldhal assays, N was estimated by titration following distillation (Goyal et al., 2022).Total P was evaluated as per Singh et al. (2022).Total K was determined based on Goyal et al. (2022).Determinations were each carried out twice.

Statistical analyses
The results were statistically analyzed using CoStat software 6.4.Data were checked for normality before applying the analyses of variance.Treatment means were compared using Tukey's HSD test (P ≤ 0.05).

Antagonistic potential of the isolated diazotrophic bacteria against R. solani
Eight isolates of diazotrophic bacteria were recovered from the collected soil samples.Results of the in vitro antifungal assays against R. solani showed that inhibition of R. solani mycelial growth varied among the eight isolates ranging from no inhibition to medium inhibition.The greatest level of growth inhibition (42% growth, compared to control plates) was observed with the isolate designated as YRNF (Figure 1, a and b).Growth of the R. solani colony was suppressed and the colony had an arc-shaped border with the diazotrophic bacterium YRNF1, with a clear no-growth zone in between the fungus and bacterium, indicating release of antifungal metabolites by the bacterium, and an antibiosis-based mechanism of antifungal activity (Figure 1).

Detection of the nitrogenase gene (nifH) in the diazotrophic bacteria
Results obtained from PCR analyses showed that the selected isolate YRNF1 possessed the nifH nitrogenase gene, which was observed as a single band at 450 bp (Figure 3).This indicated that the isolate was a nitrogen fixing bacterium.

Molecular identification of the diazotrophic bacterium YRNF1
Results of BLAST analysis of the 16S rRNA sequence showed that the diazotrophic bacterial strain YRNF1 had 99.71% similarity with N. circulans (reference strain MH130347).The nucleotide sequence of N. circulans YRNF1 was deposited in the GenBank under accession number OP703372.The phylogenetic analysis of N. circulans YRNF1 in comparison to ten species of the genus Bacillus (Figure 4

Disease severity
Mean severity of Rhizoctonia root rot in the infected, non-treated eggplant plants was 69%, which was the greatest of the nine treatments.Control plants with either one of the two bio-inoculants had no disease.Moderate disease severity was observed for the infected plants inoculated with N. circulans YRNF1 (45%), and 32% for that inoculated with AMF.Application of both bio-inoculants reduced disease severity by 26% compared to the non-treated infected plants.This indicated additive activity of these two bio-inoculants.Treatment with fludioxonil decreased the disease severity by 25% (Figure 5).

Triggering of the resistance-related genes in eggplant roots
Transcriptional expression of the resistance genes JERF3, PAL1, C3H, CHI2, and HQT in eggplant roots inoculated with N. circulans YRNF1 and colonized with AMF is shown in Figure 6.Compared to the control treatment, all of the experimental treatments elicited expression of JERF3.Combined application of N. circulans YRNF1 and AMF up-regulated the JERF3 gene more than the single treatments.The greatest gene expression (29.4-fold) was for infected eggplant roots after inoculation with N. circulans YRNF1 and colonization by the AMF.Both applied bioagents increased the expression of PAL1, compared to the control plants.Greatest gene expression (17-fold) was for infected eggplant roots inoculated with N. circulans YRNF1 and colonized with the AMF.This was the same for C3H, where all the treatments induced expression of this gene, compared to the control treatment.Overexpression (8.6fold) was detected for infected eggplant roots inoculated with N. circulans YRNF1 and colonized with the AMF.The applied treatments up-regulated expression of CHI2   at varying levels compared to the untreated non-infected eggplants, with greatest expression of this gene recorded value showing a 15.9-fold increase.Application of all the treatments led to overexpression of HQT, compared to the control plants.The infected eggplant roots inoculated with N. circulans YRNF1 and colonized with the AMF had maximum expression of 11.7-fold.

Effects of bioagents on phenol levels and antioxidant enzyme activities in treated eggplant roots
Eggplants challenged with N. circulans YRNF1 and AMF and inoculated with R. solani had increased level in total phenol contents, and increased POD and PPO enzyme potential (Table 3).At 30 dpp, mean phenolic content increased in the infected, non-treated eggplant roots, to 673.2 mg g -1 f wt compared to the control plants (415.7 mg g -1 f wt).Phenolic content of the noninfected eggplant roots treated with N. circulans YRNF1 was 579.0 mg g -1 f wt, and for roots treated with AMF was 594.4 mg g -1 f wt.Combination of both bio-inoculants increased phenolic content of the non-infected eggplant roots to 763.3 mg g -1 f wt.Treatment of the infected eggplant roots with a combination of both bioagents resulted in increased phenolic content up to 1197.4 mg g -1 f wt.Application of fludioxonil increased phenolic content to 668.2 mg g -1 f wt.
Compared to the control plants, POD enzyme activity increased in the infected non-treated eggplant roots to 1.53 ∆A 470 min -1 g -1 f wt, and for PPO activity increased to 1.34 ∆A 420 min -1 g -1 f wt.Enhancements in POD and PPO potential were recorded after treatments of plants with the two bio-agents, either singly or in combination.Greatest activity of both enzymes was recorded in the infected eggplant roots treated with N. circulans YRNF1 (2.32 ∆A 470 min -1 g -1 f wt) and AMF (1.86 ∆A 420 min -1 g -1 f wt).Compared to R. solaniinoculated non-treated plants, activity of both enzymes increased after treatment of infected roots with both bio-inoculants.However, the increases in enzyme activity attributable to the combined treatment (B+M+P) was higher than each single treatment.Application of the fungicide resulted in a recognizable increase in the POD and PPO activity, compared to the control plants.

Mycorrhization of eggplants after treatment with Niallia circulans YRNF1
Effects of N. circulans YRNF1 on mycorrhizal colonization of eggplant roots infected with R. solani are summarized in Table 4. Eggplant roots un-treated with AMF did not develop mycorrhizal colonization.In contrast, all eggplant roots treated with AMF showed varying levels of mycorrhization.Greatest colonization parameters were recorded for roots treated with N. circulans YRNF1 and AMF, which gave means of 88.7% colonization frequency, 56.1% colonization intensity, and 39.7% arbuscule formation frequency, while the eggplants colonized by AMF only had 76.3% colonization frequency, 45.3% colonization intensity, and 27.4% arbuscule formation frequency.These results indicated the compatibility between the two potential bio-agents.However, R. solani inoculation of the mycorrhizal eggplants reduced the mycorrhization levels, compared to the mycorrhizal eggplants not inoculated by the pathogen.This was also the case for the mycorrhizal-infected roots treated with N. circulans YRNF1.
Eggplants growth in response to application of N. circulans

YRNF1 and AMF
Results from treatments of R. solani inoculated eggplants with N. circulans YRNF1 and AMF showed increases in most of the measured plant growth parameters (Table 5).Compared to experimental controls, the R. solani-inoculated had 20.5% reduction in mean shoot height and 26.3% reduction in mean root length.Eggplant roots colonized by AMF had 54.6% greater shoot height and 54.7% greater root length.Treating the non-R.solani inoculated plants with N. circulans YRNF1 and AMF increased shoot height by 71.4% and root length by 41.8%.Treatment of the R. solani inoculated eggplants with both bio-inoculants, increased shoot height by 53.7% and root length 66.5%.Treatment with fludioxonil also increased shoot height and root length of the R. solaniinoculated eggplants.Compared to controls, inoculation of the plants reduced shoot dry weight by 41.0% and root dry weight by 60.0%.Treatment of the non-infected eggplants with N. circulans YRNF1 and AMF increased shoot dry weight 79.5 and root dry weight by 130%.Application of both bio-agents to infected plants increased shoot dry weight by 51.3% and root dry weight by 90%.Treatment of infected plants with fludioxonil also increased shoot and root dry weights.Inoculation of plants with R. solani also reduced mean leaf area by 34.3%.Application of both bio-inoculants increased the leaf area by 115.7%.Combined treatment of the infected eggplants with both bioagents increased mean leaf area by 85.8%.Treatment of the infected eggplants with fludioxonil increased leaf area, compared to the untreated infected plants.

Influence of Niallia circulans YRNF1 and AMF on the macronutrient contents in the eggplant leaves
Total nitrogen (TN) contents in eggplant leaves varied across the different treatments (Table 6).Compared to experimental controls, all the treatments caused increased TN.Greatest mean TN increases resulted for R. solani-inoculated plants colonized by AMF (3.9%), inoculated with N. circulans YRNF1 (3.6%) or treated with fludioxonil (3.6%).Greatest total phosphorus (TP: 2.55%) was measured in non-inoculated plants colonized with AMF, and in R. solani-inoculated plants colonized with AMF (TP = 2.26%).Most of the applied treatments also increased total potassium (TK) contents in the eggplant leaves, compared to control.Measured N/P ratios were increased in the R. solani inoculated plants either treated with the fludioxonil (mean N/P ratio = 4.29), inoculated with N. circulans YRNF1 (N/P = 3.86), or colonized with AMF (N/P = 3.85).

DISCUSSION
Rhizoctonia solani is a damaging pathogen that infects several economically important host plants.In this study, isolation of diazotrophic bacteria from the soil samples resulted in the recovery of eight bacterial isolates.Results of in vitro antagonism assays of these isolates against R. solani showed that one strain YRNF1, molecularly identified as N. circulans, considerably inhibited R. solani growth in culture.This was probably because of antifungal compounds production (antibiosis) by N. circulans.Analysis of N. circulans YRNF1 culture filtrate using GC-MS indicated occurrence of 19 metabolites.Butyric acid and ethylene glycol were the major components of the culture filtrate.Previous studies have reported antifungal activity of butyric acid, which is a short chain fatty acid produced by bacteria (e.g.Lactobacilli spp.), and this compound is widely used as a bio-preservative in dairy products due to its high antifungal activity (Garnier et al., 2020).Ethylene glycol is widely used as an effective antifungal component in the hand sanitizers (Vuai et al., 2022).The in vitro antifungal potential of N. circulans YRNF1 may be due to additive actions of butyric acid and ethylene glycol.It is possible that ethylene glycol, thanks to its amphiphilic nature, facilitates contact between butyric acid, which is a hydrophobic and antifungal compound, and R. solani cells, whose contents are hydrophilic (Abouloifa et al., 2022).
Nitrogen fixation is an important biological process as nitrogen (N) is a limiting nutrient for crop growth.The nitrogen fixing ability of N. circulans YRNF1 was confirmed by the presence of nifH gene in its DNA, which provides marker for N fixation ability (Young, 1992).Nitrogen fixation by N. circulans YRNF1 is probably the main mechanism of the observed promotion of the eggplant growth, as shown by the increases of NPK contents in leaves of the treated plants.Nitrogen is the most important element in plant nutrition, and nitrogen nutrition via nitrogen fixation increases plant vegetative vigor.Phosphorus and potassium are also vital for plant biochemical processes, such as photosynthesis and for the nitrogen fixation process itself (Abdelraouf et al., 2020).Potassium has an important function in activating enzymes including nitrogenase (Rashad et al., 2023).Coskun et al. (2017) highlighted that the form of available N, particularly NH 4 + and NO 3 -, and the transformation of N in soil, also affect K uptake by plants.These authors reported that administration of NH 4 + to barley plants enhanced K + uptake.Phosphorus nutrition is also important for N fixation processes, where conversion of N 2 to NH 4 + catalyzed by nitrogenase depends on ATP (Bello et al. 2023).Nitrogen fertilization also positively affects P uptake by plant roots (Krouk and Kiba, 2020).
The results obtained in the present study from the greenhouse assays showed that application of N. circulans YRNF1 suppressed disease severity caused by R. solani in the treated eggplants.The bio-control efficacy of N. circulans YRNF1 was probably due to its ability to produce metabolites (particularly butyric acid and ethylene glycol) with antifungal potential.Other possible mechanisms of action include the ability to inhibit the growth of R. solani and limit severity of root rot through colonization of root infection sites, competitive exclusion of the fungal pathogen, and secretion of antifungal and/or cell wall hydrolyzing enzymes (Lugtenberg et al., 2009;Berendsen et al., 2012).Systemic immunity may also be triggered by N. circulans in treated eggplants.All the above mentioned results are promising for the potential use of N. circulans YRNF1 as an effective biocontrol agent.Disease severity may also be reduced by AMF colonization of eggplants infected by R. solani, which triggers host defense responses in plants under stress caused by plant pathogens (Rashad et al. 2020b).AMF can also enhance plant resistance, by promoting plant growth and vigor via improvement of plant nutrition (El-Sharkawy et al., 2023) and production of plant hormones (Song et al., 2020).In the present study, co-inoculation of infected plants with the two biocontrol agents decreased disease severity by 26%, indicating an additive action of the N. circulans YRNF1 and AMF.Both potential biocontrol agents affected expression of the defense genes JERF3, PAL1, C3H, CHI2, and HQT.The JERF3 gene was particularly expressed in response to treatment of eggplants with N. circulans YRNF1 and AMF.The gene JERF3 controls several plant defense genes (Rashad et al., 2022b).PAL1 regulates the main step in the polyphenolic biosynthetic pathway, through conversation of phenylalanine to t-cinnamic acid (Mouradov and Spangenberg, 2014).C3H has an important role in biosynthesis of monolignols that constitute lignin (Tao et al., 2015), where lignification of the infected cell walls is a pivotal physical mechanism preventing pathogen penetration of hosts and cell to cell proliferation.CHI2 catalyzes the bioconversion of coumaroyl CoA to naringenin involved in the biosynthesis of several fungitoxic compounds, including flavonoids and phytoalexins (Zhou et al., 2018).HQT regulates biosynthesis of chlorogenic acid from caffeoyl CoA (André et al., 2009).Triggering expression of these defense genes indicates induction of effects of the combined application of N. circulans YRNF1 and AMF for eggplant resistance.
Increases in phenolics and in activities of POD and PPO were detected in response to applying N. circulans YRNF1 and AMF.This is similar to the results of Singh et al. (2016).Increments of phenolic compounds content is a plant defense response associated with induced resistance (IR) in plants infected by pathogens (Chin et al., 2022).POD and PPO important antioxidant enzymes, which catalyze formation of lignin, thus contributing to structural reinforcement of plant cells and formation of physical barriers against invading pathogens (Nasr-Esfahani et al., 2020).In addition, POD and PPO scavenge the oxidative ROS generated within infection processes.
Results from the present study have demonstrated that the frequency of colonization of eggplant roots by AMF, the level of colonization, and the frequency of arbuscule formation were greater after treating eggplants with a combination of N. circulans YRNF1 and AMF.This revealed the inducing effect of N. circulans YRNF1 on mycorrhizal colonization, which indicates compatibility of the two potential biocontrol agents.
In the present study, the two applied potential biocontrol agents promoted eggplant growth.Colonization of plant roots by two or more AMF species has previously been reported to provide a spectrum of advantages, and has more benefits to host plants than colonization by one species (Sharma and Kapoor, 2023).The strain N. circulans E9 has been found to promote plant growth (Sarmiento-López et al., 2022), which is associated with production of IAA and promotion of growth through stimulation of cell division and increased nutrient and water uptake, resulting in increased crop yield and quality (Sarmiento-López et al., 2022).In the present study, increases in the growth parameters of R. solani-infected eggplants were detected after individual treatments with each of the two bio-inoculants.This may be due to the biocontrol potential of N. circulans YRNF1 and growth promotion from each of the bio-inoculants.The recorded enhancement of eggplant growth after colonization with AMF was also similar to effects reported by Han et al. (2023), where treating lettuce by AMF increased the plant height by 30% and root length by 64%.Colonization by AMF is known to improve the host plants through multiple modes of action, including increases in active and passive nutrient and water uptake, and increased hyphal networks on root radicles, which magnify the hydraulic conductivity of water from the soil (Rashad et al., 2020c).In addition, AMF can produce hydrolyzing phosphatases, pectinases, and cellulases, as well as organic acids, which assist in nutrient utilization and improve their plant availability (Liu et al., 2021).AMF also produce phytohormones (e.g.abscisic acid and strigolactones) that enhance plant growth (Pozo et al., 2015).
Niallia circulans YRNF1 also enhanced eggplant nitrogen nutrition via the N-fixation, which probably promoted eggplant growth, whether or not colonized by AMF.The enhancements of eggplant growth parameters were increased after applying the two bio-gents, suggesting their additive action.This observation is consistent with that of El-Sharkawy et al. (2022), who showed that co-treating of pea plants by Streptomyces viridosporus HH1 and the mycorrhizal fungus Rhizophagus irregularis increased pea growth, compared to individual treatments with these two microorganisms.The AMF supply host plants with mineral nutrients, mainly phosphorus, which play prominent roles in plant growth (Adolfsson et al., 2015).The present results also demonstrated that combined application of N. circulans YRNF1 and mycorrhizal colonization improved the NPK contents in eggplant leaves, which was probably due to a cumulative effect of N. circulans YRNF1 and AMF.These data were similar to those of Hafez et al. (2022), who reported that combined application of growth-promoting rhizobacteria with organic fertilizers increased total N, P, and K in treated plant tissues, compared to untreated plants, and to individual applications of the mineral fertilizers.
The present study is the first to report effective use and the additive effects of the diazotrophic bacterium N. circulans and AMF, as biofungicides and biofertilizers, for potential management of Rhizoctonia root rot and growth-promotion in eggplant.

Figure 2 .
Figure 2. GC-MS chromatogram of the secondary metabolites detected in culture filtrates of Niallia circulans YRNF1.

Figure 3 .
Figure 3. Agarose gel showing the amplified DNA product of the nitrogenase gene (nifH) of the diazotrophic bacterium YRNF1 as a single band (450 bp).

Figure 5 .
Figure 5. Mean root rot disease severities of eggplant plants treated with the diazotrophic bacterium Niallia circulans YRNF1 and/or AMF.Columns accompanied by the same superscript letters are not significantly different (Tukey's HSD test, P ≤ 0.05).Error bars are Standard deviations (± SD).Treatments were: C, non-mycorrhizal, untreated and uninfected; P, non-mycorrhizal, untreated and infected; B, non-mycorrhizal, treated with N. circulans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, nonmycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, non-mycorrhizal, treated with the fungicide and infected; and B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected.

Figure 6 .
Figure 6.Mean transcriptional expression of resistance genes (JERF3, PAL1, C3H, CHI2, and HQT) in eggplant roots treated with Niallia circulans YRNF1 and colonized with AMF and inoculated with Rhizoctonia solani.For each gene, columns accompanied by the same superscript letter are not different (P ≤ 0.05), according to Tukey's HSD test.The error bars are the standard deviations of the means.The experimental treatments were: C, non-mycorrhizal, untreated and uninfected; P, non-mycorrhizal, untreated and infected; B, non-mycorrhizal, treated with N. circulans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, non-mycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, non-mycorrhizal, treated with the fungicide and infected; and B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected.
4 Si 2 Biocontrol of Rhizoctonia root-rot of eggplant

Table 3 .
Mean phenolic contents and antioxidant enzyme (POD or PPO) activities in eggplant roots 30 d after inoculation with Rhizoctonia solani and treatments with Niallia circulans YRNF1 and/or mycorrhizae.
*Means followed by different superscript letters are significantly different (P ≤ 0.05), according to Tukey's HSD tests.The data are means of five replicates ± SD.Treatments applied were: C, nonmycorrhizal, untreated and uninfected; P, non-mycorrhizal, untreated and infected; B, non-mycorrhizal, treated with N. circulans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, non-mycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, non-mycorrhizal, treated with the fungicide and infected; B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected, and peroxidase (POD) and polyphenol oxidase (PPO).lans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, non-mycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, nonmycorrhizal, treated with the fungicide and infected; and B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected.

Table 5 .
Mean eggplant shoot heights, root lengths, shoot and root dry weights, and leaf areas 45 d after inoculation with Rhizoctonia solani and treatments of Niallia circulans YRNF1 and/or mycorrhizal colonization.Means accompanied by the same superscript letter are not significantly different (P ≤ 0.05), according to Tukey's HSD test.The values are means of five replicates ± SD.Treatments applied were: C, non-mycorrhizal, untreated and uninfected; P, non-mycorrhizal, untreated and infected; B, non-mycorrhizal, treated with N. circulans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, non-mycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, non-mycorrhizal, treated with the fungicide and infected; and B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected.Biocontrol of Rhizoctonia root-rot of eggplant *

Table 6 .
Mean contents of nitrogen, phosphorus, and potassium, and N/P ratios 45 d after treatments of eggplants with Niallia circulans YRNF1 and/or AMF.Means accompanied by the same superscript letter are not significantly different (P ≤ 0.05), according to Tukey's HSD test.The values are means of five replicates ± SD.Treatments applied were: C, non-mycorrhizal, untreated and uninfected; P, non-mycorrhizal, untreated and infected; B, non-mycorrhizal, treated with N. circulans YRNF1 and uninfected; M, mycorrhizal, untreated and uninfected; B+M, mycorrhizal, treated with N. circulans YRNF1 and uninfected; B+P, non-mycorrhizal, treated with N. circulans YRNF1 and infected; M+P, mycorrhizal, untreated and infected; F+P, nonmycorrhizal, treated with the fungicide and infected; and B+M+P, mycorrhizal, treated with N. circulans YRNF1 and infected. *