Antagonistic Potential of Novel Endophytic Bacillus Strains and Mediation of Plant Defense against Verticillium Wilt in Upland Cotton

Verticillium wilt caused by Verticillium dahliae is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic Bacillus strains isolated from cotton roots exhibited inhibitory activity against V. dahliae strain VD-080 in a dual culture assay. B. altitudinis HNH7 and B. velezensis HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, was considerably higher in plants treated with endophytic Bacillus strains and inoculated with VD-080 as compared to control.


Introduction
Cotton is an important cash crop worldwide, but the crop is vulnerable to the attack of Verticillium dahliae, affecting the quality and quantity of the produce. V. dahliae associated with verticillium wilt is considered as the most destructive pathogen of cotton, causing a loss of 2.5 million hectares annually in China [1,2]. Although a few tolerant cotton varieties are available, they do not possess sufficient ability to protect the crop from economic damage. Some chemicals, such as fungicide benomyl and the plant defense activator acibenzolar-S-methyl, seem to be effective but they are not environmentally friendly [3]. The biological control using beneficial bacteria is a safer and better approach to protect the crop from economic yield loss.
The rhizosphere contains a variety of microbial communities which can protect plants through the suppression of soil-borne pathogens. Among these microbes, endophytic bacteria reside inside the plant and colonize an ecological niche like phytopathogens and can effectively be used to suppress the attack of various phytopathogens. Therefore, these endophytic bacteria are strong candidates for the biological control of many plant diseases [4][5][6][7][8][9]. Bacillus species such as B. amyloliquefaciens, B. velezensis,

Antagonistic Activity of the Bacterial Isolates against VD-080
Nine bacterial endophytes isolated from cotton roots were evaluated for their antagonistic activity against VD-080 in a dual culture assay. Results showed that all the isolated endophytic Bacillus strains were able to inhibit the growth of VD-080, but B. altitudinis HNH7 and B. velezensis HNH9 showed the highest inhibition of fungal growth. The diameter of inhibition zones was 10.5 and 9.3 mm for HNH7 and HNH9, respectively ( Table 1). The results showed that the nine endophytic Bacillius strains could devour V. dahliae (VD-080), with variable antagonistic effects ( Figure 2).

Antagonistic Activity of the Bacterial Isolates against VD-080
Nine bacterial endophytes isolated from cotton roots were evalua against VD-080 in a dual culture assay. Results showed that all the is were able to inhibit the growth of VD-080, but B. altitudinis HNH7 an highest inhibition of fungal growth. The diameter of inhibition zones and HNH9, respectively ( Table 1). The results showed that the nine e devour V. dahliae (VD-080), with variable antagonistic effects (Figure 2

Antagonistic Activity of the Bacterial Isolates against VD-080
Nine bacterial endophytes isolated from cotton roots were evaluated for their antagonistic activity against VD-080 in a dual culture assay. Results showed that all the isolated endophytic Bacillus strains were able to inhibit the growth of VD-080, but B. altitudinis HNH7 and B. velezensis HNH9 showed the highest inhibition of fungal growth. The diameter of inhibition zones was 10.5 and 9.3 mm for HNH7 and HNH9, respectively ( Table 1). The results showed that the nine endophytic Bacillius strains could devour V. dahliae (VD-080), with variable antagonistic effects ( Figure 2)

Antagonistic Activity of the Bacterial Isolates against VD-080
Nine bacterial endophytes isolated from cotton roots were evaluated for their antagonistic activity against VD-080 in a dual culture assay. Results showed that all the isolated endophytic Bacillus strains were able to inhibit the growth of VD-080, but B. altitudinis HNH7 and B. velezensis HNH9 showed the highest inhibition of fungal growth. The diameter of inhibition zones was 10.5 and 9.3 mm for HNH7 and HNH9, respectively ( Table 1). The results showed that the nine endophytic Bacillius strains could devour V. dahliae (VD-080), with variable antagonistic effects ( Figure 2)

Antagonistic Activity of the Bacterial Isolates against VD-080
Nine bacterial endophytes isolated from cotton roots were evaluated for their antagonistic activity against VD-080 in a dual culture assay. Results showed that all the isolated endophytic Bacillus strains were able to inhibit the growth of VD-080, but B. altitudinis HNH7 and B. velezensis HNH9 showed the highest inhibition of fungal growth. The diameter of inhibition zones was 10.5 and 9.3 mm for HNH7 and HNH9, respectively ( Table 1). The results showed that the nine endophytic Bacillius strains could devour V. dahliae (VD-080), with variable antagonistic effects ( Figure 2). B. aryabhattai. B. amyloliquefaciens HNH8 5.9 ± 0.09 e B. velezensis HNH9 9.3 ± 0.09 b *CK 0.0 ± 0.00 g Each value represents a mean ± SE of three replicates. *CK = control. Corresponding results of ANOVA. F = 1161, p = 0.000, dF = 9. Values in the columns followed by the same letters (a-g) are not significantly different from each other according to LSD test (p ≤ 0.05).

Interaction of Extracted Lipopeptides (LPs) with VD-080
Methanolic extracts (LPs) of bacterial strains performing best in dual culture assay were evaluated for their antifungal activity against VD-080. Results demonstrated that the methanolic extracts of both endophytic strains HNH7 and HNH9 significantly inhibited the hyphal growth of VD-080 compared to control. The growth restriction of the pathogenic fungi around the holes containing crude extracts of bacterial LPs can be observed easily, while the holes of only methanol in the control plate are fully covered by the fungal mycelium ( Figure 3).

Interaction of Extracted Lipopeptides (LPs) with VD-080
Methanolic extracts (LPs) of bacterial strains performing best in dual culture assay were evaluated for their antifungal activity against VD-080. Results demonstrated that the methanolic extracts of both endophytic strains HNH7 and HNH9 significantly inhibited the hyphal growth of VD-080 compared to control. The growth restriction of the pathogenic fungi around the holes containing crude extracts of bacterial LPs can be observed easily, while the holes of only methanol in the control plate are fully covered by the fungal mycelium ( Figure 3).

B. amyloliquefaciens HNH8
5.9 ± 0.09 e B. velezensis HNH9 9.3 ± 0.09 b *CK 0.0 ± 0.00 g Each value represents a mean ± SE of three replicates. *CK = control. Corresponding results of ANOVA. F = 1161, p = 0.000, dF = 9. Values in the columns followed by the same letters (a-g) are not significantly different from each other according to LSD test (p ≤ 0.05).

Interaction of Extracted Lipopeptides (LPs) with VD-080
Methanolic extracts (LPs) of bacterial strains performing best in dual culture assay were evaluated for their antifungal activity against VD-080. Results demonstrated that the methanolic extracts of both endophytic strains HNH7 and HNH9 significantly inhibited the hyphal growth of VD-080 compared to control. The growth restriction of the pathogenic fungi around the holes containing crude extracts of bacterial LPs can be observed easily, while the holes of only methanol in the control plate are fully covered by the fungal mycelium ( Figure 3).

LC-MS Analysis of Antimicrobial Compounds
The results of PCR amplification of antimicrobial genes were further validated through LC-MS analysis. The LC-MS results confirmed that both bacterial strains were able to produce corresponding products of the antimicrobial genes identified based on their mass to charge ratio (m/z). The peaks for fengycin were detected in B. altitudinis HNH7 at 1491.83 and 1477.80 m/z ( Figure

LC-MS Analysis of Antimicrobial Compounds
The results of PCR amplification of antimicrobial genes were further validated through LC-MS analysis. The LC-MS results confirmed that both bacterial strains were able to produce corresponding products of the antimicrobial genes identified based on their mass to charge ratio (m/z). The peaks for fengycin were detected in B. altitudinis

Hyphal Anomalies in VD-080 by Methanolic Extracts of Endophytic Bacillus Strains
The structural deformities in VD-080 hyphae caused by the antimicrobial compounds produced by both endophytic Bacillus strains were studied under scanning electron microscope (Figure 7). The hyphae treated with methanolic extracts of HNH7 and HNH9 strains showed pore formation, breakdown of hyphae, curling, shirking and removal of cytoplasmic content from the hyphae. Meanwhile, the hyphae in the control treatment were long, cylindrical and healthy with no structural

Hyphal Anomalies in VD-080 by Methanolic Extracts of Endophytic Bacillus Strains
The structural deformities in VD-080 hyphae caused by the antimicrobial compounds produced by both endophytic Bacillus strains were studied under scanning electron microscope (Figure 7). The hyphae treated with methanolic extracts of HNH7 and HNH9 strains showed pore formation, breakdown of hyphae, curling, shirking and removal of cytoplasmic content from the hyphae. Meanwhile, the hyphae in the control treatment were long, cylindrical and healthy with no structural abnormalities. The results indicated that the LPs of the Bacillius strains could damage the cell structure of VD-080.

Biocontrol Effects of the Isolates on Cotton Verticillium Wilt
The ability of endophytic bacterial strains to inhibit verticillium wilt disease in cotton plants was evaluated in a greenhouse experiment. Disease severity was recorded 30 days post-inoculation by observing vascular browning. The highest disease severity (83.33%) was observed in the treatment which was inoculated with VD-080 only. Furthermore, there was a significant reduction in disease severity in treatments which were administered with endophytic bacterial strains HNH7 and HNH9 and challenged with VD-080 compared to the treatments inoculated with VD-080 only. HNH7 showed higher reduction of disease than HNH9 as the disease severity recorded was 36.55% by HNH7 and 42.08% by HNH9 compared to control (Figure 8).

Biocontrol Effects of the Isolates on Cotton Verticillium Wilt
The ability of endophytic bacterial strains to inhibit verticillium wilt disease in cotton plants was evaluated in a greenhouse experiment. Disease severity was recorded 30 days post-inoculation by observing vascular browning. The highest disease severity (83.33%) was observed in the treatment which was inoculated with VD-080 only. Furthermore, there was a significant reduction in disease severity in treatments which were administered with endophytic bacterial strains HNH7 and HNH9 and challenged with VD-080 compared to the treatments inoculated with VD-080 only. HNH7 showed higher reduction of disease than HNH9 as the disease severity recorded was 36.55% by HNH7 and 42.08% by HNH9 compared to control (Figure 8).

Expression Profiling of Plant Defense-Linked Genes
The expression profiling of six defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, in cotton plants was studied through qRT-PCR. According to the results, both bacterial endophytes elicited the defense response in cotton plants as the expression of defense-linked genes in plants treated with bacterial endophytes was significantly higher compared to the plants inoculated with water only. Interestingly, the expression of defense-linked genes was much higher in plants treated with bacterial antagonists and challenged with VD-080 also. However, the expression of genes was higher in the plants treated with endophytic bacterial strains HNH7 compared to HNH9. The highest expression was recorded in the case of PAL and PPO encoding Letters (a-c) above the columns represent significant differences between treatment means at p ≤ 0.05.

Expression Profiling of Plant Defense-Linked Genes
The expression profiling of six defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, in cotton plants was studied through qRT-PCR. According to the results, both bacterial endophytes elicited the defense response in cotton plants as the expression of defense-linked genes in plants treated with bacterial endophytes was significantly higher compared to the plants inoculated with water only. Interestingly, the expression of defense-linked genes was much higher in plants treated with bacterial antagonists and challenged with VD-080 also. However, the expression of genes was higher in the plants treated with endophytic bacterial strains HNH7 compared to HNH9. The highest expression was recorded in the case of PAL and PPO encoding genes. Moreover, the expression of all defense-linked genes was found to be downregulated in the plants challenged with the pathogen only ( Figure 9). Plants 2020, 9, x FOR PEER REVIEW 9 of 17 genes. Moreover, the expression of all defense-linked genes was found to be downregulated in the plants challenged with the pathogen only ( Figure 9).

Discussion
The plants endophytes of Pseudomonas, Serratia and Bacillus spp. have been analyzed as efficient biocontrol agents against verticillium wilt both in vitro and in vivo [7,22]. The plant root colonized bacteria apply different built-in tools to initiate defense mechanisms which progressively devour the pathogenic fungi. The potential of plant growth promoting rhizobacteria (PGPRs) to produce hydrolytic enzymes, siderophores, a range of different antibiotics, aggressive colonization and induced systemic resistance, which are the basic mechanisms to prevent the proliferation of plant pathogens [23]. However, endophyte-allied antibiotics and ISR have positive effects on competitive interactions with plants and the plant pathogens which inhibit the synthesis of pathogen cell walls and cell membrane structures and disrupt ribosomal subunits [24]. The bacterial strains 41B-1 and SQR9 have shown significant biocontrol activity against V. dahliae in greenhouse and field trials [7,25].
In this study, we characterized nine endophytic Bacillus strains isolated from cotton roots. These endophytic strains include B. amyloliquefaciens, B. subtilis, B. velezensis, B. pumilis, B. altitudinis and B.   Figure 9. Expression profiling of 6 defense-linked genes in cotton was upregulated in treatments with the isolates. The mean values were computed and separated by LSD test at p ≤ 0.05 after one-way ANOVA. The error bars indicate the standard error ± SE of the mean values. Letters above the columns (a-e) represent significant differences between treatment means at p ≤ 0.05.

Discussion
The plants endophytes of Pseudomonas, Serratia and Bacillus spp. have been analyzed as efficient biocontrol agents against verticillium wilt both in vitro and in vivo [7,22]. The plant root colonized bacteria apply different built-in tools to initiate defense mechanisms which progressively devour the pathogenic fungi. The potential of plant growth promoting rhizobacteria (PGPRs) to produce hydrolytic enzymes, siderophores, a range of different antibiotics, aggressive colonization and induced systemic resistance, which are the basic mechanisms to prevent the proliferation of plant pathogens [23]. However, endophyte-allied antibiotics and ISR have positive effects on competitive interactions with plants and the plant pathogens which inhibit the synthesis of pathogen cell walls and cell membrane structures and disrupt ribosomal subunits [24]. The bacterial strains 41B-1 and SQR9 have shown significant biocontrol activity against V. dahliae in greenhouse and field trials [7,25].
In this study, we characterized nine endophytic Bacillus strains isolated from cotton roots. These endophytic strains include B. amyloliquefaciens, B. subtilis, B. velezensis, B. pumilis, B. altitudinis and B. aryabhattai. All these strains show variable antagonistic effects to VD-080. Two of the endophytes, B. altitudinis (HNH7) and B. velezensis (HNH9), exhibited high biocontrol efficacy. The composition of endophytic strains which have antifungal effects on V. dahliae may be related to bacterial antibiotics and induce resistance in cotton cultivars against verticillium wilt. Endophytic bacteria have shown antagonistic activity against several phytopathogens such as Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, F. oxysporum f. sp. niveum, F. oxysporum f. sp. cubense and V. dahliae [22,26,27].
Bacillus species can produce several antibiotics such as surfactin, fengycin, bacilysin, bacillibactin, bacillomycin and iturin [28,29]. The antimicrobial compounds produced by Bacillus species either directly hamper the growth of the pathogen or induce systemic resistance in plants [19,30]. The isolates HNH7 and HNH9 were found to possess bacillibactin, bacillomycin, surfactin, and fengycin encoding genes, while both were lacking in iturin producing genes. One siderophore producing (bacillibactin), two antifungal (bacillomycin and fengycin) and one multifunctional (antifungal plus antibacterial) metabolite (surfactin) encoding genes were successfully detected in the genome of HNH7 and HNH9. Fengycin, surfactin, bacillibactin and bacillomycin have been reported previously to exhibit antagonistic activity against fungal pathogens including V. dahliae [7], which is consistent with our Microbial lipopeptides (LPs) can cause morphological and ultrastructural deformities in fungal hyphae such as pore formation, curling, plasmolysis or removal of cytoplasmic content and breakdown or disintegration of fungal hyphae [19,[31][32][33]. Scanning electron microscopy (SEM) analysis has previously supported the hypothesis that the loss of turgidity and structural alterations in the cell wall of fungal hyphae is the core mechanism involved in the antifungal action of LPs [32,33]. In our study, the SEM analysis revealed disruption of V. dahliae (VD-080) hyphae when treated with the LPs of HNH7 and HNH9 compared to control treatment and these morphological changes in the hyphae explain the possible deleterious role of LPs produced by the two endophytes.
Bacillus spp. protect plants against phytopathogens through the induction of systemic resistance and subsequent upregulation of defense-linked genes [7,9,20,21]. Our study demonstrated that the disease severity on cotton plants treated with endophytes was significantly reduced compared to the infected control treatment, plus the expression of defense-linked genes HMGR, MPK3, GST, PAL, PPO and SOD was significantly upregulated in cotton plants treated with endophytes + VD-080, compared to the treatments inoculated with HNH7 and HNH9. Interestingly, the activities of defense-linked genes were higher in HNH-7 and HNH-9 treated plants compared to controls, indicating that the endophytes were responsible for defense elicitation in cotton plants. Our results are in conformation with the findings of Chandrasekaran and Chun [34], where the expression of defense-linked genes SOD, CAT, POD, PPO, PAL and β-1,3-glucanase was significantly increased in Bacillus subtilis + pathogen-treated tomato plants. SOD is responsible for H 2 O 2 accumulation in plants. According to several previous studies, H 2 O 2 has a significant role in disease resistance against pathogens [35,36]. It can be concluded that SOD expression induced by endophytes provided protection against VD-080. PAL contributes to the oxidation of phenolic compounds and defense stimulation against the pathogens [35]. In addition to SOD and PAL, the activity of PPO was also upregulated in our study. According to Li and Steffens [37], the overexpression of PPO in the plants leads to enhanced resistance against the disease.
The high expression of MPK3, HMGR and GST genes in the present study indicated their possible role in defense elicitation. These genes and their corresponding products play several important roles in the plant defense system. Mitogen-activated protein kinases (MPK) are a cascade of signaling molecules that play an important role in signal transduction to activate plant defense against the pathogens [38,39]. According to a previous study, the enhanced expression of MPK3 gene has been involved in providing basal resistance against Botrytis cinerea [40]. Glutathione S-transferase (GST) is an enzyme with detoxifying properties that is responsible for stress modulation in plants and provides increased resistance against several biotic and abiotic stresses [41,42]. HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) HMGR is an enzyme that catalyzes the first step in the mevalonate (MVA) pathway responsible for the formation of isoprenoid [43,44]. Isoprenoids are significant in the elicitation of plant defense against biotic and abiotic stresses.
Based on the present study and previous reports, we suggest that the enhanced expression of defense-related genes is responsible for the strong biological control activity of HNH-7 and HNH-9 and plays a vital role in plant defense against Verticillium dahliae.

Isolation of Endophytic Bacterial Isolates
Endophytic Bacillus strains were isolated from cotton roots following the procedure given by Khan et al. [45]. Briefly, cotton roots of cultivar "Jimian 11", grown at Liuhe Experimental Station, Nanjing, Jiangsu Province, were washed with tap water, excised into small pieces of 5 mm, surface sterilized with 2.5% sodium hypochlorite (NaClO) for 10 min and then rinsed four times with double distilled sterile water to remove the contaminants. Afterwards, the root was ground in 1 mL of double distilled water using mortar and pestle. Post-grinding, an aliquot of 200 µL was spread onto LB agar plate and incubated at 37 ± 2 • C for 12 h. Post-incubation, the bacterial colonies were observed and the colonies differing in size and shape were selected, picked and cultured in LB medium.

16S rDNA Identification
The endophytic bacterial isolates were characterized by sequencing the 16S rDNA gene [46]. DNA of endophytic isolates was extracted by using DNA extraction kit (Omega Bio-tek, Norcross, GA, USA). The concentration and purity of extracted DNA was recorded on NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). The 16s rDNA primers forward 531F (5 -TGGAGAGTTT GATCCTGGCTCAG-3 ) and reverse 531R (5 -TACCGCGGCTGCTGGCAC-3 ) were used for amplification of the 16S rDNA gene in bacterial endophytes. PCR amplification was processed using 2× Rapid Taq Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China). The reaction conditions were as follows: initial denaturation at 95 • C for 5 min, followed by 32 cycles of denaturation at 95 • C for 15 sec, annealing at varied temperatures according to the set of primers, elongation at 72 • C for 15 sec and, finally, a cycle of final extension for 5 min at 72 • C. Additionally, a blank (CK) containing sterilized distilled water rather than genomic DNA was also included in the PCR reaction. The amplified product (5 µL) was visualized on 1% agarose gel stained with ethidium bromide and remaining product was sequenced using the Sanger dideoxy sequencing method (Genscript Co. Ltd. Nanjing, Jiangsu, China). The obtained sequences were compared with the previously available sequences in the NCBI database and the phylogenetic relationship was studied [47]. The homologous sequences were analyzed using MEGA X software [48]. Multiple sequence alignments of the sequences were performed by ClustalW and a neighbor joining phylogenetic tree was constructed using bootstrap test (1000 replicates) and evolutionary distances were calculated using Tamura Nei model [49]. A gamma distribution (shape parameter = 5) model was used to determine the rate variation among sites with partial deletion of gaps and missing data.

Antagonistic Activity of Endophytes against V. dahliae (VD-080)
The endophytic bacterial isolates were evaluated for their inhibitory effect against VD-080 in a dual culture experiment [7]. The fungus was inoculated at the center of the Petri plate (9 cm) containing Potato dextrose agar medium (PDA) and incubated for 3 days at 27 ± 2 • C. Endophytic bacterial isolates (5 µL) from an overnight culture (OD600 = 2.5) were inoculated on two sides of the fungal colony 2.5 cm away from the center. The plates were then wrapped with a parafilm and incubated at 27 ± 2 • C for seven days and the zone of inhibition was measured. The experiment was repeated thrice with three replicates.

Antifungal Activity of Secondary Metabolites
The bacterial strains HNH7 and HNH9 were individually inoculated in Landy medium [50] from an overnight culture of each strain and incubated for 3 days at 30 • C and 180 rpm. Post-incubation, the cultures were centrifuged at 10,000 rpm and 4 • C for 15 min. The cell free supernatants were collected in new tubes and each supernatant was incubated at 4 • C for 12 h after adjusting pH to 2 by adding concentrated HCl. Afterwards, the precipitates were collected through centrifugation and dissolved in 5 mL of HPLC grade methanol [51]. The crude extract of bacterial secondary metabolites (pH 7.0) was passed through 0.22 µm syringe filter to remove impurities and used for the determination of antifungal activity against VD-080. Fungus was grown on PDA for two days and then 10 µL of bacterial crude extract was pipetted in two sides 2.5 cm away from the fungus. In control treatment, methanol was used instead of crude extract.

Identification of Antifungal Genes Using Molecular Markers
Molecular markers reported by Farzand et al. [15] were used for amplification of genes, viz., iturin, bacillomycin, surfactin, bacillibactin and fengycin, responsible for producing corresponding antimicrobial secondary metabolites (Table 2). These genes were amplified through PCR in a thermal cycler (Bio-Rad, Hercules, CA, USA).

LC-MS Analysis of Antimicrobial Compounds
The crude extract of bacterial endophytes was passed through a 0.22 µm syringe filter and used for the detection of antimicrobial compounds. A surveyor LC-MS-system G2 QTof-XS, a Waters (Santa Clara, CA, USA) was employed following the protocol described by Hajji et al. [52].

Scanning Electron Microscopy
Crude extract of endophytic bacterial isolates was evaluated for its ability to cause structural deformities in fungal mycelium through scanning electron microscope following the protocol of Gu et al. [53]. V. dahliae (VD-080) was cultured for 3 days at 25 • C and 50 rpm in potato dextrose broth.
Afterward, the hyphae were harvested in 2 mL Eppendorf tube and treated with crude methanolic extract (50 µg/mL) of HNH7 and HNH9 for 12 h at 27 ± 2 • C and 180 rpm. Hyphae in the control treatment were treated with HPLC grade methanol only. Furthermore, the Eppendorf tubes were centrifuged, and the hyphae were washed three times with 1× PBS buffer. Finally, the hyphae were preserved in 2.5% (v/v) glutaraldehyde solution. Structural abnormalities of VD-080 were observed through scanning electron microscope [31].

Expression Analysis of Defense-Linked Genes in Cotton Plants through qRT-PCR
The expression of defense-linked genes in cotton plants (Table 3) was studied through qRT-PCR. Cotton leaves from each treatment were used to extract total RNA using total plant RNA extraction kit (Omega Bio-Tek, Norcross, GA, USA). NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA) was employed to measure the concentration and purity of extracted RNA. First-strand cDNA was synthesized using Evo M-MLV reverse transcriptase (Accurate Biology, Hunan, China). The expression of defense-linked genes, viz., HMGR, MPK3, GST, PAL, PPO and SOD, was studied using the SYBR Green Premix Taq HS qPCR kit (Accurate Biology, Hunan, China) in qRT-PCR (QuantStudio-6, California, USA). Sequences of defense-linked genes were obtained from NCBI [56].. The expression of genes was normalized by using actin as a housekeeping gene. Final expression of the target genes was quantified in three repeats using 2 −∆∆Ct method derived from Livak and Schmittgen [57].

Statistical Analysis
All the experiments were carried out in a completely randomized design (CRD) and repeated thrice. Data were subjected to one-way ANOVA using SPSS software. The treatment means were compared and separated using LSD test at p ≤ 0.05. Mitogen-activated protein kinase 3 MPK3-F AAATACCCTAAGCCATCCACC MPK3-R CCAACCCAATTCCCATTTGTG 3 Glutathione-S-transferase GST-F TCAGTGCTTTCCTACCCTTTG GST-R ATACCCAACAGAGCTAGCAAC 4 Phenylalanine ammonia-lyase PAL-F ATGTTTGCTCAGTTTTCGGAAC PAL-R GGCACTTTGAACATGGTTGG 5 Polyphenol oxidase PPO-F GAGTCAAGGTTCGTGATAGCC PPO-R GGTGATGTTCTTTGTTTCGGC 6 Superoxide dismutase SOD-F CTGCCTCTGTCTCGATCATTG SOD-R ACCTTTCTGAATAGCCTCATGG 7 Actin Actin-F CGAGACATTGGGACAGGTATTG Actin-R GAGATCACGACCAGCAAGG

Conclusions
Conclusively, the cotton endophytes B. altitudinis and B. velezensis (HNH7 and HNH9) possessed the powerful capabilities to synthesize different kinds of antimicrobial metabolites and elicitation of ISR to show strong antagonistic activities against Verticillium dahliae. Moreover, transcriptional profiling of defense-linked genes, viz., HMGR, MPK3, GST, PAL, PPO and SOD, through RT-qPCR showed induction of the plant defense mechanism. These results indicated that interactions between HNH7 and HNH9 provide efficient antibiotic-mediated inhibition of the VD-080 pathogen. These strains can be used to formulate biopesticides in the future for controlling verticillium wilt disease.