Suppression of Pepper Root Rot and Wilt Diseases Caused by Rhizoctonia solani and Fusarium oxysporum

Pepper is vulnerable to soil-borne fungal pathogens such as Rhizoctonia solani and Fusarium oxysporum. The potential of beneficial rhizosphere microorganisms to control R. solani and F. oxysporum f.sp. capsici was evaluated in pepper plants. Paenibacillus polymyxa and Trichoderma longibrachiatum were isolated from rhizospheric soil samples of healthy pepper plants. In vitro, both isolates caused clear reductions in the radial growth of root rot and wilt pathogens. Scanning electron microscopy displayed lysis and abnormal shape of the pathogens in dual cultures with P. polymyxa and T. longibrachiatum. The incidence and severity of root rot and wilt diseases were significantly reduced in pepper plants treated with the growth-promoting fungi (PGPF isolates; Fusarium equiseti GF19-1, Fusarium equiseti GF18-3, and Phoma sp. GS8-3), P. polymyxa, or T. longibrachiatum in comparison to the control. Moreover, the induction treatments led to increased pepper growth compared with their control. The defense related gene (CaPR4) expression was shown to be significantly higher in the treated plants than in the control plants. In conclusion, the antagonistic isolates (P. polymyxa and T. longibrachiatum) and PGPF isolates have a clear impact on the prevention of root rot and wilt diseases in pepper plants incited by R. solani and F. oxysporum f.sp. capsici. The expression of the CaPR4 gene added to the evidence that PGPF isolates generate systemic resistance to pathogen infections.


Introduction
Pepper (Capsicum annuum L.) is a commonly cultivated crop, from the family Solanaceae, in Egypt and all over the world. In agricultural production, soil-borne pathogens are the main problems causing reductions in crop yield and quality. The most dreaded diseases of vegetables are root rot, damping-off, charcoal rot, and wilt caused by Rhizoctonia solani, Life 2022, 12, 587 3 of 15 utilized for molecular identification. DNA was extracted by using the protocol of GeneJet genomic DNA purification Kit (Thermo K0721) as described by Elsharkawy and El-Sawy (2019). PCR was performed using the primer F: AGA GTT TGA TCC TGG CTC AG, R: GGT TAC CTT GTT ACG ACT T, Maxima Hot Start PCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) (Thermo K1051) and GeneJET™ PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA USA) (Thermo K0701) following manufacturer protocol. Sequencing was done using ABI 3730xl DNA sequencer as described by Elsharkawy and El-Sawy [12].

Effect of Bacterial and Fungal Antagonists on Root Rot and Wilt Pathogens In Vitro
For antagonistic bacterial isolates, PDA plates were inoculated in the center with agar discs (5 mm) bearing mycelium of 7-days old cultures of R. solani (R) and F. oxysporum f.sp. capsici (F). Plates were concurrently inoculated by bacteria (four isolates per dish). Non-inoculated plates served as control. Plates were kept at 19-22 • C until the control treatment was fully covered with PDA. The diameter of inhibition zone was measured, and the relative power of antibiosis (RPA) was determined using the formula: RPA = Z/C [14]. Z: The diameter of the inhibition zone. C: The diameter of spotted antagonistic isolate.
For fungal antagonists, the dual culture technique was used for testing the antagonistic activity of fungal isolates. PDA plates were inoculated with discs (5 mm) of the pathogenic fungi (one-week old cultures), and on the opposite side, the discs of the antagonistic fungi were placed. Plates containing the pathogens alone have been considered as control. Plates were kept at 18-20 • C until the full growth in control plates. The degree of antagonisms was determined as elucidated by Bell et al. [15,16].

Microscopic Examination
Scanning electron microscopy (SEM) has been utilized to evaluate the effects of Paenibacillus polymyxa and Trichoderma longibrachiatum (the most effective bio-agents in the experiments compared with other bioagents in vitro) on the growth of F. oxysporum f.sp. capsici and R. solani [12]. The samples were immersed for fixation in a modified Karnovsky [17] solution (2.5% buffered glutaraldehyde +2% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4). The tissues have been incubated overnight at 4 • C followed by washing three times in 0.1 M sodium phosphate buffer and 0.1 M sucrose. The tissues were postfixed in 2% sodium phosphate with osmium tetroxide pH 7.4 for 90 min. Sodium phosphate buffer pH 7.4 (0.1 M) was used for washing the samples for three times. Serial dilutions of ethanol were utilized for the dehydration of samples. The samples were put in a critical point drying and specimens were coated with gold-palladium membranes and observed in a Jeol JSM-6510L.V SEM. The microscope was operated at 30 KV in EM Unit, Mansoura University, Egypt.

Preparation of Bacterial Inoculum
Inoculum of the antagonistic isolate was prepared by growing on nutrient broth media in conical flasks (500 mL) at 28 • C for 5 days using shaking incubator (100 rpm). Cell suspension was diluted and adjusted to 10 8 cfu/mL of P. polymyxa.

Plant Growth Conditions
Pot experiments were carried out during two consecutive seasons for studying the effect of the five isolates on root-rot and wilt diseases of pepper. Plastic pots (24 cm in Life 2022, 12, 587 4 of 15 diameter) were filled with autoclave sterilized sandy loam soil infested with one of the two pathogens, F. oxysporum f.sp. capsici or R. solani, at the rate of 2% (w/w, inoculum/soil). Inoculum of each pathogen was mixed separately with soil. Infested pots were irrigated and kept for 5 days before transplanting [4]. Pots were planted with pepper seedlings (3 seedlings per pot).

Induction Treatments a-Seed treatment
Pepper seeds were treated by soaking in 10 mL of the bacterial suspension (10 8 cfu/mL) or fungal spore suspension (2 × 10 6 spore/mL) for 2 h before planting in seedling trays [19]. Seeds were air-dried in sterile petri plates. Three seeds were sown in each well of the tray. Untreated pepper seeds were utilized as a control.
b-Seedling treatment A seedling tray was filled with potting soil and sown with sterilized pepper seeds (using 5% sodium hypochlorite). The 19-d-old seedlings, raised in the tray, were soaked in the suspension of each isolate for 2 h and transferred to small pots (5 cm in diameter) filled with the potting soil. Twenty days later, the seedlings were transplanted in pots artificially infested with the pathogen (2% w/w). As a control treatment, autoclaved PDB was used instead of bioagent suspensions.

Disease Assessment
Survived seedlings were removed, washed, and scored for R. solani as described by O'Sullivan and Kavanagh [20]. For the assessment of Fusarium wilt disease severity, foliar symptoms were evaluated as explained by Horinouchi et al. [5]. Discoloration severity of vascular tissues was assessed as described by Horinouchi et al. [5]. Plant growth parameters such as plant height, number of leaves, and shoot (fresh and dry) weight were determined [6].

Assessment of Defense Enzymes and Total Phenol Contents
Using a liquid nitrogen-cooled mortar and pestle, 1 g of freshly inoculated pepper leaves (3 days after inoculation) were crushed into powder. Subsequently, the obtained powder was macerated for 30 s and homogenized with sodium phosphate buffer (3 mL, pH 6.8, 0.01 M). The filtrates were centrifuged (15 min, 6000 rpm, 4 • C) after filtering the triturated tissues through four layers of cheese cloth. A sample of the clear supernatant was collected for enzyme extraction.
Peroxidase and polyphenol oxidase activities in the collected samples were estimated (min −1 g −1 ) by spectrophotometric analysis [9]. Estimation of total phenol contents (expressed as milligram per gram of sample) was determined [21].

Molecular Investigation of Pathogenesis-Related Genes Expression
Leaves were harvested at 2 days after pathogen inoculation. RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA USA) was used to extract RNA. The extracted RNA was converted to complementary DNA (cDNA) using revert Aid H minus reverse transcriptase. Real-time PCR with SYBR Green was used to measure the expression of mRNAs of the target gene (CaPR4, a gene associated with defense response and cell death), with CaActin as an internal reference as described by Elsharkawy and El-Khateeb [9].

Re-Isolation Frequency
At 7 weeks after planting, root colonization of pepper plants with PGPF isolates was assessed for both PGPF and control treatments. The roots were picked from random plants and cleaned three times using sterile-distilled water before drying with a paper towel. Afterwards, they were sliced into 1-cm pieces and placed onto PDA. The frequency of PGPF was assessed as described by Elsharkawy et al. [6].

Statistical Analysis
The experimental data from trials were combined for analysis of variance. The experiments had been done three times. Means were separated by Fisher's LSD test using XLSTAT Software (Addinosoft).

Isolation and Screening of Fungal and Bacterial Antagonists In Vitro
Five fungal isolates of Trichoderma spp. (T1, T2, T3, T4, and T5) as well as 32 bacterial isolates were isolated from different rhizosphere samples of healthy pepper plants. The isolate T. longibrachiatum (T1) proved to have the highest antagonistic effect on the pathogens ( Figure 1A,B and Table 1). Among 32 bacterial isolates, six bacterial isolates had significant antagonistic effects on the linear growth of F. oxysporum f.sp. capsici in Petri dish ( Table 2). The highest inhibitory spectrum between the isolates was P. polymyxa (B25) ( Figure 1C). Six bacterial isolates had significant antagonistic effects on the linear growth of R. solani. The highest inhibitory spectrum of the isolates was P. polymyxa (B25) ( Figure 1D). assessed for both PGPF and control treatments. The roots were picked from random plant and cleaned three times using sterile-distilled water before drying with a paper towe Afterwards, they were sliced into 1-cm pieces and placed onto PDA. The frequency o PGPF was assessed as described by Elsharkawy et al. [6].

Statistical Analysis
The experimental data from trials were combined for analysis of variance. The exper iments had been done three times. Means were separated by Fisher's LSD test usin XLSTAT Software (Addinosoft).

Isolation and Screening of Fungal and Bacterial Antagonists In Vitro
Five fungal isolates of Trichoderma spp. (T1, T2, T3, T4, and T5) as well as 32 bacteria isolates were isolated from different rhizosphere samples of healthy pepper plants. Th isolate T. longibrachiatum (T1) proved to have the highest antagonistic effect on the patho gens ( Figure 1A,B and Table 1). Among 32 bacterial isolates, six bacterial isolates had sig nificant antagonistic effects on the linear growth of F. oxysporum f.sp. capsici in Petri dis ( Table 2). The highest inhibitory spectrum between the isolates was P. polymyxa (B25) (Fig  ure 1C). Six bacterial isolates had significant antagonistic effects on the linear growth o R. solani. The highest inhibitory spectrum of the isolates was P. polymyxa (B25) ( Figure 1D) subtilis is B31, and Paenibacillus polymyxa is B25.

Identification of the Most Efficient Fungal and Bacterial Isolates
The phylogenetic tree exhibited that the Trichoderma isolate T1 was strongly related to the species longibrachiatum. It showed the highest sequence similarities with T. longibrachiatum (GenBank accession number OM666052) ( Figure 2). On the other hand, the phylogenetic tree showed the relation between Paenibacillus isolate and the related bacterial species (Figure 3). It can be clearly seen that the Paenibacillus isolate B25 was highly related to the species polymyxa. It showed the highest sequence similarities with P. polymyxa strain B25 (GenBank accession number OM666057).

Identification of the Most Efficient Fungal and Bacterial Isolates
The phylogenetic tree exhibited that the Trichoderma isolate T1 was strongly related to the species longibrachiatum. It showed the highest sequence similarities with T. longibrachiatum (GenBank accession number OM666052) ( Figure 2). On the other hand, the phylogenetic tree showed the relation between Paenibacillus isolate and the related bacterial species (Figure 3). It can be clearly seen that the Paenibacillus isolate B25 was highly related to the species polymyxa. It showed the highest sequence similarities with P. polymyxa strain B25 (GenBank accession number OM666057).

Scanning Electron Microscopy (SEM)
The SEM examination showed a complete fungal growth of F. oxysporum f.sp. capsici ( Figure 4A,B). Full coalescence was developed from the mycelia in their ideal form in control. However, the dual culture with T. longibrachiatum showed morphological anomaly and coiling of F. oxysporum f.sp. capsici ( Figure 4C

Scanning Electron Microscopy (SEM)
The SEM examination showed a complete fungal growth of F. oxysporum f.sp. capsici ( Figure 4A,B). Full coalescence was developed from the mycelia in their ideal form in control. However, the dual culture with T. longibrachiatum showed morphological anomaly and coiling of F. oxysporum f.sp. capsici ( Figure 4C,D). At the same time, P. polymyxa demonstrated morphological abnormality such as degradation and lysis in the fungal mycelia ( Figure 4E,F).  Typical morphological characteristics of R. solani was observed using SEM ( Figure  5A,B). In contrast, overgrowth and lysis were observed in dual cultures of R. solani and T. longibrachiatum ( Figure 5C,D), while lysis and atrophy were observed in dual cultures of R. solani and P. polymyxa ( Figure 5E,F). Typical morphological characteristics of R. solani was observed using SEM ( Figure 5A,B). In contrast, overgrowth and lysis were observed in dual cultures of R. solani and T. longibrachiatum ( Figure 5C,D), while lysis and atrophy were observed in dual cultures of R. solani and P. polymyxa ( Figure 5E,F).  Table 3 showed that all the examined bioagents significantly reduced disease incidence and severity of wilt and root rot in comparison to their control as a result of seed and seedlings treatments. The most successful treatment for Fusarium wilt severity was GF19-1 as a seedling treatment, which gave disease severity 27.8% and 25.0% in the first and second seasons, respectively. The most helpful treatments for disease incidence caused by F. oxysporum f.sp. capsici were GF19-1 and GF18-3 as seedling treatments, which gave the same disease incidence in both seasons (27.8%). However, the most successful treatment on R. solani was GF18-3 as seedling treatment, which gave a disease severity percentage of 27.77% in both seasons. Likewise, the most useful treatment for disease incidence of Rhizoctonia root rot was GF18-3 as seedling treatment, which gave a disease incidence of 27.8%. Discoloration severity was estimated at the end of the experiment. Discoloration severity was reduced in the treatments with GF19-1 and GF18-3 as seedling treatments recording 14.8% in the second season (Table 4).   Table 3 showed that all the examined bioagents significantly reduced disease incidence and severity of wilt and root rot in comparison to their control as a result of seed and seedlings treatments. The most successful treatment for Fusarium wilt severity was GF19-1 as a seedling treatment, which gave disease severity 27.8% and 25.0% in the first and second seasons, respectively. The most helpful treatments for disease incidence caused by F. oxysporum f.sp. capsici were GF19-1 and GF18-3 as seedling treatments, which gave the same disease incidence in both seasons (27.8%). However, the most successful treatment on R. solani was GF18-3 as seedling treatment, which gave a disease severity percentage of 27.77% in both seasons. Likewise, the most useful treatment for disease incidence of Rhizoctonia root rot was GF18-3 as seedling treatment, which gave a disease incidence of 27.8%. Discoloration severity was estimated at the end of the experiment. Discoloration severity was reduced in the treatments with GF19-1 and GF18-3 as seedling treatments recording 14.8% in the second season (Table 4).

Effect of Different Bioagents on Some Plant Growth Parameters
A significant increase in plant height, number of leaves, fresh and dry weights (g)/plant was verified due to biotic inducers. All treatments significantly improved growth parameters in plants infected with F. oxysporum ( Table 5). The most successful treatment was GF19-1 (SL, seedling treatment) in the plant height and the number of leaves in both seasons. The treatment with GF18-3 (SL, seedling treatment) was the best for increasing fresh and dry weights/plant in comparison to their control in both seasons. However, in the case of R. solani infection, the most useful treatment was GF18-3 as seedling treatment in increasing plant height, the number of leaves, fresh and dry weights/plant compared with untreated control in both seasons (Table 6).

Effect of the Biological Inducers on the Activation of Defense Enzymes
Significant differences were found in phenol, peroxidase and polyphenol oxidase ( Table 7). Application of GF19-1 as seedling treatment produced the highest enzyme values (peroxidase and polyphenol oxidase) and total phenols in case of F. oxysporum. However, in case of R. solani, the highest activities of peroxidase, polyphenol oxidase and total phenols were achieved by the treatments GF19-1 and GF18-3 as seedling treatment. Control treatment (pathogen only) showed the lowest values.

Effect of Induction Treatments on the Relative Expression of PR4 Gene
Our results revealed a significant (p ≤ 0.05) increase of CaPR4 gene expression level in treated pepper plants compared to the control. Plants treated with GS8-3 showed the highest expression levels in the case of F. oxysporum and R. solani. In addition, plants infected with F. oxysporum and treated with GF18-3 resulted in the lowest up-regulation of CaPR4. However, under the infection with R. solani, treatment with GF19-1 presented the lowest gene expression ( Figure 6).

Discussion
Pepper (Capsicum annuum L.) is a major commercial crop all over the world, with a huge societal and economic impact. It is one of Egypt's most popular vegetable crops, cultivated either in open fields or in plastic greenhouses under a protected farming sys-

Discussion
Pepper (Capsicum annuum L.) is a major commercial crop all over the world, with a huge societal and economic impact. It is one of Egypt's most popular vegetable crops, cultivated either in open fields or in plastic greenhouses under a protected farming system. Pepper plants may be infected with various pathogens, including soil-borne pathogens [22]. In the greenhouse and fields of pepper plants, many fungal isolates from the genera Fusarium, Macrophomina, Rhizoctonia, Verticilium, Pythium, and Sclerotinia frequently cause damping-off, root rot, and wilt diseases [23,24]. Many researchers have observed that Fusarium wilt of pepper, caused by Fusarium spp., has resulted in significant reductions in pepper production in many countries around the world [22]. The use of a sustainable disease management strategy is necessary to reduce the impact of these diseases. Biological control is a long-term approach for disease management and healthy pepper production. P. polymyxa and T. longibrachiatum were isolated and identified by morphological and molecular biology techniques. Similarly, P. putida strain F1 was successfully identified based on the sequence of 16S rDNA comparing the sequence similarities with the related bacterial species in Gene bank [12]. In terms of antagonistic ability, the obtained findings showed that all of the tested bio-agents could significantly decrease the linear growth of F. oxysporum f.sp. capsici and R. solani. The results are in agreement with Sahii and Khalid [25], who found that the mycelial growth of F. oxysporum was hampered as a response to the antagonistic effect of Trichoderma sp. The R. solani mycelial diameter, as well as infection with root rot and damping-off diseases, were substantially decreased by T. harzianum isolate [26].
Many researchers have been interested in biological control mechanisms in recent decades. Pathogens are directly challenged by bioagents via hyperparasitism, antibiotic synthesis, and lytic enzyme production, as well as indirectly through competition for space and nutrients, developing systemic resistance, and encouraging plant development [27]. Using a scanning electron microscope, we discovered overgrowth and lysis of F. oxysporum f.sp. capsici and R. solani in dual cultures with T. longibrachiatum, as well as morphological anomalies such as atrophy and lysis using P. polymyxa in both fungal mycelia. Parasitism of pathogen fungi was reported by Trichoderma species in other studies [13]. Scanning electron microscopic analysis revealed that T. harzianum strains antagonist with R. solani [13]. T. harzianum Th-9 isolate overgrew and coiled around the R. solani cells, invading and damaging the host hypha. Through the mechanical activity, the host cells are penetrated. Secretion of antifungal compounds has been found to prevent the growth of different plant pathogens [7].
Plant growth promoting fungi (PGPF) is a kind of saprophyte that lives in the soil and promotes plant development. As a consequence of seed and seedling treatments, all of the evaluated biocontrol agents substantially decreased the severity of Fusarium wilt and root rot caused by F. oxysporum f.sp. capsici and R. solani compared to the control treatment. In several plants-PGPF combinations, colonization of roots with PGPF leads to a state of resistance in the whole plant known as induced systemic resistance. ISR in different plant species was introduced such as Arabidopsis thaliana, cucumber, and tobacco by PGPF application [6]. Curiously, PGPF isolates of Penicillium simplicissmum GP17-2, Trichoderma asperellum SKT-1, Phoma sp. GS 8-3, F. equiseti GF18-3, and Phoma sp. GS8-1 were highly effective in reducing the disease severity of white rot disease of onions [9]. In this study, the protective method of both types of biocontrol agents as individual treatments resulted in a substantial decrease in the disease. Cucua et al. [28] evaluated the efficacy of two biological control agents (BCAs) in suppressing F. oxysporum f.sp. lycopersici (Bacillus subtilis QST 713 and Trichoderma spp. TW2). Additionally, P. polymyxa NSY50 application on cucumber plants infected with F. oxysporum successfully decreased the incidence of Fusarium wilt [29]. In this study, the increase of phenolics and PR-Proteins such as peroxidase (PO), and polyphenoloxidase (PPO) inside pepper roots may have helped to limit F. oxyporum and R. solani infections. The accumulation levels of defense enzymes and the transcription levels of PR1 and PR5 genes were increased in cucumber plants treated with T. atroviride (TRS25) and led to better resistance against R. solani [30]. The fact that induction treatments substantially increased CaPR4 gene expression suggests that this gene is involved in systemic resistance to F. oxysporum and R. solani. JA and ET activated PR4, PR5, and PDF1.2 in a synergistic manner [6]. Induced systemic resistance mediated by P. simplicisimum GP17-2 in Arabidopsis and tobacco enhanced the expression of different pathogenesis-related genes [6].
All treatments significantly enhanced growth characters in plants relative to the control infected with F. oxysporum and R. solani. PGPF has been shown to improve plant growth and disease control [6]. Several studies showed that Trichoderma isolates were considered proper biofertilizers, since they could improve the capacity of nutrients uptake in plants and the resistance toward plant pathogen [9]. Furthermore, P. polymyxa NMA1017 promoted plant growth through nitrogen fixation and siderophore synthesis, which led to increased crop production [31].

Conclusions
There is a growing request for safe biocontrol agents to improve management strategies of soil-borne diseases to reduce the poisonous effects of pesticides. The results indicated that the antagonistic isolates from healthy pepper plants and PGPF isolates can effectively control the pathogens, F. oxysporum f.sp. capsici and R. solani, as well as increasing growth parameters in pepper plants. The activities of oxidative enzymes (Peroxidase and polyphenol oxidase), phenol contents, and the expression levels of CaPR4 were stimulated in the treated pepper plants, leading to induced resistance against F. oxysporum f.sp. capsici or R. solani. The ability to use these treatments to manage root rot and wilt diseases of pepper was already improved as a result of increased pepper growth.