Biological Control of the Root-Lesion Nematode Pratylenchus penetrans in Peanut Using Trichoderma koningii and Pseudomonas chlororaphis

Biological Control of the Root-Lesion Nematode Pratylenchus penetrans in Peanut Using Trichoderma koningii and Pseudomonas chlororaphis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biological Control of the Root-Lesion Nematode Pratylenchus penetrans in Peanut Using Trichoderma koningii and Pseudomonas chlororaphis Alaa G. Hassan, Abd-El-Moneim M. H. Amein1, Aida M. El-Zawahry, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9399046/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background The root-lesion nematode Pratylenchus penetrans is one of the most damaging plant-parasitic nematodes, inducing severe root injury and quantitatively reducing yield of peanut ( Arachis hypogaea L.). With the recognition of environmental and health risks, the growing restrictions on chemical nematicides have emphasized the search for more sustainable and environment-friendly alternatives. The objective of this study was to assess antagonistic fungal and bacterial bioagents originated from peanut rhizosphere as biological control agents of P. penetrans in laboratory and greenhouse conditions. Results Nematicidal activities of seven fungal and ten bacterial isolates were tested in vitro against P. penetrans at various concentrations and exposure times. The nematode mortality by the tested isolates was higher especially at concentration of 10⁹ cfu ml⁻¹ after 72 hours of exposure, with Trichoderma koningii and Pseudomonas chlororaphis the most effective. These two agents significantly reduced mean root-lesion severity by 64.19% and nematode population to the level of 535.56 individuals per100g of soil under greenhouse, while T. koningii gave even better control of root-lesion (75.31%) and lower nematode number (684.44 individuals) than that infested alone on peanut seedlings as compared with nematode control being infected on them. There was significant increase by both bioagents in plant growth parameters, while P. chlororaphis showed the maximum increase in root weight (14.44 g) and plant height (36.67 cm) followed by T. koningii when compared with infected plants. Conclusion These results suggest that T. koningii and P. chlororaphis are potentially viable and environmentally sound BCAs for controlling P. penetrans in peanut. The dual roles in the suppression of nematode populations and plant growth promotion make them prospective components to sustainable programs aiming at managing nematodes without chemical nematicides. Pratylenchus penetrans Trichoderma koningii Pseudomonas chlororaphis- biological control - identification Figures Figure 1 Figure 2 Figure 3 Background Pratylenchus penetrans , a root lesion nematode, is a nematode pest of peanut ( Arachis hypogaea L.) and other crops (Singh et al., 2017 ). Trichoderma koningii and P. chlororaphis are naturally occurring microorganisms that control the nematode in peanuts. T. koningii infects nematodes and inhibits the hatching of eggs and the second stage juvenile stage (Abraham, 2005 ), and P. chlororaphis causes 83–96 percent juvenile mortality. Both microorganisms are compatible and can be applied together to maximize efficacy (Alattas et al., 2024 ). The peanut root-lesion nematode ( P. penetrans ) cause significant yield losses in peanuts grown in tropical and subtropical regions. P. penetrans is often considered secondary, the root-lesion nematode causes substantial yield losses (Saikai and MacGuidwin, 2022 ). The frequency and distribution of P. penetrans are increasing due to continuous peanut cropping and reduced use of fumigants. The phenomenon is especially acute in the Republic of South Africa, where the nematode has been recorded for the first time (Eapen et al., 2009 ). Biocontrol of P. penetrans has been achieved with the use of Pochonia chlamydosporia and Pasteuria penetrans on black pepper. (Nagachandrabose et al., 2024 ). P. chlororaphis , a biocontrol agent of P. penetrans and other fungi and bacteria, has exhibited effects favouring biocontrol of individual pathogen or pest populations. Both single and dual applications of these biocontrol agents in peanuts have led to reduced coffee soil nematodes (Shi et al., 2024 ). P. chlororaphis and T. koningii are readily commercially available in South Africa. T. koningii , available as biocontrol, is registered for use on soil-borne fungi controlling damping-off, Fusarium root rot, and Pythium in a variety of crops. P. chlororaphis is also commercially available in preparations registered in South Africa for a variety of diseases. Low application rates of 1% have been registered (Tyagi et al., 2024 ). T. koningii suppresses root–knot nematodes through different mechanisms, notably the production of metabolite(s) that activates plant defenses. It produces metabolites targeting pathogens, inducing protective mechanisms upon host contact, and acts as a plant-growth promoter (Liu et al., 2022 ). Growing at humidity levels and temperatures tolerated by P. penetrans, T. koningii may stimulate competition among nematodes and plant pathogens. Most P. chlororaphis products already co-apply T. koningii , benefiting stakeholders directly (Vinothini et al., 2024 ). This study aims to use eco-friendly alternatives for mitigating nematode population density, improving plant growth and enhancing crop yield. Furthermore, reducing the use of nematicides is essential, as they have side effects on humans and the environment, whereas these alternatives are readily available. On the other hand, biological control is considered a safe and efficient method for nematode suppression. Methods Nematode Extraction The initial population of P. penetrans was obtained from infected peanut stock cultures maintained at the Faculty of Agriculture's greenhouse, Department of Plant Pathology, Assiut University. To isolate the nematodes, soil samples were homogenized, and a 100 g subsample was processed using the wet sieving technique with 400-mesh sieves (Goodey, 1957 ). The resulting nematode-bearing suspension was then placed in a Baermann pan lined with tissue paper to facilitate the migration of motile individuals. Following an incubation period of 24 to 48 hours, the active nematode suspension was retrieved and concentrated to a final volume of 10 ml within a glass vial using a 400-mesh sieve (Baermann, 1917 ). For population quantification, the density of the nematodes was determined by examining 1 ml aliquots of the suspension. Individual counts were performed using a Hawksley counting slide under a binocular microscope (Reichert-AUSTRIA Nr. 257596) at 100x magnification. Biological control using bioagents on P. penetrans Isolation of bioagents from the peanut rhizosphere Rhizosphere samples were obtained from healthy peanut plants across four distinct sites within the Assiut Governorate. Ten samples were collected by carefully excavating the root systems and transporting them in sealed polyethylene bags to the Plant Pathology Laboratory at the Faculty of Agriculture, Assiut University, for microbial isolation. To prepare the soil for screening, 10 g of each moist soil sample was added to 90 ml of sterile distilled water in a 250 ml flask and agitated on a rotary shaker for 20 to 30 minutes. A serial dilution series was then established, ranging from 10 6 -10 1 For the isolation of fungal species, 0.1 ml aliquots from the 10 3 and 10 4 dilutions were spread onto Potato Dextrose Agar (PDA). Bacterial isolation was conducted by plating 0.1 ml from the 10 5 and 10 6 dilutions onto Nutrient Sucrose Agar (NSA) medium, which was adjusted to a neutral pH of 7.0 (Dowson, 1957). All cultures were incubated at 27°C for a period of 48 hours to allow for colony development. Following incubation, individual colonies were purified and the resulting pure isolates were maintained at 4°C for subsequent experimental use. Preparation of Microbial Agents and In vitro Assays To evaluate the nematicidal potential of seven fungal isolates against P. penetrans , the isolates were first cultured on PDA for one week. From these active cultures, 5 mm disks were inoculated into 250 ml flasks containing 100 ml of Potato Dextrose Broth (PDB). These were incubated for seven days at 25°C under constant agitation (240 rpm). Post-incubation, the culture filtrates were obtained by centrifuging at 10,000 rpm for 10 minutes and passing the resulting supernatant through a 0.2 µm Millipore filter to ensure cell-free conditions. These filtrates were maintained at 4°C until further use (Nitao et al., 1999 ; Meyer et al., 2000 ). For the bacterial agents, 48-hour-old cultures grown on NSA medium were processed to isolate the cells. The cultures were centrifuged at 10,000 rpm for 10 minutes; the supernatant was removed, and the remaining pellets underwent three wash cycles with sterile distilled water via centrifugation (Abo-Elyousr et al., 2010 ). Bacterial density was standardized using a UV-visible spectrophotometer to an optical density (OD) of 0.2 at A 360, corresponding to concentrations of 10 5 − 10 7 and 10 9 CFU/ml. Effect of fungal culture filtrate on P. penetrans in vitro The nematicidal activity of seven fungal isolates against P. penetrans was systematically assessed. Specifically, 5 ml of cell-free culture filtrate from each isolate—originally derived from cultures standardized to 10 5 , 10 7 , and 10 9 propagules—was added to Petri dishes containing a population of 100 adult females per 5 ml. To provide a baseline for natural mortality, distilled water was utilized as a control treatment. Each experimental set was monitored to determine the suppressive effect of the fungal metabolites on nematode viability. Effect of bacterial bioagents on P. penetrans in vitro The ability tests of ten bacterial isolates to suppress P. penetrans. For the mortality tests, 5 ml of bacterial suspension from each isolate, containing 10 5 -10 7 and 10 9 CFU/ml, was introduced separately to each Petri dish containing 100 female/5 ml. The control consisted of distilled water. Three replicates were used in this experiment and the Petri dishes were incubated at 25 ± 2°C. Identification of fungal bioagents Identification of the antagonistic fungus that reduced P. penetrans was carried out at the Mobasher Mycological Centre (AUMMC), Assiut University, verification was based on the physical characteristics of mycelium and spores as stated by (Leslie and Summerell, 2006 ). Identification of bacterial bioagents Molecular identification of bacterial bioagents The bacterial bioagent was then identified based on physiological and morphological characteristics. Genomic DNA was extracted from 48h old bacterial colony (Sambrook and Russell, 2001 ), and subsequently, the chromosomal DNA was amplified for 16S rDNA using a pair of primers63F (5'-CAGGCCTAACACATGCAAGTC-3') and1387R (5'-GGGCGGWGTGTACAAGGC-3') [Marchesi et al. 1998 ). A standard PCR reaction was done in a thermal cycler (iCyclerTM) where the final volume of PCR reaction mixture in each tube included 50 µl consisting of 1µl forward primer, 1 µl reverse primer, the genomic DNA template 2 µl (2 N). 5 µl of 10x standard Taq reaction buffer, and dNTPs; 4 µl or at least final concentration is approximately.25 nM, and Taq Polymerase: half a microliter: Table 1. The PCR reaction was performed as follows: 95°C for 5 min; 35 cycles of (95°C, 1 min; 56°C, 40s; and72°C,1 min) followed by a final extension step at72°C for10min. The reaction was incubated at 4°C for several minutes (Li et al. 2012 ). The PCR products were resolved on 2% agarose gel in 1×Tris–acetate (TAE) buffer and then stained for 10 min with 0.5µg ethidium bromide solution. Results were recorded using the Alfa-imager TM gel imager system and products sequenced (Macrogen Gangnam-gu, Seoul, Korea) The nucleotide sequences were then compared with the publicly available sequence in the NCBI gene bank. To examine the closely related sequences of Pseudomonas, CLUSTAL X was used to compose several alignments, and based on these 16S rDNA sequences, A phylogenetic tree for a selected isolate was constructed with MEGA 4.0 software using Neighbor-Joining (NJ) method (Kim et al., 1993 ), and evaluation of phylogenetic tree topology was performed according to the method outlined by Felsenstein ( 1985 ). Effect of fungal and bacterial bioagents on P. penetrans under greenhouse conditions The experiment was carried out under Greenhouse, Plant Pathology Dep., Faculty of Agri., Assiut Univ., Egypt. Seedlings of peanut (cv. Ismailia 2) plants were grown in clay pots (20 cm diam.) containing 2 kg of sterilized soil (silt 1:1 clay) where each pot containing three seedlings. Fifteen days later, plants were inoculated with 1000 females per plant. Fifteen days after of inoculating, 50 ml from 10 9 suspensions of T . koningii and P. chlororaphis were added separately. The suspension was added around the root of each plant. In addition to control healthy (without nematode) and infected control (only nematode). Each treatment was replicated three times. Three months later, data were recorded as nematode and plant parameters. Plants were uprooted and water was used to remove the soil that was sticking to the roots. Population density of nematodes in 100 g of soil. Statistical analysis All experimental data were analyzed using MSTATC statistical software. Analysis of variance (ANOVA) (Gomez and Gomez, 1984 ) was performed; means were compared by compared with Duncan’s test (Duncan, 1955 ). Results Effect of fungal culture filtrates on P. penetrans mortality% in vitro Seven fungal isolates were isolated from the rhizosphere of peanut healthy plants. The culture filtrates were tested in vitro for their ability to suppress P. penetrans. Nematode mortality was assessed at three concentrations 10 5 , 10 7 and 10 9 and the exposure period (24, 48 and 72h). Figure (1) showed that all of the tested fungal isolates were able to suppress P. penetrans with different concentrations and periods, compared to the control. Results indicated that there were significant differences between the fungal isolates. Isolate No. 4 gave the highest effect on P. penetrans with average mortality (22.74%), followed by isolate No. 1 (16.29%) then isolate No. 3 (13.52%). The lowest effect on P. penetrans was found with isolate No. 6 (0.62%). All fungal culture filtrates at concentration 10 9 after 72h showed the highest effect on nematode mortality rate in vitro . Effect of bacterial bioagents on P. penetrans mortality% in vitro Ten bacterial isolates were isolated from the rhizosphere of healthy peanut plants. The suspensions were tested to suppress P. penetrans in vitro. The nematodes mortality was assessed at concentrations 10 5 , 10 7 and 10 9 for 24, 48 and 72h. Data in Figure (2) showed the effectiveness of bacterial isolates against P. penetrans . Results canfield that bacterial isolate influenced the mortality rate of P. penetrans , isolate No. 3 had the highest effect on J2 with an average of (21.92%) followed by isolate No. 8 with an average (15.88%), then isolate No. 4 with an average (13.22%) and isolate No. 2 with an average (13.29%). The lowest effect on P. penetrans was found with isolate No. 7 with an average (7.51). Nematode mortality rate was affected by the increase in the exposure period (72h), compared to the control. Identification of the highest antagonistic isolate of fungi Identification of the highest antagonistic isolate of fungi, isolate No. 4 of fungi had the highest effect on P. penetrans mortality. The isolate was identified as Trichoderma koningii based on the morphological feature of mycelia and spores as stated by (Leslie and Summerell, 2006 ) and confirmed by Mobasher Mycological Center in Assiut University (AUMMC). Identification of the highest antagonistic isolate of bacteria Molecular identification of bacterial bioagents No. 3 Based on phylogenetic result analysis, the isolate (No. 3) was identified as pseudomonas chlororaphis. Moreover, 16S rDNA nucleotide sequence of the identified isolate was deposited in the NCBI gene bank database with an accession number MW740157.1 Pseudomonas chlororaphis Figure (2). Effect of bioagents on P. penetrans under greenhouse conditions Data in Table (1) explained that treating infected peanut plants with each antagonistic fungal isolate (T. koningii) and bacterial isolate ( P. chlororaphis ) significantly reduced root- lesion and J2s compared with control (only nematode). Results explored that P. chlororaphis reduced the root-lesion to (64.19%) and J2s to (535.56), while T. koningii reduced the root-lesion to (75.31%) and J2s to (684.44), compared with control (only nematode). P. chlororaphis and T. koningii at concentration 10 9 were used on root weight and plant height infected by P. penetrans under greenhouse conditions. Table (2) explained the effect of P. chlororaphis and T. koningii on peanut plants growth (root weight and plant height). Results showed that the highest root weight and plant height was observed in treatment of P. chlororaphis (14.44 g and 36.67 cm) followed by T. koningii (11.11 g and 31.56 cm) respectively compared with the infected control (21.67 g and 6.67 cm). Discussion Plant-parasitic nematodes pose a severe risk to global agricultural productivity across diverse cropping systems. The environmental and health risks associated with the high toxicity of synthetic nematicides have necessitated the advancement of innovative management strategies. Promising ecological alternatives include the use of biocontrol agents (BCAs) and plant growth-promoting bacteria (PGPB) In laboratory assessments of seven fungal and ten bacterial isolates, Trichoderma koningii and Pseudomonas chlororaphis were identified as the most effective agents for inducing mortality in P. penetrans . Under greenhouse conditions, these specific isolates significantly reduced both root-lesion severity and the overall population density of P. penetrans while simultaneously improving plant growth metrics, findings that align with prior research (Ashoub and Amara, 2010 ). Specifically, in vitro evaluations of fungal culture filtrates revealed that Trichoderma koningii achieved the highest mortality rates (Haggag and Amin, 2001 ; Samuels et al., 2006 ; Kiriga et al., 2018 ). Trichoderma species function as multifaceted BCAs by deploying several survival strategies; they combat nematodes directly through parasitism, the secretion of lytic enzymes, and the release of antibiotic or paralyzing compounds. Indirectly, they optimize plant vigor by enhancing water and nutrient assimilation and modifying root architecture to favor beneficial rhizosphere interactions. Furthermore, these fungi trigger systemic resistance by activating hormone-mediated defense pathways-including salicylic acid, jasmonic acid, and strigolactones-which prompts the production of defensive enzymes and secondary metabolites. Because Trichoderma species colonize only the outer root layers without invading vascular tissues, they establish a highly effective symbiotic relationship with the host (Hermosa et al., 2012 ; Poveda, 2020). Similarly, rhizobacteria employ a diverse array of mechanisms to suppress nematode populations. Direct interference includes the production of volatile organic compounds, crystal proteins, and lytic enzymes, alongside direct parasitism. Indirectly, these bacteria outcompete nematodes for resources and induce systemic resistance (ISR). A critical indirect pathway involves the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which lowers plant ethylene levels and enhances stress tolerance. Genera such as Agrobacterium , Bacillus , Paenibacillus , and Pseudomonas are particularly noted for these capabilities (Lugtenberg, 2009 ; Gamalero and Glick, 2019 ; Mhatre et al., 2019 ; Glick, 2020 ). Ultimately, this study demonstrates that P. chlororaphis effectively suppresses P. penetrans juveniles (J2) in vitro and significantly limits root-lesion development and population growth in vivo , while fostering robust plant development (Lee et al., 2011 ; Anderson and Kim, 2018 ; Kang et al., 2018 ; Nam et al., 2018 ; Anderson and Kim, 2020 ). P. penetrans is a plant-parasitic nematode that causes yield losses in peanut ( Arachis hypogaea ) and several other crops. Biological control agents such as Trichoderma koningii (strain HOL) and Pseudomonas chlororaphis (strain PCL1391) have the potential to mitigate P. penetrans damage through a variety of direct and indirect mechanisms. By harnessing these naturally occurring microbial agents, peanut nematode management can be achieved using non-pesticidal products, which is important for residue-sensitive crops. A comprehensive understanding of the mechanisms employed by T. koningii and P. chlororaphis to reduce P. penetrans damage, as well as the relative efficacy of these agents against P. penetrans in peanut, is important for clarifying nematode management strategies based on these agents and identifying ways to optimize their use (Ain et al., 2025 ). Trichoderma species are well-documented antagonists of plant-parasitic nematodes. They can invade and destroy nematodes directly through mycoparasitism or immobilize nematodes quickly through trap formation. Secondary metabolites such as chitinases and β-glucanases can directly degrade nematode cuticles, and nematode-induced production of lytic enzymes has been noted (Guzmán-Guzmán et al., 2023 ). Other indirect mechanisms that reduce migratory and sedentary nematodes include the production of antibiotics, competition for nutrients, and enhancement of host resistance. Inoculating Trichoderma species can also enhance nematode management through indirect pathways, including positive health effects on the target plant, enhancement of soil properties such as water holding capacity or nutrient availability, suppression of soil-borne diseases, and promotion of beneficial microbes (Eapen et al., 2009 ). The degree of influence evidently varies according to the microbial strains, targets, and environmental conditions. In studies evaluating the biocontrol potential of T. koningii against sedentary and migratory nematodes in various crops, mycotoxin production of T. koningii was found to be highly activity-dependent. When barley seeds were used, the agent stimulated plant growth and established a mutualistic relationship with its host (Ismaiel et al., 2023 ). Pseudomonas sp. strains produce several metabolites containing bacteriotropic and fungitropic properties, including phenazines, pyoluteorin, and 2,4-diacetylphloroglucinol. These metabolites cause alterations of physiological processes in neighbouring microorganisms, thereby controlling plant-attacking fungal pathogens. Major actinobacteria inhibiting plant pathogenic fungi also produce a variety of antagonistic metabolites (Kumar et al., 2023 ). Both T. koningii (T34) and P. chlororaphis (PCL1391) substantially lowered P. penetrans populations when combined with peanut ( Arachis hypogaea ). Apparent damage symptoms on roots from the nematode and associated fungus were limited to a few single eyes and were similar to those on non-infested controls, contrasting with extensive lesions in infested controls. While efficacy against P. penetrans has been reported for both T. koningii and P. chlororaphis , most literature focuses on P. incognita –host systems (Pocurull et al., 2020 ) and the relatively few T. koningii–P. penetrans reports emphasize different hosts (Eapen et al., 2009 ). Root-knot nematodes constitute major parasitic nematodes in economically important crops, but several nematicidal products exhibit limited effectiveness, especially in perennial crops. P. penetrans ranks among the top three nematodes in black pepper and other economically significant perennial crops in Southeast Asia and Pacific Island nations. Consequently, P. chlororaphis , initially isolated from nicotine-contaminated sites and checked against fungal antagonism, was selected as an additional biocontrol agent. Soil application of P. chlororaphis PCL1606 induces soil suppressiveness and enriches specific microbial communities potentially responsible for disease suppression. This bacterium colonizes plant roots and produces antifungal metabolites, including hydrogen cyanide, which contribute to biocontrol against soilborne plant pathogens (Wei et al., 2024 ). The phenazine-producing rhizobacterium P. chlororaphis 2B1-2 reduces the root-knot nematode Meloidogyne javanica in tomato and improves fruit quality (Wei et al., 2024 ). For P. chlororaphis , various mechanisms involve competition for space and nutrients, production of bioactive metabolites, the synthesis of antifungal and antibacterial compounds, and induction of resistance in host plants. Bioactive volatile and non-volatile organic compounds with strong biocontrol activities help prevent soilborne and aerial outbreaks of fungal and bacterial diseases (Li et al ., 2025). Management of P. penetrans on peanut and other hosts requires the suppression of secondary pathogens, enabling complete biocontrol through direct inactivation of the target nematode. Production of secondary metabolites such as the diacetylphloroglucinol-like compound, pyoluteorin, and other antimicrobial compounds with widespread efficacy against fungi and bacteria may be likewise implicated in the control of P. penetrans ( Pires et al., 2022 ). These metabolites may represent an ideal mechanism for control in soil, in which bioluminescent signalling could be detected free of any direct antagonistic action on the target nematode. Systems approach using sapphire direct-like illumination well suited for soil environments permits the screening of proximate antimicrobials (Trejo-Meléndez and Contreras‐Garduño, 2025 ). Persistence tests indicate that T. koningii can be effective against P. penetrans in pea, cowpea, and peanut (Pocurull et al., 2020 ). T. harzianum T-22 significantly reduced P. penetrans numbers and root galls in peanut. In small-scale on-farm tests, an emulsion of T. koningii applied 10–15 days post planting reduced nematodes by 66% compared with untreated plots. Similar results were obtained in chickpea (Cicer arietinum) tested with T-22 and other Trichoderma spp. Population reductions in T. koningii treatments were confirmed by nematode extraction from root galls (Eapen et al., 2009 ). Effects of P. chlororaphis on P. penetrans in peanut were significant, with mean nematode counts reduced by 81% in the field after a single seed treatment. Nematode lesion lengths also decreased considerably (Rakh et al., 2011 ). Seed bacterization in pot experiments markedly increased root and shoot length of groundnut seedlings in soil infected with S. rolfsii ; T. koningii applications in soil with a high population of P. penetrans showed no beneficial effect but reduced gall formation when applied before infection. When several biological control agents are applied at once, it is important to ensure that they do not exert negative interactions that could diminish their ability to control a pest (Roberts et al., 2005 ). The interaction between two control agents is considerably influenced by various agronomic factors, such as soil texture, chemistry, and moisture, that affect their establishment and activity (Eapen et al., 2009 ). Peanut cropping systems naturally experience moisture stress during the crop life cycle, but irrigation can extend this period and, under less stressful conditions, improve nematicide effect. Furthermore, intensive annual peanut cultivation can cause significant chemical alteration of raw and sterilized sandy soils, which, alongside certain geographical conditions, limit the viability of T. koningii and P. chlororaphis . Thus, the agronomic and environmental conditions should be investigated during pilot studies, which have revealed that the two agents do not interfere with nematode suppression, and the peanut plant–root also remains intact under a range of these conditions (Zhang et al., 2024 ). Abbreviations 16S rDNA (16s Ribosomal DNA) ANOVA (Analysis of variance) BCAs (Biocontrol agents) CFU/ml (Colony forming units per milligram) DNA (Deoxyribonucleic acid) dNTP (Deoxy nucleoside Triphosphate) ISR (Induce systemic resistance). J2s (Second juveniles) NJ (Neighbor-joining) NSA (Nutrient Sucrose Agar) OD (Optical density) PCR (Polymerase chain reaction) PDA (potato dextrose Agar) plant growth-promoting bacteria (PGPB), Potato Dextrose Broth (PDB). rpm (Rotary per minute) Tris–acetate (TAE) Declarations Ethics approval and consent to participate Not applicable. This study did not involve human participants or animals requiring ethical approval. Consent for publication Not applicable. Availability of data and materials This article includes all data generated or analyzed during the study. Competing interests The authors declare no competing interests. Funding This research did not receive any external funding Authors’ contributions AME, AMHA and AEF planned the study; AGH, HMMKB, RGM conducted the laboratory experiments; AGH, AME, HMMKB and KAMA wrote the manuscript. Acknowledgements The authors express their gratitude to Assiut University for providing the necessary facilities to carry out this work. 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Curr Microbiol., 62:746–751 Leslie JF, Summerell BA (2006) Wiley Blackwell publishing, 388 Li L, Ma J, Li Y, Wang Z, Gao T, Wang Q (2012) Screening and partial characterization of Bacillus with potential applications in biocontrol of cucumber Fusarium wilt. Crop Prot 35:29–35 Liu Y, He P, He P, Munir S, Ahmed A, Wu Y, Yang Y, Lu J, Wang J, Yang J, Pan X, Tian Y, He Y (2022) Potential biocontrol efficiency of Trichoderma species against oomycete pathogens. ncbi.nlm.nih.gov Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. Annu Rev Microbiol 63(1):541–556 Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rDNA. Appl Environ Microbiol 64:795–799 Meyer SLF, Massoud SI, Chitwood DJ, Roberts DP (2000) Evaluation of Trichoderma virens and Burkholderia cepacia for antagonistic activity against root-knot nematode. Meloidogyne incognita Nematology 2(8):871–879 Mhatre PH, Karthik C, Kadirvelu K, Divya KL, Venkatasalam EP, Srinivasan S, Ramkumar G, Saranya C, Shanmuganathan R (2019) Plant growth promoting rhizobacteria (PGPR): A potential alternative tool for nematodes bio-control. Biocat Agricul Biotechnol 17:119–128 Nagachandrabose S, Sivathanu L, Pon SM, Kalimuthu R, Somasundaram P, Subramanian KS (2024) Nano-emulsion formulation of nematode egg parasitic fungus, Pochonia chlamydosporia to control Meloidogyne incognita infecting tomato. Biocontrol Sci Technol 34(1):1–17. https://doi.org/10.1080/09583157.2023.2294216 Nam HS, Anderson AJ, Kim YC (2018) Biocontrol Efficacy of Formulated Pseudomonas chlororaphis O6 against Plant Diseases and Root-Knot Nematodes. Plant Pathol J 34(3):241–249 Nitao JK, Meyer SLF, Chitwood DJ (1999) In vitro assays of Meloidogyne incognita and Heterodera glycines for detection of nematode-antagonistic fungal compounds. Journal of Nematology, 31(2): 172–183 Pires D, Vicente CS, Menéndez E, Faria JM, Rusinque L, Camacho MJ, Inácio ML (2022) The fight against plant-parasitic nematodes: Current status of bacterial and fungal biocontrol agents. Pathogens 11(10):1178 Pocurull M, Fullana A, Ferro M, Valero P, Escudero N, Saus E, Gabaldón T, Sorribas JF (2020) Commercial Formulates of Trichoderma Induce Systemic Plant Resistance to Meloidogyne incognita in Tomato and the Effect Is Additive to That of the Mi-1.2 Resistance Gene. ncbi.nlm.nih.gov Poveda J, Abril-Urias P, Escobar C (2020) Biological control of plant-parasitic nematodes by filamentous fungi inducers of resistance; Trichoderma , Mycorrhizal and endophytic fungi. Front Microbiol 11(992):1–13 Rakh RR, Raut LS, Dalvi SM, Manwar AV (2011) Biological control of Sclerotium rolfsii , causing stem rot of groundnut by Pseudomonas cf. monteilii 9. Recent Res Sci Technol, 3(3) Roberts DP, Lohrke SM, Meyer SL, Buyer JS, Bowers JH, Baker CJ, Li W, de Souza JT, Lewis JA, Chung S (2005) Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber. Crop Prot 24(2):141–155. https://doi.org/10.1016/j.cropro.2004.07.004 Saikai K, MacGuidwin AE (2022) Impact of Pratylenchus penetrans on soybean grown in Wisconsin, USA. Plant Dis 106(11):2904–2910. https://doi.org/10.1094/PDIS-09-21-1888-RE Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sambrook J, Russell DW (2001) Rapid isolation of yeast DNA Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratory, New York, pp 631–632 Samuels GJ, Dodd SL, Lu B, Petrini O, Schroers H, Druzhinina IS (2006) The Trichoderma koningii aggregate species. Studies in mycology, 56: 67–133 Shi XQ, Zhu DH, Chen JL, Qin YY, Li XW, Qin S, Xing K (2024) Growth promotion and biological control of fungal diseases in tomato by a versatile rhizobacterium, Pseudomonas chlororaphis subsp. aureofaciens SPS-41. Physiol Mol Plant Pathol 131:102274 Singh R, Kumar M, Mittal A, Mehta PK (2017) Microbial metabolites in nutrition, healthcare and agriculture. Biotech 7:15 Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA), 101: 11030–11035 Trejo-Meléndez VJ, Contreras‐Garduño J (2025) Master of Puppets: How Microbiota Drive the Nematoda Ecology and Evolution. Ecol Evol, 15(8), e71549 Tyagi A, Tamang LT, Kashtoh H, Mir RA, Mir ZA, Manzoor S, Ali S (2024) A review on biocontrol agents as sustainable approach for crop disease management: applications, production, and future perspectives. Horticulturae 10(8):805 Vinothini K, Nakkeeran S, Saranya N, Jothi P, Richard JI, Perveen K, Mastinu A (2024) Rhizosphere engineering of biocontrol agents enriches soil microbial diversity and effectively controls root-knot nematodes. Microb Ecol 87(1):120 Wei D, Zhu D, Zhang Y, Yang Z, Hu Y, Song C, Chang X (2024) Pseudomonas chlororaphis IRHB3 assemblies beneficial microbes and activates JA-mediated resistance to promote nutrient utilization and inhibit pathogen attack. Front Microbiol 15:1328863 Zhang L, Zhu J, Zhang Y, Xia K, Yang Y, Wang H, Cui J (2024) Maize, Peanut, and Millet Rotations Improve Crop Yields by Altering the Microbial Community and Chemistry of Sandy Saline–Alkaline Soils. Plants 13(15):2170 Tables Table (1): Effect of P. chlororaphis and T. koningii on P. penetrans under greenhouse conditions Bioagents Root-lesion % nematodes/ 100 g soil P. chlororaphis 64.19 c* 535.56 c T. koningii 75.31 b 684.44 b Control (only nematode) 87.65 a 783.33 a *Means within each column followed by the same letter are not significantly different according to Duncan multiple range test at P < 0.05. Table (2): Effect of P. chlororaphis and T. koningii on root weight and plant height of peanut plants infected with P. penetrans under greenhouse conditions Bioagents Root weight (g) Plant height (cm) P. chlororaphis 14.44 b* 36.67 b T. koningii 11.11 c 31.56 c Control (only nematode) 6.67 d 27.00 d Control (without nematode) 21.67 a 39.00 a *Means within each column followed by the same letter are not significantly different according to Duncan multiple range test at P < 0.05.s Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 05 May, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 13 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9399046","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638710771,"identity":"9e231f4f-f552-40a7-9942-154daa30a2fe","order_by":0,"name":"Alaa G. Hassan","email":"","orcid":"","institution":"Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Alaa","middleName":"G.","lastName":"Hassan","suffix":""},{"id":638710772,"identity":"58acfb90-69f5-432d-9dbd-16f7087d75dd","order_by":1,"name":"Abd-El-Moneim M. H. Amein1","email":"","orcid":"","institution":"Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Abd-El-Moneim","middleName":"M. H.","lastName":"Amein1","suffix":""},{"id":638710773,"identity":"a9339b45-185f-48ae-b039-7e59a0f6d24b","order_by":2,"name":"Aida M. El-Zawahry","email":"","orcid":"","institution":"Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Aida","middleName":"M.","lastName":"El-Zawahry","suffix":""},{"id":638710774,"identity":"84a5f55a-928a-4666-a500-69cd5df67a18","order_by":3,"name":"Ameer E. Elfarash","email":"","orcid":"","institution":"Department of Genetics, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Ameer","middleName":"E.","lastName":"Elfarash","suffix":""},{"id":638710775,"identity":"d3968c56-c0a7-4bf3-9203-bc7f4119346b","order_by":4,"name":"Hadeel M. M. K. Bagy1","email":"","orcid":"","institution":"Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Hadeel","middleName":"M. M. K.","lastName":"Bagy1","suffix":""},{"id":638710776,"identity":"a47650bf-af5c-46c5-ae19-fbd8e5355953","order_by":5,"name":"Radwa G, Mostafa","email":"","orcid":"","institution":"Plant Pathology Research Institute, Agriculture Research Center, Giza, Egypt","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"Radwa","lastName":"G","suffix":""},{"id":638710779,"identity":"284c4533-8496-4f4e-b4e0-8a8415d0bad5","order_by":6,"name":"Kamal Abo-Elyousr","email":"data:image/png;base64,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","orcid":"","institution":"Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt","correspondingAuthor":true,"prefix":"","firstName":"Kamal","middleName":"","lastName":"Abo-Elyousr","suffix":""}],"badges":[],"createdAt":"2026-04-13 05:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9399046/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9399046/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109192027,"identity":"9119b9ad-0e07-41aa-a0a7-483356e9ca07","added_by":"auto","created_at":"2026-05-13 12:15:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77751,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of fungal culture filtrates on \u003cem\u003eP. penetrans\u003c/em\u003e mortality%\u003cem\u003e in vitro. \u003c/em\u003eA), \u003cem\u003eIn vitro\u003c/em\u003e evaluation after 24 h from treatments B), after 48 hr and C after 72 hr with three replicates (\u003cem\u003en\u003c/em\u003e= 3). Values followed by different letters indicates that means are significantly different from each other according to fisher’s least significant difference test at \u003cem\u003ep\u003c/em\u003e ≤ 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9399046/v1/c5e37758bf1be026db6f4c45.png"},{"id":109192028,"identity":"bafb4dff-f556-457d-92ac-2ce9f560686b","added_by":"auto","created_at":"2026-05-13 12:15:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90192,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bacterial bioagents on \u003cem\u003eP. penetrans\u003c/em\u003emortality%\u003cem\u003e in vitro. \u003c/em\u003eA), \u003cem\u003eIn vitro\u003c/em\u003e evaluation after 24 h from treatments B), after 48 hr and C after 72 hr with three replicates (\u003cem\u003en\u003c/em\u003e= 3). Values followed by different letters indicates that means are significantly different from each other according to fisher’s least significant difference test at \u003cem\u003ep\u003c/em\u003e ≤ 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9399046/v1/5a937c07c2372b9cbc48d99a.png"},{"id":109192026,"identity":"c8db2d28-0a62-40bb-a1b1-d41fa5ab4411","added_by":"auto","created_at":"2026-05-13 12:15:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure (2): \u003c/strong\u003ePhylogenetic tree of \u003cem\u003ePseudomonas chlororaphis \u003c/em\u003eisolate from data of 16s rDNA gene\u003c/p\u003e","description":"","filename":"image14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9399046/v1/41fbdf1d54c6827d71a7a4de.jpeg"},{"id":109192064,"identity":"b545af60-fcb2-4792-8148-ccdddc4e0444","added_by":"auto","created_at":"2026-05-13 12:15:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":566824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9399046/v1/a6ded8b3-9e8b-46bb-9b59-0dc8fb60f8b5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological Control of the Root-Lesion Nematode Pratylenchus penetrans in Peanut Using Trichoderma koningii and Pseudomonas chlororaphis","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003ePratylenchus penetrans\u003c/em\u003e, a root lesion nematode, is a nematode pest of peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.) and other crops (Singh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eTrichoderma koningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e are naturally occurring microorganisms that control the nematode in peanuts. \u003cem\u003eT. koningii\u003c/em\u003e infects nematodes and inhibits the hatching of eggs and the second stage juvenile stage (Abraham, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and \u003cem\u003eP. chlororaphis\u003c/em\u003e causes 83\u0026ndash;96 percent juvenile mortality. Both microorganisms are compatible and can be applied together to maximize efficacy (Alattas et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe peanut root-lesion nematode (\u003cem\u003eP. penetrans\u003c/em\u003e) cause significant yield losses in peanuts grown in tropical and subtropical regions. \u003cem\u003eP. penetrans\u003c/em\u003e is often considered secondary, the root-lesion nematode causes substantial yield losses (Saikai and MacGuidwin, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The frequency and distribution of \u003cem\u003eP. penetrans\u003c/em\u003e are increasing due to continuous peanut cropping and reduced use of fumigants. The phenomenon is especially acute in the Republic of South Africa, where the nematode has been recorded for the first time (Eapen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiocontrol of \u003cem\u003eP. penetrans\u003c/em\u003e has been achieved with the use of \u003cem\u003ePochonia chlamydosporia\u003c/em\u003e and \u003cem\u003ePasteuria penetrans\u003c/em\u003e on black pepper. (Nagachandrabose et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eP. chlororaphis\u003c/em\u003e, a biocontrol agent of \u003cem\u003eP. penetrans\u003c/em\u003e and other fungi and bacteria, has exhibited effects favouring biocontrol of individual pathogen or pest populations. Both single and dual applications of these biocontrol agents in peanuts have led to reduced coffee soil nematodes (Shi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eP. chlororaphis\u003c/em\u003e and \u003cem\u003eT. koningii\u003c/em\u003e are readily commercially available in South Africa. \u003cem\u003eT. koningii\u003c/em\u003e, available as biocontrol, is registered for use on soil-borne fungi controlling damping-off, Fusarium root rot, and \u003cem\u003ePythium\u003c/em\u003e in a variety of crops. \u003cem\u003eP. chlororaphis\u003c/em\u003e is also commercially available in preparations registered in South Africa for a variety of diseases. Low application rates of 1% have been registered (Tyagi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. koningii\u003c/em\u003e suppresses root\u0026ndash;knot nematodes through different mechanisms, notably the production of metabolite(s) that activates plant defenses. It produces metabolites targeting pathogens, inducing protective mechanisms upon host contact, and acts as a plant-growth promoter (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Growing at humidity levels and temperatures tolerated by \u003cem\u003eP. penetrans, T. koningii\u003c/em\u003e may stimulate competition among nematodes and plant pathogens. Most \u003cem\u003eP. chlororaphis\u003c/em\u003e products already co-apply \u003cem\u003eT. koningii\u003c/em\u003e, benefiting stakeholders directly (Vinothini et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aims to use eco-friendly alternatives for mitigating nematode population density, improving plant growth and enhancing crop yield. Furthermore, reducing the use of nematicides is essential, as they have side effects on humans and the environment, whereas these alternatives are readily available. On the other hand, biological control is considered a safe and efficient method for nematode suppression.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNematode Extraction\u003c/h2\u003e \u003cp\u003eThe initial population of \u003cem\u003eP. penetrans\u003c/em\u003e was obtained from infected peanut stock cultures maintained at the Faculty of Agriculture's greenhouse, Department of Plant Pathology, Assiut University. To isolate the nematodes, soil samples were homogenized, and a 100 g subsample was processed using the wet sieving technique with 400-mesh sieves (Goodey, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). The resulting nematode-bearing suspension was then placed in a Baermann pan lined with tissue paper to facilitate the migration of motile individuals. Following an incubation period of 24 to 48 hours, the active nematode suspension was retrieved and concentrated to a final volume of 10 ml within a glass vial using a 400-mesh sieve (Baermann, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1917\u003c/span\u003e). For population quantification, the density of the nematodes was determined by examining 1 ml aliquots of the suspension. Individual counts were performed using a Hawksley counting slide under a binocular microscope (Reichert-AUSTRIA Nr. 257596) at 100x magnification.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiological control using bioagents on\u003c/b\u003e \u003cb\u003eP. penetrans\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of bioagents from the peanut rhizosphere\u003c/h3\u003e\n\u003cp\u003eRhizosphere samples were obtained from healthy peanut plants across four distinct sites within the Assiut Governorate. Ten samples were collected by carefully excavating the root systems and transporting them in sealed polyethylene bags to the Plant Pathology Laboratory at the Faculty of Agriculture, Assiut University, for microbial isolation. To prepare the soil for screening, 10 g of each moist soil sample was added to 90 ml of sterile distilled water in a 250 ml flask and agitated on a rotary shaker for 20 to 30 minutes.\u003c/p\u003e \u003cp\u003eA serial dilution series was then established, ranging from 10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e1\u003c/sup\u003e For the isolation of fungal species, 0.1 ml aliquots from the 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e4\u003c/sup\u003e dilutions were spread onto Potato Dextrose Agar (PDA). Bacterial isolation was conducted by plating 0.1 ml from the 10\u003csup\u003e5\u003c/sup\u003e and 10\u003csup\u003e6\u003c/sup\u003e dilutions onto Nutrient Sucrose Agar (NSA) medium, which was adjusted to a neutral pH of 7.0 (Dowson, 1957). All cultures were incubated at 27\u0026deg;C for a period of 48 hours to allow for colony development. Following incubation, individual colonies were purified and the resulting pure isolates were maintained at 4\u0026deg;C for subsequent experimental use.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of Microbial Agents and\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eAssays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the nematicidal potential of seven fungal isolates against \u003cem\u003eP. penetrans\u003c/em\u003e, the isolates were first cultured on PDA for one week. From these active cultures, 5 mm disks were inoculated into 250 ml flasks containing 100 ml of Potato Dextrose Broth (PDB). These were incubated for seven days at 25\u0026deg;C under constant agitation (240 rpm). Post-incubation, the culture filtrates were obtained by centrifuging at 10,000 rpm for 10 minutes and passing the resulting supernatant through a 0.2 \u0026micro;m Millipore filter to ensure cell-free conditions. These filtrates were maintained at 4\u0026deg;C until further use (Nitao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Meyer et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the bacterial agents, 48-hour-old cultures grown on NSA medium were processed to isolate the cells. The cultures were centrifuged at 10,000 rpm for 10 minutes; the supernatant was removed, and the remaining pellets underwent three wash cycles with sterile distilled water via centrifugation (Abo-Elyousr et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Bacterial density was standardized using a UV-visible spectrophotometer to an optical density (OD) of 0.2 at A 360, corresponding to concentrations of 10\u003csup\u003e5\u003c/sup\u003e \u0026minus;\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/ml.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of fungal culture filtrate on\u003c/b\u003e \u003cb\u003eP. penetrans in vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe nematicidal activity of seven fungal isolates against \u003cem\u003eP. penetrans\u003c/em\u003e was systematically assessed. Specifically, 5 ml of cell-free culture filtrate from each isolate\u0026mdash;originally derived from cultures standardized to 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e propagules\u0026mdash;was added to Petri dishes containing a population of 100 adult females per 5 ml. To provide a baseline for natural mortality, distilled water was utilized as a control treatment. Each experimental set was monitored to determine the suppressive effect of the fungal metabolites on nematode viability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of bacterial bioagents on\u003c/b\u003e \u003cb\u003eP. penetrans in vitro\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe ability tests of ten bacterial isolates to suppress \u003cem\u003eP. penetrans.\u003c/em\u003e For the mortality tests, 5 ml of bacterial suspension from each isolate, containing 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/ml, was introduced separately to each Petri dish containing 100 female/5 ml. The control consisted of distilled water.\u003c/p\u003e\u003cp\u003eThree replicates were used in this experiment and the Petri dishes were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eIdentification of fungal bioagents\u003c/h3\u003e\n\u003cp\u003eIdentification of the antagonistic fungus that reduced \u003cem\u003eP. penetrans\u003c/em\u003e was carried out at the Mobasher Mycological Centre (AUMMC), Assiut University, verification was based on the physical characteristics of mycelium and spores as stated by (Leslie and Summerell, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eIdentification of bacterial bioagents\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular identification of bacterial bioagents\u003c/h2\u003e \u003cp\u003eThe bacterial bioagent was then identified based on physiological and morphological characteristics. Genomic DNA was extracted from 48h old bacterial colony (Sambrook and Russell, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and subsequently, the chromosomal DNA was amplified for 16S rDNA using a pair of primers63F (5'-CAGGCCTAACACATGCAAGTC-3') and1387R (5'-GGGCGGWGTGTACAAGGC-3') [Marchesi et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). A standard PCR reaction was done in a thermal cycler (iCyclerTM) where the final volume of PCR reaction mixture in each tube included 50 \u0026micro;l consisting of 1\u0026micro;l forward primer, 1 \u0026micro;l reverse primer, the genomic DNA template 2 \u0026micro;l (2 N). 5 \u0026micro;l of 10x standard Taq reaction buffer, and dNTPs; 4 \u0026micro;l or at least final concentration is approximately.25 nM, and Taq Polymerase: half a microliter: Table\u0026nbsp;1. The PCR reaction was performed as follows: 95\u0026deg;C for 5 min; 35 cycles of (95\u0026deg;C, 1 min; 56\u0026deg;C, 40s; and72\u0026deg;C,1 min) followed by a final extension step at72\u0026deg;C for10min. The reaction was incubated at 4\u0026deg;C for several minutes (Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The PCR products were resolved on 2% agarose gel in 1\u0026times;Tris\u0026ndash;acetate (TAE) buffer and then stained for 10 min with 0.5\u0026micro;g ethidium bromide solution. Results were recorded using the Alfa-imager TM gel imager system and products sequenced (Macrogen Gangnam-gu, Seoul, Korea) The nucleotide sequences were then compared with the publicly available sequence in the NCBI gene bank. To examine the closely related sequences of Pseudomonas, CLUSTAL X was used to compose several alignments, and based on these 16S rDNA sequences, A phylogenetic tree for a selected isolate was constructed with MEGA 4.0 software using Neighbor-Joining (NJ) method (Kim et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), and evaluation of phylogenetic tree topology was performed according to the method outlined by Felsenstein (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eEffect of fungal and bacterial bioagents on\u003c/b\u003e \u003cb\u003eP. penetrans\u003c/b\u003e \u003cb\u003eunder greenhouse conditions\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe experiment was carried out under Greenhouse, Plant Pathology Dep., Faculty of Agri., Assiut Univ., Egypt.\u003c/p\u003e \u003cp\u003eSeedlings of peanut (cv. Ismailia 2) plants were grown in clay pots (20 cm diam.) containing 2 kg of sterilized soil (silt 1:1 clay) where each pot containing three seedlings. Fifteen days later, plants were inoculated with 1000 females per plant. Fifteen days after of inoculating, 50 ml from 10\u003csup\u003e9\u003c/sup\u003e suspensions of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ekoningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e were added separately. The suspension was added around the root of each plant. In addition to control healthy (without nematode) and infected control (only nematode). Each treatment was replicated three times.\u003c/p\u003e \u003cp\u003eThree months later, data were recorded as nematode and plant parameters. Plants were uprooted and water was used to remove the soil that was sticking to the roots. Population density of nematodes in 100 g of soil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed using MSTATC statistical software. Analysis of variance (ANOVA) (Gomez and Gomez, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) was performed; means were compared by compared with Duncan\u0026rsquo;s test (Duncan, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1955\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of fungal culture filtrates on\u003c/b\u003e \u003cb\u003eP. penetrans\u003c/b\u003e \u003cb\u003emortality%\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeven fungal isolates were isolated from the rhizosphere of peanut healthy plants. The culture filtrates were tested \u003cem\u003ein vitro\u003c/em\u003e for their ability to suppress \u003cem\u003eP. penetrans.\u003c/em\u003e Nematode mortality was assessed at three concentrations 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e and the exposure period (24, 48 and 72h).\u003c/p\u003e \u003cp\u003eFigure (1) showed that all of the tested fungal isolates were able to suppress \u003cem\u003eP. penetrans\u003c/em\u003e with different concentrations and periods, compared to the control. Results indicated that there were significant differences between the fungal isolates. Isolate No. 4 gave the highest effect on \u003cem\u003eP. penetrans\u003c/em\u003e with average mortality (22.74%), followed by isolate No. 1 (16.29%) then isolate No. 3 (13.52%). The lowest effect on \u003cem\u003eP. penetrans\u003c/em\u003e was found with isolate No. 6 (0.62%). All fungal culture filtrates at concentration 10\u003csup\u003e9\u003c/sup\u003e after 72h showed the highest effect on nematode mortality rate \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of bacterial bioagents on\u003c/b\u003e \u003cb\u003eP. penetrans\u003c/b\u003e \u003cb\u003emortality%\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTen bacterial isolates were isolated from the rhizosphere of healthy peanut plants. The suspensions were tested to suppress \u003cem\u003eP. penetrans in vitro.\u003c/em\u003e The nematodes mortality was assessed at concentrations 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e for 24, 48 and 72h.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eData in Figure (2) showed the effectiveness of bacterial isolates against \u003cem\u003eP. penetrans\u003c/em\u003e. Results canfield that bacterial isolate influenced the mortality rate of \u003cem\u003eP. penetrans\u003c/em\u003e, isolate No. 3 had the highest effect on J2 with an average of (21.92%) followed by isolate No. 8 with an average (15.88%), then isolate No. 4 with an average (13.22%) and isolate No. 2 with an average (13.29%). The lowest effect on \u003cem\u003eP. penetrans\u003c/em\u003e was found with isolate No. 7 with an average (7.51). Nematode mortality rate was affected by the increase in the exposure period (72h), compared to the control.\u003c/p\u003e\n\u003ch3\u003eIdentification of the highest antagonistic isolate of fungi\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIdentification of the highest antagonistic isolate of fungi, isolate No. 4 of fungi had the highest effect on \u003cem\u003eP. penetrans\u003c/em\u003e mortality. The isolate was identified as \u003cem\u003eTrichoderma koningii\u003c/em\u003e based on the morphological feature of mycelia and spores as stated by (Leslie and Summerell, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and confirmed by Mobasher Mycological Center in Assiut University (AUMMC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the highest antagonistic isolate of bacteria\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eMolecular identification of bacterial bioagents No. 3\u003c/h2\u003e \u003cp\u003eBased on phylogenetic result analysis, the isolate (No. 3) was identified as \u003cem\u003epseudomonas chlororaphis.\u003c/em\u003e Moreover, 16S rDNA nucleotide sequence of the identified isolate was deposited in the NCBI gene bank database with an accession number MW740157.1 \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e Figure (2).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eEffect of bioagents on\u003c/b\u003e \u003cb\u003eP. penetrans\u003c/b\u003e \u003cb\u003eunder greenhouse conditions\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eData in Table\u0026nbsp;(1) explained that treating infected peanut plants with each antagonistic fungal isolate \u003cem\u003e(T. koningii)\u003c/em\u003e and bacterial isolate (\u003cem\u003eP. chlororaphis\u003c/em\u003e) significantly reduced root- lesion and J2s compared with control (only nematode). Results explored that \u003cem\u003eP. chlororaphis\u003c/em\u003e reduced the root-lesion to (64.19%) and J2s to (535.56), while \u003cem\u003eT. koningii\u003c/em\u003e reduced the root-lesion to (75.31%) and J2s to (684.44), compared with control (only nematode).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. chlororaphis\u003c/em\u003e and \u003cem\u003eT. koningii\u003c/em\u003e at concentration 10\u003csup\u003e9\u003c/sup\u003e were used on root weight and plant height infected by \u003cem\u003eP. penetrans\u003c/em\u003e under greenhouse conditions.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;(2) explained the effect of \u003cem\u003eP. chlororaphis\u003c/em\u003e and \u003cem\u003eT. koningii\u003c/em\u003e on peanut plants growth (root weight and plant height). Results showed that the highest root weight and plant height was observed in treatment of \u003cem\u003eP. chlororaphis\u003c/em\u003e (14.44 g and 36.67 cm) followed by \u003cem\u003eT. koningii\u003c/em\u003e (11.11 g and 31.56 cm) respectively compared with the infected control (21.67 g and 6.67 cm).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant-parasitic nematodes pose a severe risk to global agricultural productivity across diverse cropping systems. The environmental and health risks associated with the high toxicity of synthetic nematicides have necessitated the advancement of innovative management strategies. Promising ecological alternatives include the use of biocontrol agents (BCAs) and plant growth-promoting bacteria (PGPB) In laboratory assessments of seven fungal and ten bacterial isolates, \u003cem\u003eTrichoderma koningii\u003c/em\u003e and \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e were identified as the most effective agents for inducing mortality in \u003cem\u003eP. penetrans\u003c/em\u003e. Under greenhouse conditions, these specific isolates significantly reduced both root-lesion severity and the overall population density of \u003cem\u003eP. penetrans\u003c/em\u003e while simultaneously improving plant growth metrics, findings that align with prior research (Ashoub and Amara, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecifically, \u003cem\u003ein vitro\u003c/em\u003e evaluations of fungal culture filtrates revealed that \u003cem\u003eTrichoderma koningii\u003c/em\u003e achieved the highest mortality rates (Haggag and Amin, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Samuels et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kiriga et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eTrichoderma\u003c/em\u003e species function as multifaceted BCAs by deploying several survival strategies; they combat nematodes directly through parasitism, the secretion of lytic enzymes, and the release of antibiotic or paralyzing compounds. Indirectly, they optimize plant vigor by enhancing water and nutrient assimilation and modifying root architecture to favor beneficial rhizosphere interactions. Furthermore, these fungi trigger systemic resistance by activating hormone-mediated defense pathways-including salicylic acid, jasmonic acid, and strigolactones-which prompts the production of defensive enzymes and secondary metabolites. Because \u003cem\u003eTrichoderma\u003c/em\u003e species colonize only the outer root layers without invading vascular tissues, they establish a highly effective symbiotic relationship with the host (Hermosa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Poveda, 2020).\u003c/p\u003e \u003cp\u003eSimilarly, rhizobacteria employ a diverse array of mechanisms to suppress nematode populations. Direct interference includes the production of volatile organic compounds, crystal proteins, and lytic enzymes, alongside direct parasitism. Indirectly, these bacteria outcompete nematodes for resources and induce systemic resistance (ISR). A critical indirect pathway involves the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which lowers plant ethylene levels and enhances stress tolerance. Genera such as \u003cem\u003eAgrobacterium\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePaenibacillus\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e are particularly noted for these capabilities (Lugtenberg, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gamalero and Glick, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mhatre et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Glick, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ultimately, this study demonstrates that \u003cem\u003eP. chlororaphis\u003c/em\u003e effectively suppresses \u003cem\u003eP. penetrans\u003c/em\u003e juveniles (J2) \u003cem\u003ein vitro\u003c/em\u003e and significantly limits root-lesion development and population growth \u003cem\u003ein vivo\u003c/em\u003e, while fostering robust plant development (Lee et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Anderson and Kim, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nam et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Anderson and Kim, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. penetrans\u003c/em\u003e is a plant-parasitic nematode that causes yield losses in peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e) and several other crops. Biological control agents such as \u003cem\u003eTrichoderma koningii\u003c/em\u003e (strain HOL) and Pseudomonas chlororaphis (strain PCL1391) have the potential to mitigate P. penetrans damage through a variety of direct and indirect mechanisms. By harnessing these naturally occurring microbial agents, peanut nematode management can be achieved using non-pesticidal products, which is important for residue-sensitive crops. A comprehensive understanding of the mechanisms employed by \u003cem\u003eT. koningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e to reduce \u003cem\u003eP. penetrans\u003c/em\u003e damage, as well as the relative efficacy of these agents against \u003cem\u003eP. penetrans\u003c/em\u003e in peanut, is important for clarifying nematode management strategies based on these agents and identifying ways to optimize their use (Ain et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTrichoderma species are well-documented antagonists of plant-parasitic nematodes. They can invade and destroy nematodes directly through mycoparasitism or immobilize nematodes quickly through trap formation. Secondary metabolites such as chitinases and β-glucanases can directly degrade nematode cuticles, and nematode-induced production of lytic enzymes has been noted (Guzm\u0026aacute;n-Guzm\u0026aacute;n et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other indirect mechanisms that reduce migratory and sedentary nematodes include the production of antibiotics, competition for nutrients, and enhancement of host resistance. Inoculating Trichoderma species can also enhance nematode management through indirect pathways, including positive health effects on the target plant, enhancement of soil properties such as water holding capacity or nutrient availability, suppression of soil-borne diseases, and promotion of beneficial microbes (Eapen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The degree of influence evidently varies according to the microbial strains, targets, and environmental conditions.\u003c/p\u003e \u003cp\u003eIn studies evaluating the biocontrol potential of \u003cem\u003eT. koningii\u003c/em\u003e against sedentary and migratory nematodes in various crops, mycotoxin production of \u003cem\u003eT. koningii\u003c/em\u003e was found to be highly activity-dependent. When barley seeds were used, the agent stimulated plant growth and established a mutualistic relationship with its host (Ismaiel et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pseudomonas sp. strains produce several metabolites containing bacteriotropic and fungitropic properties, including phenazines, pyoluteorin, and 2,4-diacetylphloroglucinol. These metabolites cause alterations of physiological processes in neighbouring microorganisms, thereby controlling plant-attacking fungal pathogens. Major actinobacteria inhibiting plant pathogenic fungi also produce a variety of antagonistic metabolites (Kumar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth \u003cem\u003eT. koningii\u003c/em\u003e (T34) and \u003cem\u003eP. chlororaphis\u003c/em\u003e (PCL1391) substantially lowered \u003cem\u003eP. penetrans\u003c/em\u003e populations when combined with peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e). Apparent damage symptoms on roots from the nematode and associated fungus were limited to a few single eyes and were similar to those on non-infested controls, contrasting with extensive lesions in infested controls. While efficacy against \u003cem\u003eP. penetrans\u003c/em\u003e has been reported for both \u003cem\u003eT. koningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e, most literature focuses on \u003cem\u003eP. incognita\u003c/em\u003e\u0026ndash;host systems (Pocurull et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and the relatively few \u003cem\u003eT. koningii\u0026ndash;P. penetrans\u003c/em\u003e reports emphasize different hosts (Eapen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Root-knot nematodes constitute major parasitic nematodes in economically important crops, but several nematicidal products exhibit limited effectiveness, especially in perennial crops. \u003cem\u003eP. penetrans\u003c/em\u003e ranks among the top three nematodes in black pepper and other economically significant perennial crops in Southeast Asia and Pacific Island nations. Consequently, \u003cem\u003eP. chlororaphis\u003c/em\u003e, initially isolated from nicotine-contaminated sites and checked against fungal antagonism, was selected as an additional biocontrol agent.\u003c/p\u003e \u003cp\u003eSoil application of \u003cem\u003eP. chlororaphis\u003c/em\u003e PCL1606 induces soil suppressiveness and enriches specific microbial communities potentially responsible for disease suppression. This bacterium colonizes plant roots and produces antifungal metabolites, including hydrogen cyanide, which contribute to biocontrol against soilborne plant pathogens (Wei et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The phenazine-producing rhizobacterium \u003cem\u003eP. chlororaphis\u003c/em\u003e 2B1-2 reduces the root-knot nematode \u003cem\u003eMeloidogyne javanica\u003c/em\u003e in tomato and improves fruit quality (Wei et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eP. chlororaphis\u003c/em\u003e, various mechanisms involve competition for space and nutrients, production of bioactive metabolites, the synthesis of antifungal and antibacterial compounds, and induction of resistance in host plants. Bioactive volatile and non-volatile organic compounds with strong biocontrol activities help prevent soilborne and aerial outbreaks of fungal and bacterial diseases (Li \u003cem\u003eet al\u003c/em\u003e., 2025). Management of \u003cem\u003eP. penetrans\u003c/em\u003e on peanut and other hosts requires the suppression of secondary pathogens, enabling complete biocontrol through direct inactivation of the target nematode. Production of secondary metabolites such as the diacetylphloroglucinol-like compound, pyoluteorin, and other antimicrobial compounds with widespread efficacy against fungi and bacteria may be likewise implicated in the control of \u003cem\u003eP. penetrans (\u003c/em\u003ePires et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These metabolites may represent an ideal mechanism for control in soil, in which bioluminescent signalling could be detected free of any direct antagonistic action on the target nematode. Systems approach using sapphire direct-like illumination well suited for soil environments permits the screening of proximate antimicrobials (Trejo-Mel\u0026eacute;ndez and Contreras‐Gardu\u0026ntilde;o, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePersistence tests indicate that \u003cem\u003eT. koningii\u003c/em\u003e can be effective against \u003cem\u003eP. penetrans\u003c/em\u003e in pea, cowpea, and peanut (Pocurull et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eT. harzianum\u003c/em\u003e T-22 significantly reduced \u003cem\u003eP. penetrans\u003c/em\u003e numbers and root galls in peanut. In small-scale on-farm tests, an emulsion of \u003cem\u003eT. koningii\u003c/em\u003e applied 10\u0026ndash;15 days post planting reduced nematodes by 66% compared with untreated plots. Similar results were obtained in chickpea (Cicer arietinum) tested with T-22 and other \u003cem\u003eTrichoderma\u003c/em\u003e spp. Population reductions in \u003cem\u003eT. koningii\u003c/em\u003e treatments were confirmed by nematode extraction from root galls (Eapen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEffects of \u003cem\u003eP. chlororaphis\u003c/em\u003e on \u003cem\u003eP. penetrans\u003c/em\u003e in peanut were significant, with mean nematode counts reduced by 81% in the field after a single seed treatment. Nematode lesion lengths also decreased considerably (Rakh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Seed bacterization in pot experiments markedly increased root and shoot length of groundnut seedlings in soil infected with \u003cem\u003eS. rolfsii\u003c/em\u003e; \u003cem\u003eT. koningii\u003c/em\u003e applications in soil with a high population of \u003cem\u003eP. penetrans\u003c/em\u003e showed no beneficial effect but reduced gall formation when applied before infection.\u003c/p\u003e \u003cp\u003eWhen several biological control agents are applied at once, it is important to ensure that they do not exert negative interactions that could diminish their ability to control a pest (Roberts et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The interaction between two control agents is considerably influenced by various agronomic factors, such as soil texture, chemistry, and moisture, that affect their establishment and activity (Eapen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Peanut cropping systems naturally experience moisture stress during the crop life cycle, but irrigation can extend this period and, under less stressful conditions, improve nematicide effect. Furthermore, intensive annual peanut cultivation can cause significant chemical alteration of raw and sterilized sandy soils, which, alongside certain geographical conditions, limit the viability of \u003cem\u003eT. koningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e. Thus, the agronomic and environmental conditions should be investigated during pilot studies, which have revealed that the two agents do not interfere with nematode suppression, and the peanut plant\u0026ndash;root also remains intact under a range of these conditions (Zhang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e16S rDNA (16s\u0026nbsp;Ribosomal DNA)\u003c/p\u003e\n\u003cp\u003eANOVA (Analysis of variance)\u003c/p\u003e\n\u003cp\u003eBCAs\u0026nbsp;(Biocontrol agents)\u003c/p\u003e\n\u003cp\u003eCFU/ml (Colony forming units per milligram)\u003c/p\u003e\n\u003cp\u003eDNA (Deoxyribonucleic acid)\u003c/p\u003e\n\u003cp\u003edNTP (Deoxy nucleoside Triphosphate)\u003c/p\u003e\n\u003cp\u003eISR\u0026nbsp;(Induce systemic resistance).\u003c/p\u003e\n\u003cp\u003eJ2s (Second juveniles)\u003c/p\u003e\n\u003cp\u003eNJ\u0026nbsp;(Neighbor-joining)\u003c/p\u003e\n\u003cp\u003eNSA (Nutrient Sucrose Agar)\u003c/p\u003e\n\u003cp\u003eOD\u0026nbsp;(Optical density)\u003c/p\u003e\n\u003cp\u003ePCR (Polymerase chain reaction)\u003c/p\u003e\n\u003cp\u003ePDA (potato dextrose Agar)\u003c/p\u003e\n\u003cp\u003eplant growth-promoting bacteria (PGPB),\u003c/p\u003e\n\u003cp\u003ePotato Dextrose Broth (PDB).\u003c/p\u003e\n\u003cp\u003erpm (Rotary per minute)\u003c/p\u003e\n\u003cp\u003eTris\u0026ndash;acetate (TAE)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;This study did not involve human participants or animals requiring ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article includes all data generated or analyzed during the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any external funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAME, AMHA and \u0026nbsp;AEF planned the study; \u0026nbsp;AGH, HMMKB, RGM conducted the laboratory experiments; AGH, AME, HMMKB and KAMA wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to Assiut University for providing the necessary facilities to carry out this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbo-Elyousr KA, Khan Z, Abedel-Moneim MF (2010) Evaluation of plant extracts and \u003cem\u003ePseudomonas\u003c/em\u003e spp. for control of root-knot nematode. 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Plants 13(15):2170\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable (1):\u0026nbsp;\u003c/strong\u003eEffect of\u0026nbsp;\u003cem\u003eP. chlororaphis\u003c/em\u003e\u0026nbsp; and \u0026nbsp;\u003cem\u003eT. koningii\u0026nbsp;\u003c/em\u003eon \u003cem\u003eP. penetrans\u003c/em\u003e under greenhouse conditions\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"572\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003eBioagents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003eRoot-lesion %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;nematodes/ 100\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eg soil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. chlororaphis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003e64.19 c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 138px;\"\u003e\n \u003cp\u003e535.56 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. koningii\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003e75.31 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 138px;\"\u003e\n \u003cp\u003e684.44 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003eControl (only nematode)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 217px;\"\u003e\n \u003cp\u003e87.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 138px;\"\u003e\n \u003cp\u003e783.33 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;*Means within each column followed by the same letter are not significantly different according to Duncan multiple range test at \u003cem\u003eP\u003c/em\u003e \u003cu\u003e\u0026lt;\u003c/u\u003e 0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (2):\u0026nbsp;\u003c/strong\u003eEffect of \u003cem\u003eP. chlororaphis\u003c/em\u003e\u0026nbsp; and \u0026nbsp;\u003cem\u003eT. koningii\u003c/em\u003e on root weight and plant height of peanut plants infected with\u003cem\u003e\u0026nbsp;P. penetrans\u003c/em\u003e under greenhouse conditions\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"588\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 253px;\"\u003e\n \u003cp\u003eBioagents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 177px;\"\u003e\n \u003cp\u003eRoot weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 158px;\"\u003e\n \u003cp\u003ePlant height (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 253px;\"\u003e\n \u003cp\u003e\u003cem\u003eP. chlororaphis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 177px;\"\u003e\n \u003cp\u003e14.44 b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 158px;\"\u003e\n \u003cp\u003e36.67 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 253px;\"\u003e\n \u003cp\u003e\u003cem\u003eT. koningii\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 177px;\"\u003e\n \u003cp\u003e11.11 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 158px;\"\u003e\n \u003cp\u003e31.56 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 253px;\"\u003e\n \u003cp\u003eControl (only nematode)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 177px;\"\u003e\n \u003cp\u003e6.67 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 158px;\"\u003e\n \u003cp\u003e27.00 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 253px;\"\u003e\n \u003cp\u003eControl (without nematode)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 177px;\"\u003e\n \u003cp\u003e21.67 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 158px;\"\u003e\n \u003cp\u003e39.00 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;*Means within each column followed by the same letter are not significantly different according to Duncan multiple range test at \u003cem\u003eP\u003c/em\u003e \u003cu\u003e\u0026lt;\u003c/u\u003e 0.05.s\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"egyptian-journal-of-biological-pest-control","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ebpc","sideBox":"Learn more about [Egyptian Journal of Biological Pest Control](http://ejbpc.springeropen.com)","snPcode":"41938","submissionUrl":"https://submission.springernature.com/new-submission/41938/3","title":"Egyptian Journal of Biological Pest Control","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pratylenchus penetrans, Trichoderma koningii, Pseudomonas chlororaphis- biological control - identification","lastPublishedDoi":"10.21203/rs.3.rs-9399046/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9399046/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe root-lesion nematode\u0026ensp;\u003cem\u003ePratylenchus penetrans\u003c/em\u003e is one of the most damaging plant-parasitic nematodes, inducing severe root injury and quantitatively reducing yield of peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.). With the recognition of environmental and health risks, the growing restrictions\u0026ensp;on chemical nematicides have emphasized the search for more sustainable and environment-friendly alternatives. The objective of this study was to assess antagonistic fungal and bacterial bioagents originated from peanut rhizosphere as biological control agents of \u003cem\u003eP. penetrans\u003c/em\u003e in laboratory and greenhouse\u0026ensp;conditions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNematicidal activities of seven fungal and ten bacterial isolates were tested \u003cem\u003ein vitro\u003c/em\u003e against \u003cem\u003eP. penetrans\u003c/em\u003e at various concentrations and exposure\u0026ensp;times. The nematode mortality by the tested isolates was higher especially at concentration of 10⁹ cfu ml⁻\u0026sup1; after 72 hours\u0026ensp;of exposure, with \u003cem\u003eTrichoderma koningii\u003c/em\u003e and \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e the most effective. These two agents significantly reduced\u0026ensp;mean root-lesion severity by 64.19% and nematode population to the level of 535.56 individuals per100g of soil under greenhouse, while \u003cem\u003eT. koningii\u003c/em\u003e gave even better control of root-lesion (75.31%) and lower nematode number (684.44 individuals) than that infested alone on peanut seedlings as compared with nematode control being infected on them. There was significant increase by both bioagents in plant growth parameters, while \u003cem\u003eP. chlororaphis\u003c/em\u003e showed the maximum increase in root weight (14.44 g) and plant height (36.67 cm) followed by \u003cem\u003eT. koningii\u003c/em\u003e when compared with\u0026ensp;infected plants.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese results suggest that \u003cem\u003eT. koningii\u003c/em\u003e and \u003cem\u003eP. chlororaphis\u003c/em\u003e are potentially viable and environmentally sound BCAs\u0026ensp;for controlling \u003cem\u003eP. penetrans\u003c/em\u003e in peanut. The dual roles in the suppression of nematode populations and plant growth promotion make them prospective components to sustainable programs aiming at managing nematodes\u0026ensp;without chemical nematicides.\u003c/p\u003e","manuscriptTitle":"Biological Control of the Root-Lesion Nematode Pratylenchus penetrans in Peanut Using Trichoderma koningii and Pseudomonas chlororaphis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 12:14:20","doi":"10.21203/rs.3.rs-9399046/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-05T15:28:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T05:44:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T05:44:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Egyptian Journal of Biological Pest Control","date":"2026-04-13T05:21:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"egyptian-journal-of-biological-pest-control","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ebpc","sideBox":"Learn more about [Egyptian Journal of Biological Pest Control](http://ejbpc.springeropen.com)","snPcode":"41938","submissionUrl":"https://submission.springernature.com/new-submission/41938/3","title":"Egyptian Journal of Biological Pest Control","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f120f90c-ffe8-45ca-9b0d-8a4f86ffeab4","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"8","date":"2026-05-05T15:28:40+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T12:14:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 12:14:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9399046","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9399046","identity":"rs-9399046","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}