Enhancing Sustainable Control of Meloidogyne javanica in Tomato Plants: Leveraging Gamma Radiation-Induced Mutants of Trichoderma harzianum and Bacillus velezensis, with Optimal Combination Strategies

doi:10.21203/rs.3.rs-4191816/v1

This study investigates the efficacy of Trichoderma spp. and Bacillus spp., as well as their gamma radiation-induced mutants, as potential biological control agents against Meloidogyne javanica in tomato plants. The research encompasses in vitro assays, greenhouse trials, and molecular identification methodologies to comprehensively evaluate the biocontrol potential of these agents. In vitro assessments reveal significant nematicidal activity, with Bacillus spp. demonstrating notable effectiveness in inhibiting nematode egg hatching (16-45%) and inducing second-stage juvenile mortality (30-46%). Greenhouse trials further confirm the efficacy of mutant isolates, particularly when combined with chitosan, in reducing nematode-induced damage to tomato plants. The combination of mutant isolates with chitosan reduces the proliferation rate (RF) of root-knot nematodes by 94%. By optimizing soil infection conditions with nematodes and modifying the application of the effective compound, the RF of nematodes decreases by 65-76%. Molecular identification identifies Bacillus velezensis and Trichoderma harzianum as promising candidates, exhibiting significant nematicidal activity. Overall, the study underscores the potential of combined biocontrol approaches for nematode management in agricultural settings. However, further research is essential to evaluate practical applications and long-term efficacy. These findings contribute to the development of sustainable alternatives to chemical nematicides, with potential implications for agricultural practices and crop protection strategies.

Biological sciences/Microbiology

Biological sciences/Plant sciences

Biological sciences/Zoology

Earth and environmental sciences/Environmental sciences

Bacillus velezensis

biocontrol potential

combined biocontrol approaches

gamma radiation-induced mutants

Meloidogyne javanica

Trichoderma harzianum

Root-knot nematodes (Meloidogyne spp.) are a type of plant parasite that cause considerable damage to crops worldwide. Among them, Meloidogyne javanica is a particularly widespread threat that can infest a wide range of host plants and cause significant yield losses. Conventional methods of nematode control, often based on chemical nematicides, pose environmental risks and raise concerns about their long-term efficacy and safety. Alternative approaches are therefore increasingly being explored, with biological control emerging as a promising avenue. Utilizing the antagonistic properties of beneficial microorganisms offers a sustainable and environmentally friendly solution to reduce nematode damage while maintaining soil health and ecosystem integrity (Subedi, Thapa, & Shrestha, 2020).

Microorganisms are integral to soil vitality, fostering plant growth, and development, and aiding in disease prevention. Leveraging their inherent mechanisms, these microorganisms can serve as effective biocontrol agents against various soil-borne pathogens. Trichoderma spp. and Bacillus spp. are well-documented biocontrol agents known for their antagonistic activity against various plant pathogens, including nematodes. These microorganisms exert their effects through various mechanisms, such as competition for nutrients and space, production of antimicrobial compounds and stimulation of plant defense responses. In addition, their ability to colonize the rhizosphere and engage in mutualistic interactions with host plants further enhances their effectiveness in suppressing nematode populations (Bhat, Shakeel, Waqar, Handoo, & Khan, 2023). Past research has illustrated the considerable efficacy of fungal spores, hyphae, and metabolites from various strains of Trichoderma in combating root-knot nematodes, showing significant promise (TariqJaveed, Farooq, Al-Hazmi, Hussain, & Rehman, 2021). Also, Bacillus spp. has been shown to enhance plant growth parameters and reduce root-knot nematode damage(El-Nagdi & Abd-El-Khair, 2019).

In recent years, advances in genetic manipulation have enabled the development of mutant strains of microorganisms with improved biocontrol capabilities. Through targeted genetic modification, these mutants exhibit improved traits tailored to control specific plant pathogens, including nematodes. By exploiting this genetic diversity, the researchers aim to optimize the efficacy and sustainability of biological control strategies to combat plant parasites (Youssef & Hassabo, 2020). As a mutagenic agent, gamma radiation provides a powerful tool for generating genetic variability and new microbial variants with desired traits. By exposing mutant strains of Trichoderma and Bacillus to gamma radiation, researchers seek to improve their biocontrol effect against plant pathogens while ensuring the stability and safety of these modified organisms in agricultural ecosystems (Manikandan et al., 2022; Soufi, Safaie, Shahbazi, & Mojerlou, 2021).

This project offers a thorough investigation into the efficacy of mutant strains of Trichoderma harzianum and Bacillus velezensis, which have been subjected to gamma radiation, for the biological control of M. javanica in tomato plants. It aims to propose optimal application methods to effectively mitigate root-knot nematode damage. Utilizing a combination of in vitro assays, greenhouse trials, and molecular identification methodologies, we seek to demonstrate and assess their potential as sustainable alternatives to chemical nematicides.

Preparation of nematode and microorganisms

Roots infected with Meloidogyne sp. were harvested from the greenhouse in Alborz province. Employing the single egg mass technique, the nematodes were cultured on tomato (Lycopersicon esculentum Mill. cv. Early Urbana) roots. The perineal pattern of mature females served as the basis for identifying the root-knot nematode species (Eisenback, 1985). Nematode eggs were extracted from the galled roots using sodium hypochlorite (Hussey & Barker, 1973). After rinsing with tap water, the infected roots were sliced into 2–3 cm-long segments and blended with 0.5% NaOCl solution. The blending process lasted for 30 seconds at low speed, after which the mixture was sieved successively through 20-, 200-, and 500-mesh/in. sieves. Eggs retained on the 500-mesh sieve were gently rinsed with water to remove NaOCl residue and collected in a Petri dish.

Trichoderma spp. and Bacillus spp., including both wild-type strains and gamma radiation-induced mutants, were obtained from the Nuclear Science and Technology Research Institute of Iran. The antagonistic ability of isolates against some plant pathogens has been proven (Sahampoor, Zaker Tavallaie, Fani, & Shahbazi, 2020, Afsharmanesh et al., 2013). Identification was carried out through a combination of biochemical assays and morphological examinations (Miranda, Martins, & Clementino, 2008; Rifai, 1969). Purified cultures of Trichoderma were cultivated on Potato Dextrose Agar (PDA) medium and preserved at 4ºC for future applications. As for the Bacillus strain, long-term storage was facilitated at -70°C in Nutrient Broth supplemented with 30% glycerol.

Screening of microorganism's activity against nematode

In vitro evaluation of the nematicidal ability of Trichoderma spp. and Bacillus spp.

Two isolates, Trichoderma NAS120 and NAS120-M44, were cultured in Potato Dextrose Broth (PDB) in 250 ml flasks for one week at a temperature of 28°C±2 in a shaker-incubator. Subsequently, 100 ml of each culture was centrifuged for 20 minutes at 1500 rpm and filtered through 0.45 μm filters (Whatman™). The resulting filtered cultures were then assessed on a Potato Dextrose Agar (PDA) medium to ensure they were free from cells (Saharan, Patil, Yadav, Kumar, & Goyal, 2023). Filtered and cell-free cultures were used for in vitro evaluation.

To investigate the impact of bacteria (NAS-B1, NAS-B419, and NAS-B600), fungi (NAS120 and NAS120-M44), and filtered cultures of fungi on nematode egg hatching and larval mortality, 100 eggs and 100 second-stage juveniles (J2) were individually placed in one milliliter of sterile water in separate 6 cm Petri dishes. Subsequently, 5 ml of suspension containing bacteria (10⁸ CFU/ml) and fungi (10⁷ CFU/ml), along with other treatment solutions, were added. Distilled water and Abamectin 1.8% emulsifying liquid (Aria Chemical Company) served as the control in both experiments, and the number of dead larvae was recorded after 48 hours, while unhatched eggs were counted after 72 hours (Cayrol, Djian, & Pijarowski, 1989). The experiments were conducted using a completely randomized design with four replications.

Evaluation effect of Trichoderma spp. and Bacillus spp. against nematode in greenhouse condition

Greenhouse test 1

Tomato seeds (Early Urbana) were sown in 3 kg plastic pots filled with a mixture of soil (field soil and river sand, 1:2 ratio). The experiment followed a completely randomized design with four replications. When the tomato seedlings reached the four-leaf stage, they were inoculated by applying 20 ml of bacterial suspension (108 CFU/ml) and fungal suspension (107 CFU/ml), along with other treatments, to the soil of each pot. Three days later, the roots of the plants were inoculated with 6000 seeds of M. javanica. Pots containing nematodes but no treatment served as controls. Additionally, to assess the treatments' impact on plant growth, all treatments were applied without nematodes. The application of biocontrol agents was repeated every 20 days (Feyisa, Lencho, Selvaraj, & Getaneh, 2019). Throughout the experiment, pots were monitored daily and watered as needed. After sixty days following nematode inoculation, plant growth and nematode parameters were assessed (Rostami, Karegar, & Taghavi, 2021).

Greenhouse test 2

The experimental conditions and methodology mirrored those of the preceding study, albeit with distinct treatments. In this segment, a combination of potent bacteria, effective fungus, and chitosan was employed. Chitosan solution (0.1%) was prepared using the commercial chitosan formulation obtained from Sigma Aldrich. The chitosan solution was dissolved in water overnight using a stirrer until achieving a homogeneous mixture. Subsequently, after 24 hours of incubation, fungal suspension (107 CFU/ml) and potent bacterial suspension (108 CFU/ml) were introduced into the solution for further utilization (Palazzini et al., 2022). Additionally, a commercial biological nematocide (Tisan BT) from Royan Tisan Sam Company was included in the treatments to assess the efficacy of the effective microorganisms (0.1 mL/L).

Greenhouse test 3

The conditions and methodology of this experiment closely paralleled those of the previous study, albeit with distinct treatments. Two methods of plant infection with nematodes were implemented: (1) Cultivating plants in nematode-infested soil, and (2) Cultivating plants in healthy soil and subsequently inoculating them at the four-leaf stage. To prepare pots containing contaminated soil, soil from previous cultivations was assessed and nematode counts were determined. Each three-kilogram pot was then filled with soil containing 6000 nematode eggs and larvae. Pots without nematodes were filled with three kilograms of healthy soil, and upon planting, at the four-leaf stage, they were inoculated with 6000 nematode eggs and larvae. Subsequently, three-leaf plants were cultivated in all pots.

For treatments involving the application of effective bacteria, fungus, and chitosan, where plant roots needed to be immersed in the compound, roots were submerged in 50 ml of the combined solution for 30 seconds before planting. Treatments requiring the mixture to be poured at the base of the plant were administered post-inoculation. These treatments were repeated every 20 days by applying the mixture at the plant base. Pots were monitored daily and watered as necessary. After sixty days post-nematode inoculation, plant growth and nematode parameters were assessed. Figure 1 visualizes these methods.

To assess the reproducibility of results, the treatments pertaining to nematode-infested soil were replicated in the subsequent greenhouse investigation. Given that plant cultivation in contaminated soil closely mirrors natural conditions, this step was crucial. The experimental setup remained identical to previous tests, and the plants were harvested 60 days post-planting and treatment application.

Statistical analysis

The SAS statistical software (version 9.1) was employed for data analysis. Parametric indices (plant indices) were analyzed using the Proc ANOVA method, while non-parametric indices (nematode indices) were assessed using the Friedman rank test. Mean values were compared using a posthoc Tukey HSD (Honestly Significant Difference) test (P < 0.05).

Identification of effective microorganisms

Bacterial DNA extraction was carried out using the Expin™ Combo GP DNA extraction kit from GeneAll® (Tic Tech Centre, Singapore), following the manufacturer's protocol. For fungal DNA extraction, the method described by Ghasemi et al. (2020) was employed. The quality and quantity of the extracted DNAs were assessed spectrophotometrically and adjusted to a concentration of 50 ng/μL using the Nanodrop ND-100 (Nanodrop Technologies, Waltham, Massachusetts, USA).

The universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for the amplification of the complete 16S rDNA. In addition, for fungal samples, the primers 5'-CGTAGGTGAACCTGCGG-3' and 5'-TCCTCCGCTTATTGA TATGC-3' were used to amplify the ITS rDNA region, and the primers 5'-CATCGAGAAGTTCGAGAAGG-3' and 5'-TACTTGAAGGAACCCTTACC-3' were used to amplify the TEF-1α region.

For bacterial samples, the PCR reaction mixture comprised 1 μL of DNA (50 ng/μL), 1 μL each of forward and reverse primers (10 μM), 10 μL of Ampliqon® Taq DNA Polymerase Master Mix Red (Ampliqon A/S, Odense, Denmark), and 7 μL of double-distilled water. For fungal samples, PCR reactions were conducted in a 20 µL volume consisting of 10 µL of Master Mix, 0.2 µM of each primer for amplification of the TEF-1α and ITS regions, and 10 ng of DNA from each isolate (Abbasi, Safaie, Shams-Bakhsh, & Shahbazi, 2016). Subsequently, PCR products were sequenced by Microsynth Company, Switzerland.

The obtained sequences were analyzed using BLASTn (NCBI: http://blast.ncbi.nlm.nih.gov). Sequences of related species and genera were retrieved from the GenBank database, and phylogenetic analysis was conducted using MEGA version 7 (S. Kumar, Stecher, & Tamura, 2016). Sequence alignment was performed using Clustal W (Larkin et al., 2007), and the Maximum Likelihood Method was employed to construct a phylogenetic tree depicting the relationships among isolates, with percentage bootstrap values derived from 1000 replicates (Saitou, 1988).

In vitro Assessment of Nematicidal Efficacy of Trichoderma spp. and Bacillus spp.

The findings from the in vitro investigation revealed significant differences among treatments in terms of their efficacy in inhibiting nematode egg hatching and J2 mortality. Abamectin, Bacillus NAS-B1, and Bacillus NAS-B600 treatments exhibited notable effectiveness in both preventing egg hatching and reducing J2 mortality. Particularly, Bacillus NAS-B600 demonstrated the highest efficacy, reducing nematode egg hatching by 45% compared to the control. Abamectin treatment, Bacillus NAS-B419, Trichoderma NAS120, and Bacillus NAS-B1 also displayed considerable efficacy, reducing egg hatching by 34%, 31%, 24%, and 16%, respectively (Figure 2). Also, Abamectin treatment, Bacillus NAS-B1, Bacillus NAS-B600, and the filtered, cell-free culture of Trichoderma NAS120-M44 exhibited mortality rates of 59%, 46%, 30%, and 19%, respectively, among the J2 of nematode (Figure 2). These percentages were determined by subtracting the number of eggs or J2 in the respective treatment from those in the control. Figure 3 provides a visual representation of the J2 treated with bacteria and fungi.

Efficacy of Trichoderma spp. and Bacillus spp. against Nematode in Greenhouse Conditions

Greenhouse test 1

Plant Growth Parameters: Among the growth indices assessed in this experiment, only the dry weight of tomato shoots exhibited a notable difference across treatments. Notably, treatment with mutant bacteria Bacillus NAS-B419 significantly enhanced the shoot dry weight of infected plants compared to the infected control plants. Conversely, the remaining treatments yielded similar effects on the shoot dry weight of infected plants, showing no significant deviation from the control (Table 1 and Figure 4).

Nematode Parameters: Abamectin, Bacillus NAS-B419, and Trichoderma NAS120-M44 treatments elicited a substantial decrease in the number of galls, egg masses, and eggs within the root system, as well as in the presence of J2 in the soil, ultimately leading to a reduction in the overall nematode population. These treatments notably reduced the nematode reproduction factor by 77% to 82%. Additionally, Trichoderma NAS120 and Bacillus NAS-B1 treatments demonstrated efficacy in diminishing the number of J2 in the soil and the occurrence of galls within the root system, respectively (Table 1).

Greenhouse test 2

Plant Growth Parameters: Analysis of both the dry and fresh weight of tomato shoots in this experiment did not reveal any significant differences across treatments. However, a notable distinction was observed between the group infected with nematodes and the group without nematode inoculation, reaching significance at the 5% level (Figure 4).

Nematode Parameters: Abamectin, Trichoderma NAS120-M44, and Bacillus NAS-B419 treatments, along with their combinations, notably reduced the number of egg masses, eggs within the root system, and J2 in the soil, leading to a significant decrease in the overall nematode population. These treatments achieved a remarkable reduction in the nematode reproduction factor, ranging from 92% to 94%. Particularly, the combination of Bacillus NAS-B419, Trichoderma NAS120-M44, and chitosan exhibited the highest reduction in the reproduction factor (Table 2).

Greenhouse test 3

Plant Growth Indicators: Both the dry and fresh weights of tomato shoots exhibited significant differences among treatments in this experiment. Specifically, treating the roots of plants with the combination of Bacillus NAS-B419, Trichoderma NAS120-M44, and chitosan resulted in a noteworthy improvement in both the fresh and dry weights of shoots in 'four-leaf stage of plant inoculated' specimens compared to 'nematode infected soil' plants. Furthermore, applying the combination to plants cultivated in infected soil also led to an increase in both the fresh and dry weights of shoots. Conversely, other treatments demonstrated similar effects on the fresh and dry weights of infected plant shoots, showing no significant deviation from the control (Table 3 and Figure 4).

Nematode Parameters: Impregnating plant roots with the combination of Bacillus NAS-B419, Trichoderma NAS120-M44, and chitosan in nematode-infested soil, along with inoculating plants at the four-leaf stage, resulted in a significant reduction in the final nematode population. These treatments achieved a notable decrease in the nematode reproduction factor by 65% and 69%, respectively (Table 3). Given the higher rate of nematode multiplication observed in treatments related to plants grown in contaminated soil, which closely resembles natural conditions, the treatment involving the mentioned combination was repeated in contaminated soil to verify the reproducibility of the test results.

Repetition of the test showed that impregnation of plant roots with the effective combination of Bacillus NAS-B419 + Trichoderma NAS120-M44 + chitosan significantly in infected nematode soil improved the fresh and dry weight of plant shoots compared to infected control plants. (Table 4 and Figure 5).

Both treatments, involving impregnating plant roots in nematode-infested soil with the combination of Bacillus NAS-B419 + Trichoderma NAS120-M44 + chitosan, and applying the combination directly into the soil around the plant, resulted in a decrease in the final population of nematodes. Notably, dipping the roots of plants in the solution led to a higher reduction in the reproduction factor (76%), as indicated in Table 4.

Molecular Identification of Effective Bacteria and Fungi

Isolates were identified through a comparison of their 16S rDNA and ITS sequences with those archived in the GenBank database. The bacterial isolates were identified as B. velezensis, while the fungi were identified as T. harzianum. The sequences of their 16S rDNA, ITS-rDNA and, TEF-1α regions have been deposited in the GenBank (Table 5). The phylogenetic relationships among the identified bacterial isolates, fungi, and closely related species and genera are depicted in Figures 6 and 7.

The phylogenetic tree presented in Figure 6 illustrates the evolutionary relationships among various Bacillus strains, with a particular focus on the isolates NAS-B1 and NAS-B419. These isolates are prominently marked and show a close genetic relationship with other strains within the B. velezensis clade. The tree is rooted with Clostridium butyricum as an out-group, providing context for the evolutionary divergence of the studied strains.

The phylogenetic tree depicted in Figure 7 represents the evolutionary relationships among Trichoderma isolates, with a focus on ‘NAS120’ and ‘NAS120-M44’, as determined by ITS-rDNA and TEF-1α gene regions. The tree elucidates the genetic diversity within the genus, highlighting the distinct lineage of the two isolates in question.

The present study aimed to evaluate the efficacy of strains of Trichoderma spp. and Bacillus spp., and their mutants that were generated through gamma radiation, for the biological control of M. javanica in tomato plants. The investigation encompassed a comprehensive approach, integrating in vitro assays, greenhouse trials, and molecular identification methodologies, to elucidate the potential of these biocontrol agents as sustainable alternatives to chemical nematicides.

The in vitro evaluation of the nematicidal activity of Trichoderma spp. and Bacillus spp. gave promising results. Filtered cultures of Trichoderma NAS120-M44 showed significant antagonistic activity against second-stage juveniles, while bacterial strains NAS-B1, NAS-B419 and NAS-B600 showed considerable efficacy in suppressing nematode populations. These results are in agreement with previous studies emphasizing the biocontrol potential of Trichoderma and Bacillus strains against various plant pathogens, including nematodes (Basumatary, Das, Choudhury, Dutta, & Bhattacharyya, 2021; Saharan et al., 2023). The antagonistic effect of Bacillus was demonstrated in an in vitro test, that significantly inhibited the hatching of nematode eggs (Rostami et al., 2021). In addition, T. asperellum showed considerable nematicidal activity under laboratory conditions, causing high egg-hatching suppression (96.6%) and high juvenile mortality (90.3%) in M. incognita (Saharan et al., 2023). Previous studies have also highlighted the efficacy of Trichoderma spp. metabolites in reducing root-knot nematode damage (Khan et al., 2020). In particular, the cell-free culture of Trichoderma NAS120-M44 resulted in J2 mortality, suggesting that the metabolites produced by this mutant strain are effective against nematodes.

Greenhouse trials provided valuable insights into the effectiveness of biological control agents under realistic conditions. The application of mutant isolates of bacterial (B419) and fungal (NAS120-M44) suspensions significantly reduced the damage caused by nematodes to tomato plants compared to the control, resulting in a remarkable 77-92 reduction in the nematode's reproductive factor (RF). Notably, the inclusion of treatments without nematodes allowed for the assessment of the agents' impact on plant growth parameters, revealing their potential as growth-promoting agents. In subsequent greenhouse tests, these results were further elaborated and the synergistic effects of a combination of strong bacteria, effective fungi and chitosan were investigated. This combined approach resulted in a remarkable 94 reduction in nematode RF, a rate comparable to that of abamectin and higher than that of RTS, a commercial biological formulation for nematode control. Sohrabi et al. (2020) reported that the combined use of Glomus mosseae, B. subtilis, and T. harzianum has a better effect compared to their individual use. Also, other researchers have emphasized the compatibility and biocontrol potential of T. harzianum, B. subtilis, and P. fluorescens, which makes them appear as a promising tool for soilborne pathogen control (Singh, Balodi, Meena, & Singhal, 2021). In addition, chitosan, a natural biopolymer, and its derivatives have shown effective control over plant root-knot nematodes and enhancement of plant defense mechanisms against pathogens (Bibi, Ibrar, Shalmani, & Rehan, 2021; Z. Fan et al., 2020). Furthermore, the combination of chitosan with effective bacteria has been shown to reduce root-knot nematode damage (Rostami, Karegar, Taghavi, Ghasemi-Fasaei, & Ghorbani, 2023). Therefore, the combination of Bacillus NAS-B419 + Trichoderma NAS120-M44 + chitosan appears to be compatible and highly effective against nematode damage.

The exploration of effective control combinations for pathogens is crucial, but equally significant is determining how to maximize their efficacy. Various methods of utilizing these microorganisms have been documented, including seed treatment, soil treatment, and seedling plant treatment (Chinheya, 2015; El-Nagdi & Abd-El-Khair, 2019; H. Fan et al., 2020). Greenhouse Test 3 introduced variations in nematode inoculation methods, simulating different scenarios of nematode infestation in agricultural settings. These variations allowed for a more comprehensive evaluation of the biocontrol agents' adaptability and efficacy across diverse conditions. In this study, comparison between impregnation of plant roots with the evaluated combination and application of the combination onto the plant revealed that impregnation of plant roots was more effective in reducing the reproduction factor (RF) of nematodes. Additionally, as these methods simulate natural infection conditions more closely when applied in infected soil, the test was repeated in the infected soil. Impregnation of plant roots with the combination resulted in a 76% decrease in RF.

The molecular identification of effective microorganisms has significantly advanced the understanding of their taxonomic diversity and phylogenetic relationships. In this study, the identified fungi have been classified under the species T. harzianum, while the bacteria have been assigned to B. velezensis. These taxonomic classifications provide crucial information about the identities and genetic relatedness of the microorganisms under investigation.

B. velezensis is a type of gram-positive bacteria renowned for its ability to enhance plant growth. It has been documented that various strains of this species possess the capacity to inhibit the growth of microbial pathogens, spanning bacteria, fungi, and nematodes. Through genomic analysis, it has been elucidated that B. velezensis harbors strain-specific gene clusters responsible for the synthesis of secondary metabolites. These metabolites play pivotal roles in both suppressing pathogens and promoting plant growth. Specifically, B. velezensis demonstrates a robust genetic capability for producing cyclic lipopeptides (such as surfactin, bacillomycin-D, fengycin, and bacillibactin) as well as polyketides (including macrolactin, bacillaene, and difficidin). Furthermore, the secondary metabolites generated by B. velezensis have the potential to induce systemic resistance in plants. This mechanism enables plants to defend themselves against repeated assaults by harmful microorganisms, contributing to enhanced plant health and resilience (Rabbee et al., 2019). A study has presented compelling evidence regarding the effectiveness of B. velezensis VB7 as both a potent nematicide and an inducer of immune responses against root-knot nematode infestation in tomato plants. Laboratory experiments demonstrated that B. velezensis VB7 significantly impeded the hatching of RKN eggs and notably reduced the mortality of M. incognita juveniles by 87.95% and 96.66%, respectively. Additionally, when applied in nematode-infested conditions, B. velezensis VB7 triggered an immune response by inducing microbe-associated molecular pattern (MAMP)-triggered immunity, leading to the upregulation of transcription factors and defense genes. Furthermore, the study revealed the coordinated expression of various defense genes associated with immune response pathways(Kamalanathan, Sevugapperumal, & Nallusamy, 2023). Furthermore, six volatile organic compounds (VOCs) produced by B. velezensis GJ-7 demonstrated diverse modes of action against M. hapla, encompassing direct-contact nematicidal activity, fumigant activity, and repellent activity. As a result, these compounds show potential as promising biocontrol agents against root-knot nematodes (Wu et al., 2023).

Numerous research findings indicate that most Trichoderma species can produce bioactive compounds and display antagonistic properties against plant-pathogenic nematodes. Furthermore, Trichoderma is employed to enhance plant growth, optimize nutrient utilization, fortify plant resistance, and mitigate pollution from agrochemicals. The mechanisms involved in the biological control of nematode diseases comprise competitive exclusion, antibiosis, antagonistic activity, and mycoparasitism, along with the promotion of plant growth and the induction of systemic resistance in symbiosis with plants (Yao et al., 2023). The use of T. harzianum not only diminishes nematode populations and penetration rates but also improves plant growth, increases the content of nutritional elements and triggers systemic resistance in the plants. In addition, T. harzianum shows promising capabilities in the production of indoleacetic acid (IAA), exhibits remarkable ammonification activity, and shows enzymatic activities such as protease and lipase (Nafady et al., 2022). Yan et al. (2021) demonstrated that T. harzianum effectively suppressed M. incognita infestation in tomato plants, achieving a notable nematode reduction percentage of 61.88%. Their findings underscore T. harzianum's beneficial role in bolstering resistance against root-knot nematodes by stimulating secondary metabolism and enhancing the activity and transcripts of defense-related enzymes in tomato roots. Nematode infections were observed to elevate levels of reactive oxygen species (ROS) and lipid peroxidation in tomato roots; however, colonization with T. harzianum led to a significant reduction in ROS, malondialdehyde, and electrolyte leakage. This reduction was correlated with the heightened accumulation of various secondary metabolites, including flavonoids, phenols, lignin, and cellulose.

Gamma rays serve as a means to bolster the advantageous traits of biological agents against plant pathogens. This agricultural technique has proven beneficial over the years (Feldmann, Shupert, Haddock, Twardoski, & Feldmann, 2019; Piri, Babayan, Tavassoli, & Javaheri, 2011), contributing to the enhancement of biological agent properties (Mirmajlessi, Mostafavi, Loit, Najdabbasi, & Mänd, 2018). The gamma mutants of B. subtilis UTB1, M419, and M464 have better antifungal properties against Aspergillus flavus than the wild type. Production of iturin-like lipopeptides and swarm motility were increased, allowing them to colonize surfaces and reduce aflatoxin to a greater extent (Afsharmanesh, Ahmadzadeh, Javan-Nikkhah, & Behboudi, 2014). Induced gamma irradiation also resulted in increased production of biosurfactants and biofilms in mutants of B. subtilis UTB1 (Afsharmanesh et al., 2013). Furthermore, the enhancement of volatile production by gamma radiation in Lactobacillus plantarum had a promising result in controlling sapstain fungi in wood stores and infected trees (El-Fouly, Shahin, & El-Bialy, 2011). The antifungal metabolites of Trichoderma harzianum, T. viride, and T. koningii mutants were assayed by HPLC. They produced highly active exo-enzymes and had the highest isozyme band number and quantity of chitinase and beta-1,3 glucanase (Haggag & Mohamed, 2002). Moreover, the efficacy of Trichoderma against Alternaria solani, Fusarium oxysporium, and Rhizoctonia solani was improved by the use of gamma rays, and the antagonistic activity of the second-generation variants was higher than that of the first-generation (El-Bialy et al., 2019). Based on our knowledge and research, this study marks the first exploration into the biological inhibitory potential of two irradiated isolates, T. harzianum NAS120-M44 and B. subtilis NAS-B419, against root-knot nematodes. In essence, these biocontrol agents demonstrate the ability to alleviate damage caused by root-knot nematodes through irradiation-induced modifications. It appears that the fungi and bacteria investigated in this study could complement each other by activating diverse resistance pathways and targeting distinct points of effect. Consequently, the concurrent application of these biocontrol agents holds significant promise for potentially substituting fertilizers and pesticides.

In conclusion, this study investigated the efficacy of Trichoderma spp. and Bacillus spp., along with their gamma radiation-induced mutants, as potential biological control agents against Meloidogyne javanica in tomato plants. The results demonstrated promising nematicidal activity of Trichoderma and Bacillus strains in vitro, leading to significant reductions in nematode populations. Greenhouse trials further confirmed the effectiveness of mutant isolates in reducing nematode-induced damage to tomato plants, especially when combined with chitosan. Molecular identification provided valuable taxonomic insights into the effective microorganisms. Specifically, B.velezensis and T.harzianum emerged as promising candidates, exhibiting significant nematicidal activity. Overall, the study underscores the potential of combined biocontrol approaches for nematode management in agricultural settings, although further research is essential to evaluate practical applications and long-term efficacy. Future research efforts may concentrate on optimizing application methods, exploring additional synergistic combinations, and assessing long-term sustainability in agricultural ecosystems. Additionally, conducting field trials under diverse environmental conditions would validate the practical utility of these biocontrol agents, facilitating their adoption as sustainable alternatives to chemical nematicides.

Data availability

Sequence data supporting this manuscript are deposited in NCBI GenBank under accession numbers: Bacillus velezensis NAS-B1 (PP320414), Bacillus velezensis NAS-B419 (PP320416), Trichoderma harzianum NAS120 (PP316639, PP321302), and Trichoderma harzianum NAS120-M44 (PP316640, PP321301).

Ethics approval and consent to participate

The research reported here did not involve experimentation with human participants or animals. In conducting the experimental research and greenhouse studies outlined in this manuscript, we confirm that there were no specific institutional, national, or international guidelines or legislation directly applicable to the collection and use of plant materials for our study. However, it is important to note that our research was conducted under the supervision of the Nuclear Science and Technology Research Institute (NSTRI) and the Iran National Science Foundation (INSF), ensuring adherence to ethical standards and best practices in scientific research. Additionally, it's important to mention that this project did not involve the use of mutant plants for experiments..

Consent for publication

All data from this manuscript was provided by ourselves and we agree to publish these data.

Competing interests

The authors declare that they have no conflict of interest.

Funding

This work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4012799

Authors' contributions:

MR wrote the first draft and participated in the lab experiment and did data analysis. SS, RS and AG revised the final manuscript.

Abbasi, S., Safaie, N., Shams-Bakhsh, M., & Shahbazi, S. (2016). Biocontrol activities of gamma induced mutants of Trichoderma harzianum against some soilborne fungal pathogens and their DNA fingerprinting. Iranian Journal of Biotechnology, 14(4), 260.
Afsharmanesh, H., Ahmadzadeh, M., Javan-Nikkhah, M., & Behboudi, K. (2014). Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation. Crop Protection, 60, 83-92.
Afsharmanesh, H., Ahmadzadeh, M., Majdabadi, A., Motamedi, F., Behboudi, K., & Javan-Nikkhah, M. (2013). Enhancement of biosurfactants and biofilm production after gamma irradiation-induced mutagenesis of Bacillus subtilis UTB1, a biocontrol agent of Aspergillus flavus. Archives of phytopathology and plant protection, 46(15), 1874-1884.
Basumatary, B., Das, D., Choudhury, B., Dutta, P., & Bhattacharyya, A. (2021). Isolation and characterization of endophytic bacteria from tomato foliage and their in vitro efficacy against root-knot nematodes. Journal of Nematology, 53(1), 1-16.
Bhat, A. A., Shakeel, A., Waqar, S., Handoo, Z. A., & Khan, A. A. (2023). Microbes vs. nematodes: Insights into biocontrol through antagonistic organisms to control root-knot nematodes. Plants, 12(3), 451.
Bibi, A., Ibrar, M., Shalmani, A., & Rehan, T. (2021). A review on recent advances in chitosan applications. Pure and Applied Biology, 10(4), 1217-1229.
Cayrol, J.-C., Djian, C., & Pijarowski, L. (1989). Study of the nematicidal properties of the culture filtrate of the nematophagous fungus Paecilomyces lilacinus. Revue de Nematologie, 12(4), 331-336.
Chinheya, C. C. (2015). Use of trichoderma and bacillus isolates as seed treatments against the rootknot nematode, meloidogyne javanica (chitwood).
Eisenback, J. (1985). Detailed morphology and anatomy of second-stage juveniles, males, and females of the genus Meloidogyne (root-knot nematodes). An advanced treatise on Meloidogyne, 1, 47-77.
El-Bialy, H. A. A., Shahin, A. A.-F. M., El-Fouly, M. Z., Awad, M. A., Khalifa, E.-S. Z., & Fahmy, S. M. (2019). Volatiles and functional peptides compositions of Trichoderma variants induced by a new strategy of irradiation. Biocatalysis and Agricultural Biotechnology, 20, 101261.
El-Fouly, M. Z., Shahin, A. A.-F. M., & El-Bialy, H. A.-A. (2011). Biological control of sapstain fungi in Egyptian wood stores and infected trees. Annals of microbiology, 61(4), 789-799.
El-Nagdi, W. M., & Abd-El-Khair, H. (2019). Application of Bacillus species for controlling root-knot nematode Meloidogyne incognita in eggplant. Bulletin of the National Research Centre, 43, 1-10.
Fan, H., Yao, M., Wang, H., Zhao, D., Zhu, X., Wang, Y., . . . Chen, L. (2020). Isolation and effect of Trichoderma citrinoviride Snef1910 for the biological control of root-knot nematode, Meloidogyne incognita. BMC microbiology, 20, 1-11.
Fan, Z., Qin, Y., Liu, S., Xing, R., Yu, H., & Li, P. (2020). Chitosan oligosaccharide fluorinated derivative control root-knot nematode (Meloidogyne incognita) disease based on the multi-efficacy strategy. Marine Drugs, 18(5), 273.
Feldmann, F., Shupert, W. L., Haddock, E., Twardoski, B., & Feldmann, H. (2019). Gamma irradiation as an effective method for inactivation of emerging viral pathogens. The American journal of tropical medicine and hygiene, 100(5), 1275.
Feyisa, B., Lencho, A., Selvaraj, T., & Getaneh, G. (2019). Effect of Some Botanicals and Trichoderma Harzianum against Root-Knot Nematode Meloidogyne Incognita, Infecting Tomato under Green House. Academic Research Journal of Agricultural Science and Research, 7, 238-249.
Ghasemi, S., Safaie, N., Shahbazi, S., Shams-Bakhsh, M., & Askari, H. (2020). The role of cell wall degrading enzymes in antagonistic traits of Trichoderma virens against Rhizoctonia solani. Iranian Journal of Biotechnology, 18(4), e2333.
Haggag, W. M., & Mohamed, H. (2002). Enhanecment of antifungal metabolite production from gamma-ray induced mutants of some Trichoderma species for control onion white disease. Plant Pathology Bulletin, 11, 45-56.
Hussey, R., & Barker, K. (1973). A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique.
Kamalanathan, V., Sevugapperumal, N., & Nallusamy, S. (2023). Antagonistic bacteria Bacillus velezensis VB7 possess nematicidal action and induce an immune response to suppress the infection of root-knot nematode (RKN) in tomato. Genes, 14(7), 1335.
Khan, R. A. A., Najeeb, S., Mao, Z., Ling, J., Yang, Y., Li, Y., & Xie, B. (2020). Bioactive secondary metabolites from Trichoderma spp. against phytopathogenic bacteria and root-knot nematode. Microorganisms, 8(3), 401.
Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 33(7), 1870-1874.
Kumar, S. N., Mohandas, C., & Nambisan, B. (2014). Purification, structural elucidation and bioactivity of tryptophan containing diketopiperazines, from Comamonas testosteroni associated with a rhabditid entomopathogenic nematode against major human-pathogenic bacteria. Peptides, 53, 48-58.
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., . . . Lopez, R. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 2947-2948.
Manikandan, A., Johnson, I., Jaivel, N., Krishnamoorthy, R., SenthilKumar, M., Raghu, R., . . . Anandham, R. (2022). Gamma-induced mutants of Bacillus and Streptomyces display enhanced antagonistic activities and suppression of the root rot and wilt diseases in pulses. Biomolecular Concepts, 13(1), 103-118.
Miranda, C. A., Martins, O. B., & Clementino, M. M. (2008). Species-level identification of Bacillus strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis. Antonie Van Leeuwenhoek, 93, 297-304.
Mirmajlessi, S. M., Mostafavi, H. A., Loit, E., Najdabbasi, N., & Mänd, M. (2018). Application of Radiation and Genetic Engineering Techniques to Improve Biocontrol Agent Performance: A Short Review. Use of Gamma Radiation Techniques in Peaceful Applications.
Nafady, N. A., Sultan, R., El-Zawahry, A. M., Mostafa, Y. S., Alamri, S., Mostafa, R. G., . . . Hassan, E. A. (2022). Effective and promising strategy in management of tomato root-knot nematodes by Trichoderma harzianum and arbuscular mycorrhizae. Agronomy, 12(2), 315.
Palazzini, J., Reynoso, A., Yerkovich, N., Zachetti, V., Ramírez, M., & Chulze, S. (2022). Combination of Bacillus velezensis RC218 and chitosan to control fusarium head blight on bread and durum wheat under greenhouse and field conditions. Toxins, 14(7), 499.
Piri, I., Babayan, M., Tavassoli, A., & Javaheri, M. (2011). The use of gamma irradiation in agriculture. African Journal of Microbiology Research, 5(32), 5806-5811.
Rabbee, M. F., Ali, M. S., Choi, J., Hwang, B. S., Jeong, S. C., & Baek, K.-h. (2019). Bacillus velezensis: a valuable member of bioactive molecules within plant microbiomes. Molecules, 24(6), 1046.
Rifai, M. A. (1969). A revision of the genus Trichoderma.
Rostami, M., Karegar, A., & Taghavi, S. M. (2021). Biocontrol potential of bacterial isolates from vermicompost and earthworm against the root-knot nematode Meloidogyne javanica infecting tomato plants. Egyptian Journal of Biological Pest Control, 31, 1-11.
Rostami, M., Karegar, A., Taghavi, S. M., Ghasemi-Fasaei, R., & Ghorbani, A. (2023). Effective combination of arugula vermicompost, chitin and inhibitory bacteria for suppression of the root-knot nematode Meloidogyne javanica and explanation of their beneficial properties based on microbial analysis. PLoS One, 18(8), e0289935.
Saharan, R., Patil, J., Yadav, S., Kumar, A., & Goyal, V. (2023). The nematicidal potential of novel fungus, Trichoderma asperellum FbMi6 against Meloidogyne incognita. Scientific Reports, 13(1), 6603.
Saitou, N. (1988). Property and efficiency of the maximum likelihood method for molecular phylogeny. Journal of molecular evolution, 27, 261-273.
Singh, S., Balodi, R., Meena, P., & Singhal, S. (2021). Biocontrol activity of Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens against Meloidogyne incognita, Fusarium oxysporum and Rhizoctonia solani. Indian Phytopathology, 74(3), 703-714.
Sohrabi, F., Sheikholeslami, M., Heydari, R., Rezaee, S., & Sharifi, R. (2020). Investigating the effect of Glomus mosseae, Bacillus subtilis and Trichoderma harzianum on plant growth and controlling Meloidogyne javanica in tomato. Indian Phytopathology, 73, 293-300.
Soufi, E., Safaie, N., Shahbazi, S., & Mojerlou, S. (2021). Gamma irradiation induces genetic variation and boosting antagonism in Trichoderma aureoviride. Archives of phytopathology and plant protection, 54(19-20), 1649-1674.
Subedi, S., Thapa, B., & Shrestha, J. (2020). Root-knot nematode (Meloidogyne incognita) and its management: a review.
TariqJaveed, M., Farooq, T., Al-Hazmi, A. S., Hussain, M. D., & Rehman, A. U. (2021). Role of Trichoderma as a biocontrol agent (BCA) of phytoparasitic nematodes and plant growth inducer. Journal of Invertebrate Pathology, 183, 107626.
Wu, W., Zeng, Y., Yan, X., Wang, Z., Guo, L., Zhu, Y., . . . He, X. (2023). Volatile organic compounds of Bacillus velezensis GJ-7 against Meloidogyne hapla through multiple prevention and control modes. Molecules, 28(7), 3182.
Yan, Y., Mao, Q., Wang, Y., Zhao, J., Fu, Y., Yang, Z., . . . Liu, A. (2021). Trichoderma harzianum induces resistance to root-knot nematodes by increasing secondary metabolite synthesis and defense-related enzyme activity in Solanum lycopersicum L. Biological Control, 158, 104609.
Yao, X., Guo, H., Zhang, K., Zhao, M., Ruan, J., & Chen, J. (2023). Trichoderma and its role in biological control of plant fungal and nematode disease. Frontiers in Microbiology, 14, 1160551.
Youssef, M. M. A., & Hassabo, A. (2020). The role of genetic engineering in management of plant parasitic nematodes with emphasis on root-knot nematodes: A review. Pakistan Journal of Nematology, 38(2), 179-185.

Table 1- Effects of microorganisms on tomato plant growth parameters and nematode indices of Meloidogyne javanica under glasshouse conditions.

Treatments	Shoot dry weight (g)		Eggs/root	J2/pot soil	Galls/ root	Egg mass/root	Final population (Pf)	Reproduction factor (Rf)	Rf reduction (%)^*
Treatments	With nematode	Without nematode	Eggs/root	J2/pot soil	Galls/ root	Egg mass/root	Final population (Pf)	Reproduction factor (Rf)	Rf reduction (%)^*
Trichoderma NAS120	1 cd	1.62 a-d	6655 ab	750 b	329 ab	211 ab	7734 ab	1.28	73
Trichoderma NAS120-M44	1.92 a-d	2.52 ab	5399 b	900 b	222 bc	112 bc	6521 b	1.08	77
Bacillus NAS-B1	1.80 a-d	1.92 a-d	6661 ab	1950 ab	229 bc	152 abc	8840 ab	1.47	70
Bacillus NAS-B419	2.24 ab	2.75 a	4020 b	900 b	251 bc	120 bc	5171 b	0.86	82
Bacillus NAS-B600	1.67 a-d	2.10 a-c	8260 ab	1650 ab	492 ab	235 ab	10402 ab	1.73	64
Abamectin	1.9 a-d	1.52 b-d	4741 b	750 b	127 c	80 c	5618 b	0.93	81
Water (control)	0.87 d	2.07 a-c	24345 a	4350 a	628 a	306 a	29323 a	4.88	-

*: Reduction percent of nematode reproduction factor compared to the control.

Data are means of four replicates. Means within a column with the same letter are not significantly different (P < 0.05)

Table 2- Effects of Bacillus NAS-B419 and Trichoderma NAS120-M44 and their combination with chitosan on nematode indices of Meloidogyne javanica in the roots of tomato plants under glasshouse conditions.

Treatments	Eggs/root	J2/pot soil	Galls/ root	Egg mass/root	Final population (Pf)	Reproduction factor (Rf)	Rf reduction (%)^*
Bacillus NAS-B419	5220 ab	4050 ab	129 ab	98 ab	9399 ab	1.56	80
Trichoderma NAS120-M44	2207 bc	1200 bc	77 abc	56 bcd	3484 bc	0.58	92
Bacillus NAS-B419 + Trichoderma NAS120-M44	1853 c	1350 bc	87 abc	66 bc	3290 bc	0.54	93
Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan	1615 c	900 c	53 bc	32 de	2568 c	0.42	94
RTS²	3656 ab	750 c	51 c	43 cde	4457 ab	0.74	90
Abamectin	2294 bc	900 c	55 bc	22 e	3249 bc	0.54	93
Water (control)	21035 a	28200 a	234 a	234 a	49469 a	8.20	-

RTS - Commercial Biological Nematicide (Tisan BT) by Royan Tisan Sam Company

*: Reduction percent of nematode reproduction factor compared to the control.

Data are means of four replicates. Means within a column with the same letter are not significantly different (P < 0.05)

Table 3. Effect of The most effective combination of microorganisms (Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan) on tomato Plant Growth Indicators and nematode indices of Meloidogyne javanica under glasshouse conditions.

Plant Growth Indicators

Treatments

Shoot fresh weight (g)

Shoot dry weight (g)

NIS

NI4L

NIS

NI4L

Impregnation of plant roots with Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan

12.10 bc

15.62 a

2.10 abc

2.80 a

Application of Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan onto the Plant

14.30 ab

12.87 abc

2.25 ab

2.32 ab

Water (control)

11.55 c

10.50 c

1.37 c

1.55 bc

Nematode Parameters

Treatments

Eggs/root

J2/pot soil

Galls/
root

Egg mass/root

Final population (Pf)

Reproduction factor (Rf)

Rf reduction (%)^*

NIS

NI4L

NIS

NI4L

NIS

NI4L

NIS

NI4L

NIS

NI4L

NIS

NI4L

NIS

NI4L

Impregnation of plant roots with Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan

767 bc

318 cd

1950 c

2100 c

100 abc

70 bc

27 c

35 ab

2750 c

2434 c

0.45

0.40

65

69

Application of Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan onto the Plant

628 bc

219 d

2550 c

3300 bc

113 ab

52 c

57 ab

34 ab

3219 bc

3538 bc

0.53

0.58

59

55

Water (control)

1980 a

1100 ab

6750 a

6150 ab

142 a

155 abc

106 a

105 a

8784 a

7284 ab

1.46

1.25

-

Data are presented as the mean of four replicates. Means within a column with the same letter are not significantly different (P < 0.05)

*: Reduction percent of nematode reproduction factor compared to the control.

NIS: Nematode-infested soil

NI4L: Nematode inoculation to a 4-leaf plant

Table 4. Effect of the most effective combination of microorganisms (Bacillus NAS-B419 + Trichoderma NAS120-M44 + chitosan) on tomato plant growth indicators and nematode indices of Meloidogyne javanica in nematode-infected soil under glasshouse conditions.

Treatments	Shoot fresh weight (g)	Shoot dry weight (g)	Eggs/root	J2/pot soil	Gall/ root	Egg mass/root	Final population (Pf)	Reproduction factor (Rf)	Rf reduction (%)^*
Impregnation of plant roots with Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan	15.62 a	2.80 a	696 b	1350 b	78 b	29 b	2125 b	0.35	76
Application of Bacillus NAS-B419+ Trichoderma NAS120-M44 +chitosan onto the Plant	12.87 abc	2.32 ab	432 b	2100 b	79 b	33 b	2610 b	0.43	70
Water (control)	10.50 c	1.55 bc	3240 a	5550 a	167 a	97 a	8957 a	1.49	-

Data are presented as the mean of four replicates. Means within a column with the same letter are not significantly different (P < 0.05)

*: Reduction percent of nematode reproduction factor compared to the control.

Table 5. Accession numbers of sequence data for bacteria and fungi for 16S rDNA, ITS rDNA and TEF-1α regions

Regions sequence	Bacteria and Fungi	Accession no.
16S rDNA	Bacillus velezensis NAS-B1	PP320414
16S rDNA	Bacillus velezensis NAS-B419	PP320416
ITS-rDNA	Trichoderma harzianum NAS120	PP316639
ITS-rDNA	Trichoderma harzianum NAS120-M44	PP316640
TEF-1α	Trichoderma harzianum NAS120	PP321302
TEF-1α	Trichoderma harzianum NAS120-M44	PP321301

No competing interests reported.

Enhancing Sustainable Control of Meloidogyne javanica in Tomato Plants: Leveraging Gamma Radiation-Induced Mutants of Trichoderma harzianum and Bacillus velezensis, with Optimal Combination Strategies

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Abstract

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Materials And Methods

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Discussion

Conclusion

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References

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