Efficacy of endophytic bacteria as promising inducers for enhancing the immune responses in tomato plants and managing Rhizoctonia root-rot disease

Around the world, a variety of crops, including tomatoes, suffer serious economic losses due to the Rhizoctonia root-rot disease. Herein, Bacillus velezensis, Bacillus megaterium, and Herpaspirillum huttiense isolated from strawberry (Fragaria chiloensis var. ananassa) plants were pragmatic as plant growth promotors for battling the Rhizoctonia root rot disease and bringing about defense mechanisms as well as growth promotional strategies in tomato plants. These endophytic bacteria demonstrated potent antifungal activity against R. solani in vitro along in vivo. Data explained that the isolated endophytic bacteria could produce Indole acetic acid, Gibberellic acid GA, and siderophore as well as solubilize phosphate in the soil. The consortium of (Bacillus velezensis, Bacillus megaterium, and Herpaspirillum huttiense) increased the protection % against Rhizoctonia infection by (79.4%), followed by B. velezensis by (73.52%), H. huttiense by (70.5%), and B. megaterium by (67.64%), respectively. There was an increase in soluble proteins and carbohydrates in infected plants treated with a consortium of endophytic bacteria by 30.7% and 100.2% over untreated infected plants, respectively. Applying endophytic bacteria either alone or in combination lowered the level of malondialdehyde MDA and hydrogen peroxide H2O2 and improved the activities of antioxidant enzymes in both infected and uninfected plants. Also, bacterial endophytes have distinctive reactions regarding the number and concentrations of isozymes in both infected and uninfected plants. It could be recommended the commercial usage of a mixture of targeted bacterial endophyte strains as therapeutic nutrients against Rhizoctonia root-rot disease as well as plant growth inducer.


Isolation and purification of endophytes
The fresh and healthy strawberry plants were collected from Badr City, El-Behera Governorate, Egypt in January 2022.The plant collection and use were in accordance with all the relevant guidelines.All plants were immediately taken to the lab and prepared for future work.The strawberry plants have been washed with tap water to remove surface dust and then rinsed twice with distilled water.Next, the plant materials were immersed in 75% ethanol for 2 min, rinsed with 2% sodium hypochlorite (Na Cl O) for 3 min, and finally washed 3 times with sterile distilled water 36 .The plant parts were checked for any surface contamination, in which 100 µL water from the third rinsing was inoculated on three types of media (Reasoner's 2A agar, PDA, and nutrient agar).The plates were incubated at 30 °C for 2 days to determine surface sterilization efficacy.The sterilized explants were cut with a sterile blade into 0.5 to 0.3 cm pieces and placed on nutrient agar media.The plates were incubated at 30 °C for 2 days.The isolated endophytic bacteria were imperiled for morphological and microscopic description.

Quantitative determination of plant growth-promoting substances in liquid culture
Bacterial Endophyte isolates were checked for their quantitative capabilities to produce plant growth-promoting substances.The efficacy of (IAA) production is done using the colorimetric technique 37 .Determination of total Gibberellins production was done using the colorimetric technique 38 .Siderophore production was detected by the CAS plate assay method 39 .The ability of isolates to solubilize phosphate was tested by streaking the isolates in the center of Pikovyskyaya's agar medium plates containing a known amount of tri-calcium phosphate Ca 3 (PO 4 ) 2 .The plates were incubated at 37 ºC for 72 h.Phosphate solubilization was detected by the appearance of a clear halo zone around the streak of endophytic strains 40 .The ability of isolates to solubilize potassium was tested according to Zahra 41 .The formation of hydrogen cyanide (HCN) by the endophytic bacterial isolates was assessed according to the methods of Frey et al. and Daigham et al. 42,43 .

Source of pathogen
The Pathogen Rhizoctonia solani was obtained from the Plant Pathology Institute, Agriculture Research Center, Giza, Egypt.The pathogenic fungus' inoculum was established following the method of Hibar et al. 44 .

Molecular identification of endophytic bacterial isolates
The endophytic bacterial isolates (L1-L2-S7) were cultured in a nutrient broth medium 45 and incubated at 28ºC for 48 h. for DNA extraction.Patho-gene-spin DNA/RNA extraction kit afforded by Intron Biotechnology Company; Korea was applied.PCR was operated utilizing two universal primers namely 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′).The purified PCR products were reconfirmed using a size nucleotide marker (100 base pairs) by electrophoreses on 1% agarose gel.Purified PCR products were sequenced in the sense and antisense directions using 27F and 1492R primers with the incorporation of dideoxynucleosides (dd NTPs) in the reaction mixture 46 .Sequences were analyzed via the Basic Local Alignment Search Tool (BLAST) from (NCBI) website.Meg Align (DNA Star) software version 5.05 was utilized for the phylogenetic testing of sequences.

In vitro antagonistic activity of endophytic bacteria against R. solani by the dual culture assay
Antagonistic activity test of endophytic bacterial strains (L1-L2-S7) was conducted as illustrated by Rosa et al. 47 with minor modification.PDA media in Petri plates have been streaked individually by a fine line of the isolates (24 h.old) along one end of the plate and incubated at 30 °C for 24 h.Next, along the edge of the plate opposing the bacterial inoculum, a 5-mm-diameter mycelial plug of a 7-day-old culture of R. solani was applied.As a negative control, a PDA plate loaded with only a mycelial plug of R. solani at one side was employed.All treatments were employed in triplicates and then incubated for 5 days at 30 °C.Lastly, the fungal radial growth was estimated, and inhibition was calculated.

Antagonistic activity of the endophytic bacterial extract
The bacterial isolates under study (L1-L2-S7) were grown on the PDB broth and incubated on the shaker for 48 h. at 30 °C.After incubation, the samples were centrifuged at 20,000 rpm for 10 min.and the supernatant was filtered through a 0.22 µm microbiological filter.The resulting supernatant was kept in sterilized flasks.A disk of each pathogenic fungus Rhizoctonia solani was added to the surface of the extract.Incubation was done at 28 °C for 7 days, and the spread of fungal growth was observed on the surface of the extract.

Experimental design
Three weeks age tomato seedlings (Solanum lycopersicum L. var.023) were achieved from Agriculture Research Centre, Giza, Egypt.Similar seedlings were planted into plastic pots (40 × 40 cm), encompassing a combination of sand and clay (1: 3 W/W), a total of 6 kg, in a plastic greenhouse.Pots stayed in the greenhouse at Day/night temperature (22/18 °C) and relative humidity (70-85%).After planting, the seedlings were normally irrigated and left for 7 days without treatment.The pots were set up with 6 replicates in a subsequent random order : (T1) Tomato seedlings planted in soil that was recently sterilized (Healthy control);

Disease symptoms and disease index
The disease indicators were recorded, and the following equation was used to determine the severity of the disease along with the protection percentage.
Protection % = A-B/A × 100%, where A = PDI in diseased control plants B = PDI in diseased-treated plants as reported by Hashem et al. 48.

Biochemical defense indicators
Total soluble carbohydrate content in dry leaves was assessed utilizing the anthrone technique according to Irigoyen, 49 .Also, the content of total protein was measured in the dry leaves 50 .The phenol content of the dry leaves was also estimated 51 .Malondialdehyde (MDA) content in fresh leaf was assessed by the thiobarbituric acid (TBA) method conferring to Hu et al. 52 with slight modification.Hydrogen peroxide (H 2 O 2 ) levels were determined 53 .

Evaluation of antioxidant enzymes activity
Peroxidase (POD) activity was detected corresponding to Verduyn et al. 54 .The activity of polyphenol oxidase (PPO) and SOD were evaluated affording to methods of Matta and Dimond 55 and Marklund and Marklund 56 , respectively.

Isozyme electrophoresis
Peroxidase (POD) and polyphenol oxidase (PPO) isozymes were detected corresponding to Trivedi et al. 57 , while polyphenol oxidase (PPO) isozymes were assessed as stated by Knegt & Bruinsma 58 .Detection of SOD isozymes in fresh leaves was done following the method of Beauchamp & Fridovich 59 .

Molecular identification of bacterial isolates
Based on a mega blast search of the NCBIs GenBank nucleotide database, the closest hit using the sequence of the L1 strain is Bacillus velezensis.Figure 2. B. velezensis L-1 showed 99.79-100% identity and 99-100% coverage with several strains of the same species with GenBank accession no.OQ073573.On the other hand, the Phylogenetic analysis based on the 16S rRNA gene of Bacillus megaterium isolate L2 (arrowed) aligned with sequences of closely related bacterial species.B. megaterium L2 showed 99.72-100% identity and 99-100% coverage with several strains of the same species including the type of material Bacillus megaterium ATCC14581 with GenBank accession no.OQ073583.Staphylococcus aureus represents an outgroup strain Fig. 3. Additionally, based on the 16S rRNA gene of Herpaspirillum huttiense isolate S7 (arrowed) aligned with sequences of closely related bacterial species.H. huttiense isolate S7 showed 99.50-99.86%identity and 93-100% coverage with several strains of the same species including the type of material H. huttiense ATCC14670 with GenBank accession no.OQ073584.Staphylococcus aureus represents an outgroup strain, Fig. 4.

Antagonistic activity of endophytes
Varying degrees of mycelial growth inhibition of the phytopathogenic fungus R. solani using the dual culture assay were observed with antagonistic endophytic bacteria Bacillus velezensis L1, Bacillus megaterium L2 and Herpaspirillum huttiense S7.The results indicated that Herpaspirillum huttiense S7 had the maximum inhibitory effect on the mycelial growth of Rhizoctonia solani followed by Bacillus velezensis isolate L1 and then Bacillus megaterium isolate L2.The interactions between antagonistic endophytic bacteria and R. solani were shown in Fig. 5.Moreover, the extract of endophytic bacteria under study exerts variable degrees of R. solani mycelial growth inhibition, and the most effective was Bacillus velezensis isolate L1 followed by Herpaspirillum huttiense isolate S7 Table 2 and Fig. 6.

Disease symptoms and disease index
As shown in Table 3, the disease index reached 85%, due to R. solani infection.The results showed that R. solani infects the roots of the plant, causing yellowing, root rot, and eventually death.The results in Table 3 and Fig. 7 showed that both B. velezensis, H. huttiense, and B. megaterium filtrates alone were effective in reducing the severity of the Rhizoctonia root rot disease by 22.5%, 25%, and 27.5%, and increase protection by 73.52%, 70.5% and 67.64%, respectively.However, the combining treatment of B. velezensis, B. megaterium, and H. huttiense filtrates was even more effective, with a DI 17.5% and Protection 79.4%.

Biochemical defense indicators Total soluble carbohydrates and protein
Results recorded in Fig. 8A declared that infected plants established significant declines in contents of total soluble carbohydrates by 51.9% in comparison with healthy control.Relating the consequence of tested treatments on healthy plants, it was found that all filtrates alone were effective in increasing total soluble carbohydrates by 15.24%, 8.59%, and 11.14%.However, the treatment with mixed filtrates from B. velezensis, B. megaterium, and H. huttiense was even more effective in increasing total soluble carbohydrates by 29.09%.Also, all filtrates alone were effective in increasing total soluble carbohydrates by 86.95%, 87.79%, and 86.65% in infected plants.However, the mixed treatment of all filtrates was even more effective in increasing total soluble carbohydrates by 100.2%.Additionally, results in Fig. 8 B illustrated a substantial decrease in the soluble protein content of R. solani-infected plants.Application of all filtrates individual or combination, resulted in, mostly, significant increases in total soluble protein in both healthy and diseased plants.About the effect of tested treatments on the diseased plants with R. solani.It was recovered that mixed treatment with (B.velezensis, B. megaterium, and H. huttiense) filtrates showed a highly substantial rise in the soluble protein contents by 30.07%related to B. megaterium 20.8%, B. velezensis 9.8% and H. huttiense 9.3% when being compared with untreated infected control.

Phenol content
As shown from the results in Fig. 9, the phenol content increased by 52.77%, due to R. solani infection in comparison with healthy control.Application of all filtrates alone or in combination resulted in, mostly, a significant increase of phenol content in both healthy and infected plants.

Oxidative stress (MDA and H 2 O 2 )
As shown in the results represented in Fig. 10A,B

Antioxidant enzymes activity
The activities of PPO, POD, SOD, and CAT highly increased in diseased plants linked to healthy control plants Fig.

Superoxide dismutase (SOD) isozymes
Seven SOD isozymes were noticed on native PAGE in Fig. 12 and

Peroxidase (POD) isozymes
Data offered in Fig. 13 and

Discussion
Microorganisms are now used to control pathogens and pests to protect important plants 60 .The first and most important step in biological management is the identification and selection of active antagonistic microorganisms 61 .The goal of biological control is to sustain a balance in agrosystems in which the host suffers less damage in the presence of pathogens owing to the regulatory actions of non-pathogenic microorganisms that inhibit/antagonize plant pathogens.In the current work, endophytic bacteria Bacillus velezensis isolate L1, Bacillus megaterium isolate L2 and Herpaspirillum huttiense isolate S7 proved significant antifungal ability contrary to R. solani in Vitro.Our findings corresponded with those of Azevedo et al. 62 , who found that endophytic microorganisms have attracted attention for use in biological management and might be used to promote plant growth and prevent fungal infections.The isolated endophytic bacteria under study produce IAA, siderophores, HCN and solubilize phosphate in the soil.Indole acetic acid is a significant and powerful plant hormone, that controls cell growth and responds plants to light and gravity, as well as inducing plant growth and development 63,64 .Moreover, endophytic microorganisms produce bioactive compounds such as alkaloids, steroids, terpenoids, peptides, polyketides, flavonoids, quinols, and phenols.These compounds have a variety of agricultural, industrial, and therapeutic uses 30,65,66 .Endophytic bacteria have the ability to synthesize plant growth promotors, phosphate solubilization, mineral acquisition, and fixation of nitrogen 67 .Moreover, Shakirova 68 indicated that IAA has been shown to play a significant fundamental function in cell division and root stimulation, which brings in improved plant development.HCN is a potent antifungal and has a significant role in the biocontrol of plant fungal pathogens 43,69 .Endophytes may have acted in various ways against plant pathogens, and this preliminary assessment suggests a potential ability to create metabolic substances that restrict pathogen growth or directly compete for nutrients and space, simulating conditions inside the plant that hosts them 70,71 .In addition, Schulz et al. 72 proposed that endophytes are effective suppliers of secondary metabolites since they have an intimate connection to the host plants.Besides, Shukla et al. 73 indicated that endophytes have been shown to produce a variety of bioactive compounds in a single plant or microbe, making them a promising source of drugs for the treatment of various diseases, as well as having conceivable uses in agriculture.The endophytes' production of bioactive compounds has been linked to the advancement of the host microbes, which might have adopted genetic codes from higher plants, thus allowing them to more easily adapt to their host plant and fulfill certain roles, such as protection from numerous sorts of pathogens 74,75 .Similar to our findings, Devi et al. 76 reported the usage of B. velezensis as a biocontrol agent has a wide antifungal spectrum and affords a sustainable alternative for hazardous chemicals.Besides, our results agreed with Hashem et al. 48who recorded the antifungal potential of B. megaterium against phytopathogenic R. solani.It's interesting to note that, except for a few isolates rarely reported in humans as a pathogen causing bacteremia in

B. velezensis (L1) 100%
Bacillus megaterium (L2) 48 Herpaspirillum huttiense (S7) 65% Vol:.( 1234567890   leukemic persons 77,78 , the majority of the Herbaspirillum genus were mostly isolated from soil, plants, or water.The isolation of the novel endophytic bacterium Herbaspirillum camelliae sp.from Camellia sinensis L. was also described by Liu et al. 79 .The study of Andreozzi et al. 80 showed the beneficial endophyte Herpaspirillum huttiense RCA24 as a promising strain to improve rice plants in the greenhouse.Endophytic actinomycetes and bacteria are important contributors to the synthesis of bioactive substances.Moreover, Liarzi et al. 81 reported that endophytic bacteria endure most of their lives inside plant tissues without causing any evident impairment to the host plant and improve plant tolerance to various abiotic and biotic stress, as well as plant durability against pests and insects.The combination of endophytic bacteria under study increased the tomato's protection against R. solani disease by 79.4%, according to the results.B. velezensis has been shown to exert opposing effects on plant diseases by producing a variety of antimicrobial chemicals; siderophore bacillibactin [82][83][84] . Besides, R. solani infectivity causes significant decreases in the contents of soluble carbohydrates and soluble proteins in our study.R. solani caused reduced photosynthetic rate and excessive respiration rate in infected plants resulting in lower concentrations of both soluble carbohydrates and protein [85][86][87] .The results also showed that the application of (B.velezensis, B. megaterium, and H. huttiense) individually or in combinations had a substantial impact on the level of osmolytes (total soluble sugar and soluble protein), which may operate as a marker of resistance, in both healthy and diseased plants.The noticed increase in tomato plant soluble sugar and protein could be attributed to the ability of these endophytic bacteria to fix nitrogen, produce plant growth regulators; and solubilize phosphate, which enhances nutrient uptake from the root rhizosphere 88,89 .
The phenol content increased by 52.77%, due to Rhizoctonia solani infection in comparison with healthy control.These results are supported by many previous findings 26,90 .The role of phenolic compounds is fundamental in preventing the spread of root rot diseases by either making numerous metabolic products including those involved in host defense mechanisms, or by reducing the pathogen's toxicity and increasing host defense pathways 91 .Besides, phenolic chemicals were able to strengthen cellular membranes by restricting membrane flexibility, which lowers the ability of free radicals to cross membranes and causes membrane peroxidation 92,93 .Our results were supported by many previous findings 48,94 .
Herein, R. solani infection accumulated MDA by 57.6% and H 2 O 2 by 140%, compared to untreated healthy plants.These results were supported by previous findings 87,95 .By boosting antioxidant molecules that eliminate ROS and protect cell membranes against oxidative stress, the application of endophytic bacteria reduced the generation of MDA and H 2 O 2 96 .One of the most important signs of stress resistance is avoidance and reduction of oxidative stress and capture of free radicals 97 .
Our findings confirmed that plants subjected to R. solani infection had considerably higher activity levels of antioxidant enzymes.The plant displayed various defense mechanisms against infection, boosting the action of antioxidant enzymes to maintain ROS levels in plant cells low.POD and other antioxidant enzymes assist in converting H 2 O 2 to H 2 O 98 .Increasing the activity of antioxidant enzymes provides a key function in plant physiological immunity and defending cells from oxidation as a result of infection 99 .As part of the primary regulating mechanisms of metabolism within cells, several enzyme isoforms are essential for plant cellular defense versus biological stress 100,101 .The generation of these isozymes is thought to be essential for the cell's defense against oxidative damage 102,103 .In the leaf-soluble protein extracts of the tomato plant, active staining of antioxidants disclosed five PPO isozymes, seven SOD isozymes, and seven POD isozymes.The antioxidant enzyme levels in R. solani-infected plants handled with endophytic bacteria B. velezensis, B. megaterium, and H. huttiense were greater than in the control one since numerous unique bands were produced because of infection.The stressed tomato plants treated with B. velezensis, B. megaterium, and H. huttiense either in combination or alone displayed the highest bands of POD isozyme allied to another treatment.The outcomes demonstrate how treatments protect tomato plants from root rot disease in an ameliorative manner which is agreed with Hao et al. 104 .These findings are consistent with the findings of Rajendran et al. 105 , which reported increased antioxidant isozyme transcript abundances in plants.In comparison to untreated and infected control plants, the level of all SOD isozymes intensified when endophytic bacteria (B.velezensis, B. megaterium, and H. huttiense) were applied.The recent results were in the same link as prior investigations that claimed a range of proteins, including CAT and POD, may act as scavengers for these ROS 106,107 .

Conclusion
The present investigation used a promising approach that was focused on applying endophytic bacteria to promote systemic resistance in tomato plants against Rhizoctonia root-rot disease.Three endophytic bacteria; Bacillus velezensis OQ073573, Bacillus megaterium OQ073583, and Herpaspirillum huttiense OQ073584 isolated from strawberry plants boost the systemic resistance in tomato plants and reduce the severity of the Rhizoctonia rootrot disease.These endophytic bacteria demonstrated potent antifungal activity against R. solani in vitro along with in vivo.Pre-treated tomato plants with endophytic bacteria had significantly higher levels of total soluble proteins, total carbohydrates, and phenols.Interestingly, the harmful impact of Rhizoctonia root-rot disease on tomato plants was pointedly decreased and it can be clear from diminished MDA and H 2 O 2 levels.In considering this, endophytic bacteria are promising isolates for application in agriculture, as an effective biological manager against Rhizoctonia root-rot, and for the induction of healthy tomato plants.

8 Figure 1 .
Figure 1.(A) Isolation of endophytic bacteria from stem, leaves, and roots of strawberry plants.(B) HCN production by endophytic bacterial isolates.
Regarding the effect of the tested treatment on the affected plants with R. solani, the B. megaterium treatment showed a significant rise in the content of phenol by 131.2% related to the mixed treatment of B. velezensis, B. megaterium, and H. huttiense by 120.3%, B. velezensis by 64% and H. huttiense 55.2%, when being compared with untreated infected control.

Figure 3 .
Figure 3. Phylogenetic tree of Bacillus megaterium isolate L2 (arrowed) aligned with sequences of closely related bacterial species.B. megaterium L2 disclosed 99.72-100% identity and 99-100% reporting with various strains of the same species including the type of material Bacillus megaterium ATCC14581 with GenBank accession no.OQ073583.Staphylococcus aureus represents an outgroup strain.

Figure 4 .
Figure 4. Phylogenetic tree of Herpaspirillum huttiense isolate S7 (arrowed) aligned with sequences of closely related bacterial species.H. huttiense isolate S7 exhibited 99.50-99.86%identity and 93-100% reporting with various strains of the same species including the type of material H. huttiense ATCC14670 with GenBank accession no.NR_024698.Staphylococcus aureus represents an outgroup strain.

Figure 12 .
Figure 12.Consequence of R. solani infection, application of (B.velezensis, B. megaterium, and H. huttiense) and their impacts on tomato plants on (A) SOD isozyme and (B) Ideogram analysis of SOD isozyme of tomato plants.

Figure 13 .
Figure 13.Consequences of R. solani infection, application of (B.velezensis, B. megaterium, and H. huttiense) and their impacts on tomato plants on (A) POD isozyme and (B) Ideogram analysis of POD isozyme of tomato plants.

Figure 14 .Table 6 .
Figure 14.Consequences of R. solani infection, application of (B.velezensis, B. megaterium, and H. huttiense) and their impacts on tomato plants on (A) POD isozyme, and (B) Ideogram analysis of PPO isozyme of tomato plants.

Table 1 .
Determination of plant growth-promoting substances of endophytic bacterial isolates.

Table 4 at
Rf 0.515, 0.566, 0.571, 0.729, 0.803, 0.853, and 0.903.Infected tomato plants with R. solani exhibited substantially overexpressed SOD, which was detected in three bands, two low and one dense at Rf 0.515 and 0.803 and 0.729 respectively.Under infection circumstances, the application of a mixture from B. velezensis, B. megaterium, and H. huttiense, H. huttiense, and B. velezensis treatments verified the similar 6 bands at the identical Rf. and came next the B. megaterium treatment which confirmed one moderate band at Rf 0.515 and one low band at Rf 0. 729.

Table 2 .
Percentage of R. solani mycelial growth inhibition by endophytic bacterial strains.

Table 3 .
Effect of B. velezensis, H. huttiense, and B. megaterium filtrates on root rot disease of tomato plants caused by Rhizoctonia solani under pots conditions.