Metarhizium anisopliae reduces Meloidogyne incognita reproduction and promotes tomato plant growth

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The entomopathogenic fungus Metarhizium anisopliae has been recognized for both its nematicidal activity and its capacity to promote plant growth, although these two functions have rarely been demonstrated simultaneously in the same study. This work evaluated the effectiveness of M. anisopliae in stimulating tomato growth and reducing M. incognita reproduction under in vitro and greenhouse conditions. Three experiments were conducted using tomato hybrid ‘Sophia’, with treatments including fungal application, nematode inoculation, and their combination. In both experiments, M. anisopliae significantly increased plant height, shoot fresh mass, and root fresh mass compared with untreated controls. The effect of M. anisopliae was also directly evaluated on egg masses under in vitro conditions to verify the hatching rate of juveniles. When applied to nematode infested plants, the fungus reduced the nematode multiplication by 55 and 56%, lowering the reproduction factor from 4.2 and 5.2 (nematode-only) to 1.8 and 2.3. Single and double applications of M. anisopliae produced similar agronomic responses, indicating that one application was sufficient for growth promotion. Overall, the results demonstrate that M. anisopliae acts as both a plant growth promoter and an effective biological control agent against M. incognita , bringing greater prominence to the management of plant-parasitic nematodes, particularly root-knot nematodes in tomato. Southern root-knot nematode Solanum lycopersicum L. bionematicidal biostimulant Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The Brazilian agricultural sector has faced, in recent decades, an increasing demand for sustainability and efficiency, driven by environmental pressures, climate change, global food needs, and rising costs and restrictions associated with pesticide use. Within this scenario, biotic factors especially soilborne pathogens and pests stand out as major constraints to crop productivity, causing economic losses and compromising yield stability (Fontes and Valadares-Inglis 2020 ). Among these pathogens, plant parasitic nematodes are considered one of the most damaging threats to tomato ( Solanum lycopersicum L.) production, a crop of high economic and social importance in Brazil and worldwide (Agrios 2005 ; Pinheiro et al. 2014 ; Lopes and Ferraz 2016 ). It is widely recognized that root-knot nematodes ( Meloidogyne spp.) can severely reduce tomato yield, and in many cases their impact becomes devastating when infestations reach high levels. In Brazil, Meloidogyne incognita (Kofoid and White, 1919) Chitwood (1949), known as the Southern root-knot nematode, is widespread and frequently reported as one of the most critical soil pathogens affecting tomatoes and other high value crops (Pinheiro et al. 2014 ; Oliveira and Rosa 2014 ; Lopes and Ferraz 2016 ). Historically, the management of root-knot nematodes has relied on synthetic nematicides and traditional agronomic practices such as crop rotation, the use of nematode free seedlings, and when available resistant or less susceptible cultivars (Agrios 2005 ; Oliveira and Rosa 2014 ). However, increasing environmental restrictions, high input costs, and risks to human health and non-target organisms have driven the search for more sustainable alternatives, among which biological control with antagonistic or growth promoting microorganisms has gained prominence (Fontes and Valadares-Inglis 2020 ). Research on the interaction between Metarhizium anisopliae (Metschnikoff) Sorokin and plant parasitic nematodes has advanced substantially in recent decades. Early studies demonstrated the direct nematicidal action of the fungus, including its ability to reduce Meloidogyne spp. in sugarcane crop (Rossi et al. 2006 ), as well as its capacity to suppress M. javanica populations in soils enriched with organic residues (Abdollahi 2018 ). Furthermore, there are reports of M. anisopliae isolates obtained from suppressive soils being pathogenic to juveniles of other nematode species, such as Heterodera avenae , reinforcing the versatility of this entomopathogenic species as a bionematicide (Ghayedi and Abdollahi 2013 ). Complementary evidence from Brazilian agriculture indicates that M. anisopliae is effective in reducing M. incognita infestation in banana, coffee, and cotton crops (Oliveira et al. 2021 ; Paes et al. 2025 ). Beyond its biocontrol activity, M. anisopliae has also been recognized as a plant growth promoting microorganism, enhancing physiological performance and root development in several hosts, including tomato, maize, and Arabidopsis (Sasan and Bidochka 2012 ; Siqueira et al. 2020 ; González-Pérez et al. 2022 ). Despite this substantial progress, no prior study has clearly demonstrated the simultaneous expression of these two functions, nematicidal activity and plant growth promotion. Considering the magnitude of losses caused by root-knot nematodes in solanaceous crops (Oliveira and Rosa 2014 ; Pinheiro et al. 2014 ; Lopes and Ferraz 2016 ), the development of microbial agents capable of integrating pest suppression and plant stimulation aligns with the current demands of sustainable agriculture and consolidates the novelty and scientific relevance of the present study. Despite these advances, there is still a lack of studies clearly documenting the dual function of M. anisopliae. This gap represents an important research opportunity to investigate and demonstrate, for the first time, that a single isolate of M. anisopliae can reduce M. incognita infestation while also enhancing plant vigor. Therefore, the present study aimed to evaluate the impact of M. anisopliae on plant growth and nematode suppression in tomato plants inoculated with M. incognita under in vitro and greenhouse conditions. MATERIAL AND METHODS Three greenhouse experiments were conducted under controlled conditions at the Biological Institute, in the city of Campinas, SP, Brazil (22 º54’S,47º00’W and altitude of 707 m) at spring, 2025. The average temperature during the experiments was 34 ± 3 ºC. The plots consisted of 800 cm³ plastic pots (15 cm height, 4.75 cm upper radius, and 3.5 cm lower radius) filled with 750 cm³ of autoclaved commercial substrate Tropstrato Florestal® (pH of 6.0; composed of pine bark, vermiculite and single superphosphate) and tomato hybrid ‘Sophia’ (Sakata Seed Sudamerica) was used in both experiments. The seeds were first sown in seedling trays, and after 15 days the seedlings were transplanted to the pots, with one seedling per pot and 0.5g of a slow-release fertigation fertilizer (NPK 13-40-13) was applied. The experiments were conducted in a completely randomized design. Nematode inoculum To preserve nematode virulence, the isolate was periodically inoculated on different host plants. Annual identification of M. incognita was confirmed using perineal pattern morphology (Kleynhans 1986 , Jepson 1987 ) and isoenzyme electrophoresis (esterase profile), identifying the isolate as M. incognita race 3 (Alfenas and Brune 2006 ). Nematode inoculum consisted of eggs and second-stage juveniles (J2) extracted from infected roots using a modified Coolen and D’Herde ( 1972 ) protocol. Roots were blended in a 0.5% NaOCl solution for one minute, and the suspension was filtered through a series of sieves (60-200-500 mesh; 0.250-0.074-0.025 mm). The resulting suspension was used for inoculation. M. anisopliae isolate The isolate of M. anisopliae was obtained from the fungal collection maintained at the Biological Control Reference Laboratory Unit/CAPSA of Biological Institute. The isolate was freshly cultured and exhibited spore viability above 80%. The fungal isolate was preserved thorough lyophilization and reactivated on Petri dishes containing PDA (Potato dextrose agar) supplemented with an antibiotic used to prevent contamination. Once sporulation was observed, conidia were scraped from the agar surface and suspended in sterile Milli-Q water containing 0.01% of Tween 80. This suspension was then inoculated onto autoclaved rice incubated under controlled conditions (25 ºC; 12:12 h light-dark photoperiod). After approximately seven days of incubation, once intense sporulation was evident, conidia were collected from the rice substrate through a dry separation method. The colonized rice was transferred to a 20-mesh sieve (0.25 mm aperture) and shaken by hand for about three minutes. This procedure allowed the conidia to detach from the rice grains and pass through the sieve openings by gravity. The released conidia were collected in a clean plastic container placed beneath the sieve. Conidial density was then quantified using a Neubauer hemocytometer under a light microscope (Leica DMLS), and the suspension was adjusted to 1 × 10⁸ viable conidia mL⁻¹. Each plant received 30 mL of the suspension, applied in the soil near the seedlings. Experiment 1 – Greenhouse test of a single application of M. anisopliae isolate on tomato plants infested with M. incognita The first experiment consisted of 4 treatments: one non-inoculated control, one inoculated with M. incognita (Mi), one treated with M. anisopliae isolate (Ma) and one inoculated with M. incognita and treated with M. anisopliae (Mi + Ma), each with seven replicates. The inoculation of M. incognita was performed in the same day of transplanting, at sowing by 5,000 eggs and J2 nematodes into two 2 cm deep holes near the seedlings. Plants were maintained in the greenhouse throughout the tomato cycle and irrigated daily. At 60 days after transplanting (DAT), the following variables were evaluated: final nematode population in the roots (Fp); reproduction factor (Rf = Fp/initial population); nematode suppression efficiency compared to control (E%), calculated according to the Abbott’s formula, using the expression: E% =[(C - T)/C] × 100, where C represents the mean value observed in the control treatment and T the mean value in the treated group; root fresh mass (RFM); shoot dry (SDM) and fresh mass (SFM); and shoot height (SH). Nematodes were extracted from the roots using the same method as for inoculum preparation, whereas the nematode counts were performed under a light microscope (Leica DMLS) using Peters counting slides with two 1-mL sub samples. Experiment 2 – Greenhouse test of two applications of M. anisopliae isolate on tomato plants infested with M. incognita The second experiment was conducted under the same period, inoculation of M. incognita and greenhouse conditions as experiment 1. This experiment included 5 treatments: one non-inoculated control, one inoculated with M. incognita , one treated with M. anisopliae isolate, one treated with twice application of M. anisopliae (Ma2) and one inoculated with M. incognita and treated with M. anisopliae , with eight replicates per treatment. The evaluations were carried out at 60 DAT and included the same variables. Experiment 3 - Effects of M. anisopliae on second-stage juveniles hatching of M. incognita under in vitro conditions followed by a greenhouse assay Experiment 3 was conducted in two sequential stages to evaluate the effects of M. anisopliae on the hatching of second-stage juveniles (J2) of M. incognita and the persistence of its nematicidal activity over time. The stage 1 consisted of an in vitro assay, using the modified Baermann funnel (Baermann 1917 ) method for a shallow container (Whitehead and Hemming 1965 ) to assess J2 hatching over a 7-day period. Egg masses treated with distilled water (control) were compared with egg masses treated with M. anisopliae , using the same fungal concentration adopted in Experiments 1 and 2. Six egg masses extracted from symptomatic tomato plants and stained with Phloxine B to facilitate visualization, were carefully placed in each container, with absorbent paper, with six replicates per treatment. Each container received 5 mL of either distilled water or fungal suspension and was maintained in a BOD incubator at 25°C. J2 hatching was evaluated at 3, 5, and 7 days after treatment using a stereomicroscope. At each assessment time, the liquid from the bottom of the container was collected, and the number of hatched J2 was counted. The Stage 2 consisted of a bioassay in tomato plants, in which all material from each container, containing remaining eggs and hatched J2, was transferred to tomato seedlings transplanted under the same conditions described for Experiments 1 and 2. This bioassay was designed to confirm whether M. anisopliae exerts a direct effect on egg hatching and to determine whether this effect persists during the period in which the tomato plants were maintained under greenhouse conditions. After 55 days of inoculation, tomato roots were processed to determine the final nematode population (Pf) and the number of nematodes/g⁻¹ root, following the same root nematode extraction method described for Experiments 1 and 2. Nematode quantification at this stage was performed using an optical microscope with the aid of a Peters counting slide. Statistical analysis Statistical analyses were performed using the Sisvar software (Ferreira 2011 ) and treatment means compared by the Tukey test at 5% of significance. When necessary, data were log-transformed [log (x + 1)] to meet the assumptions of normality, which were verified using the Shapiro-Wilk test. RESULTS Significant differences among treatments were observed for most of the evaluated variables in both experiments (Table 1 ). In Experiment 1, tomato plants treated with M. anisopliae exhibited the greatest increases in agronomic variables, clearly indicating that the fungus stimulated plant development. Application of M. anisopliae resulted in the tallest plants (76.1 cm) and the highest shoot fresh mass (44.0 g), values statistically similar to those obtained in the combined Ma + Mi treatment (77.0 cm and 46.7 g), and both superior to the control and to plants inoculated only with M. incognita (Fig. 1 ). No significant differences were detected for shoot dry mass. Root fresh mass followed the same trend, with M. anisopliae producing the highest root mass (27.3 g), followed by Ma + Mi (24.5 g), both significantly greater than the control (14.5 g). Plants inoculated only with M. incognita exhibited reduced root mass (16.2 g) relative to the fungal treatments. Regarding nematode parameters, M. incognita alone reached a final population of 21,107 specimens per root system, resulting in the highest reproduction factor (4.2). When M. anisopliae was applied to infested plants, nematode reproduction decreased, with the Ma + Mi treatment reducing the final population to 9,189 and the reproduction factor to 1.8, corresponding to 56% efficiency compared with the nematode-only treatment. Table 1 Effects of Metarhizium anisopliae on tomato plant growth and Meloidogyne incognita reproduction in greenhouse experiments evaluated 60 days after inoculation. SH = shoot height; SFM = shoot fresh mass; SDM = shoot dry mass; RFM = root fresh mass; Fp = final nematode population; Rf = reproduction factor; E% = control efficacy. EXPERIMENT 1 Treatment SH (cm) SFM (g) SDM (g) RFM (g) Fp Rf E% Control 59.8 b 29.1 b 7.2 a 14.5 c - - - M. incognita 66.7 ab 31.2 b 7.6 a 16.2 bc 21,107 a 4.2 a - M. anisopliae 76.1 a 44.0 a 10.4 a 27.3 a - - - Ma + Mi 77.0 a 46.7 a 8.3 a 24.5 ab 9,189 b 1.8 b 56 EXPERIMENT 2 Treatment SH (cm) SFM (g) SDM (g) RFM (g) Fp Rf E% Control 65.2 c 10.4 b 6.5 b 13.4 a - - - M. incognita 75.0 bc 44.7 b 4.7 b 15.6 a 26,429 a 5.2 a - M. anisopliae 93.5 a 67.7 a 11.4 a 30.3 a - - - M. anisopliae 2 90.7 a 76.1 a 11.9 a 21.9 a - - - Ma + Mi 81.4 ab 57.8 ab 8.1 ab 18.2 a 11,746 b 2.3 b 55 Means followed by different lower case in column differ from each other by Tukey test at 0.05 significance. In Experiment 2, M. anisopliae again stimulated plant growth, reinforcing its beneficial effect on agronomic variables. The highest shoot heights were observed in plants receiving either one (93.5 cm) or two (90.7 cm) fungal applications, which also resulted in the greatest shoot fresh and dry mass accumulation. Importantly, applying M. anisopliae twice at transplanting and again 30 days later did not differ significantly from a single application for any agronomic variable, indicating that one application was sufficient to promote plant development. Although root fresh mass did not differ significantly among treatments, the fungal applications tended to produce higher values than the control. As in Experiment 2, the highest nematode reproduction occurred in plants inoculated with M. incognita alone, reaching a final population of 26,429 and a reproduction factor of 5.2. The application of M. anisopliae to infested plants significantly reduced nematode multiplication, lowering the final population to 11,746 and the reproduction factor to 2.3, resulting in 55% efficiency relative to the nematode-only treatment. Overall, M. anisopliae consistently reduced M. incognita reproduction and stimulated tomato plant development across both experiments. Finally, the Experiment 3, in the in vitro assay (Stage 1), the hatching of second-stage juveniles of M. incognita increased progressively over time in both treatments. However, egg masses treated with M. anisopliae showed consistently lower numbers of hatched juveniles compared with the control throughout the evaluation period. At 3 days after incubation, the control treatment presented 1,234 J2, whereas the treatment with M. anisopliae showed 405 J2. At 5 days, the number of hatched juveniles increased to 2,176 in the control and to 534 in the M. anisopliae treatment. At the final evaluation, 7 days after incubation, the control reached 2,371 hatched J2, while the M. anisopliae treatment resulted in 613 J2 (Fig. 3 ). In the biotest conducted in tomato plants (Stage 2), significant differences were observed between treatments for both nematode population variables evaluated at 55 days after inoculation. Plants inoculated with control treatment showed a Pf of 25,964 and 5,753 nematodes/g⁻¹ of root. In contrast, plants inoculated with the nematode treated with M. anisopliae presented lower values, with a Pf of 4,977 and 693 nematodes/g⁻¹ of root (Fig. 4 ). DISCUSSION The results of this study demonstrate that the application of M. anisopliae simultaneously promoted tomato plant growth and reduced the reproduction of M. incognita . The increases observed in plant height, shoot fresh mass, and root fresh mass clearly indicate a biostimulant effect on plant development. These findings corroborate previous studies showing the capacity of Metarhizium spp. to act as plant growth promoters. Siqueira et al. ( 2020 ) reported that the fungus produces biochemical compounds capable of modulating plant metabolism, enhancing endophytic colonization, and stimulating tomato growth. Similarly, Sasan and Bidochka ( 2012 ) demonstrated that M. robertsii can colonize plant roots and promote greater root development, improving nutrient acquisition. Although no molecular, biochemical, or colonization assays were performed in the present study, the stimulatory effect was clearly verified, as illustrated in Fig. 1 . Further support for this biostimulant role comes from González et al. (2022), who demonstrated that multiple M. anisopliae strains significantly enhanced fresh weight and chlorophyll content in Arabidopsis within seven days and stimulated primary root elongation through volatile organic compounds such as β-caryophyllene and o-cymene. These effects were also confirmed in soil-grown tomato, and maize plants. Consistent with these findings, our results showed that M. anisopliae improved several agronomic variables in tomato and effectively reduced nematode reproduction, reinforcing its dual activity as both a plant growth promoter and a biological control agent. In the present study, the M. anisopliae + M. incognita treatment significantly reduced the nematode reproduction factor in both experiments, with efficiencies of 55% and 56%. This reduction confirms the potential of the fungus as a biological control agent against plant parasitic nematodes. Similar effects have been documented by Abdollahi ( 2018 ), who observed suppression of M. javanica in soil amended with organic material colonized by M. anisopliae , and by Ghayedi and Abdollahi ( 2013 ), who reported high pathogenicity of isolates from suppressive soils against juveniles of Heterodera avenae . The present findings also align with Paes et al. ( 2025 ), who demonstrated that multiple M. anisopliae isolates significantly reduced M. incognita reproduction in cotton, indicating that the nematicidal potential of the fungus is consistent across different cropping systems. In perennial crops, Oliveira et al. ( 2021 ) found the fungus to be effective against M. incognita in banana and coffee, and Rossi et al. ( 2006 ) also reported nematicidal activity of microbial agents containing M. anisopliae in sugarcane. Collectively, these studies suggest that the suppressive effect of M. anisopliae is robust and not strongly dependent on the host plant, underscoring its potential applicability in diverse agricultural systems. An additional finding of practical relevance is that a second application of M. anisopliae performed 30 days after transplanting did not enhance plant growth or reduce nematode reproduction beyond the effects obtained with a single application. In both experiments, one application at transplanting was sufficient to get biostimulant and nematicidal responses. Although authors have demonstrated the efficacy of a single application of M. anisopliae against plant parasitic nematodes (Oliveira et al. 2021 ; Paes et al. 2025 ), none have compared single versus repeated applications. Therefore, our observation that a second application provides no additional benefit represents a contribution. This finding reinforces a more economical and operationally efficient strategy for growers, reducing labor and input use without compromising efficacy. The results obtained in Experiment 3 are consistent with previous reports describing the inhibitory effect of M. anisopliae on M. incognita egg hatching under in vitro conditions. In the present work, egg masses treated with M. anisopliae showed a reduction in the number of hatched second-stage juveniles throughout the 7-day evaluation period compared with the control, indicating a direct ovistatic or ovicidal effect of the fungus. Similar responses were reported by Youssef et al. ( 2020 ), who demonstrated that culture filtrates and spore suspensions of M. anisopliae significantly inhibited M. incognita egg hatching in vitro , with inhibition levels exceeding 70% depending on concentration and exposure time. Although methodological differences exist between the studies, particularly regarding exposure period and data expression, both sets of results converge in demonstrating that M. anisopliae negatively affects egg hatching in a time-dependent manner. Moreover, the reduced hatching observed in the present in vitro assay was reflected in the subsequent biotest on tomato plants, in which lower final nematode population (Pf) and nematode density per gram of root were recorded, reinforcing the biological relevance of early hatching inhibition and ovicidal effect, corroborating the nematicidal potential of M. anisopliae reported in previous studies. Although several biological nematicides are commercially available, mainly products based on Purpureocillium lilacinum, Pochonia chlamydosporia and Bacillus species (MAPA 2025), there are no registered nematicides formulated with M. anisopliae . Still marketed exclusively as an entomopathogenic fungus, its use against plant parasitic nematodes has not yet been incorporated. The present study reinforces its potential, showing that M. anisopliae not only suppresses nematodes but also stimulates plant growth. This dual functionality underscores M. anisopliae as a promising candidate to advance beyond current biological options and may encourage future efforts toward its registration for nematode management. Taken together, the results of this work confirmed the multifunctional role of M. anisopliae in the plant nematode system: the fungus reduced M. incognita reproduction while simultaneously stimulating plant agronomic development. These findings reinforce the relevance of M. anisopliae as a viable bioinput and the potential integration into nematode management programs as a sustainable strategy for both plant growth promotion and nematode control in tomato cultivation. CONCLUSION This work demonstrates that M. anisopliae can simultaneously promote tomato growth and suppress M. incognita under greenhouse conditions. 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Front Sustainable Food Syst 4:137. 10.3389/fsufs.2020.00137 WHITEHEAD AG, HEMMING JR (1965) A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Ann Appl Biol 55:25–38 YOUSSEF MMA, EL-NAGDI WMA, LOTFY DEM (2020) Evaluation of the fungal activity of Beauveria bassiana , Metarhizium anisopliae and Paecilomyces lilacinus as biocontrol agents against root-knot nematode, Meloidogyne incognita , on cowpea. Bulletin of the National Research Centre . 44: 112. 10.1186/s42269-020-00367-z Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor invited by journal 02 Feb, 2026 Editor assigned by journal 31 Jan, 2026 First submitted to journal 30 Jan, 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. 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Paes","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3076-6344","institution":"Instituto Biologico","correspondingAuthor":true,"prefix":"","firstName":"Bruno","middleName":"Scentinela Jacintho","lastName":"Paes","suffix":""},{"id":607076037,"identity":"8e0b2fd9-b7d9-44d2-8523-7d1f004c804a","order_by":1,"name":"Rhayane Resende Pillat","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rhayane","middleName":"Resende","lastName":"Pillat","suffix":""},{"id":607076038,"identity":"c0b5f4a8-b134-414a-9872-e21edd6f9753","order_by":2,"name":"Melissa Dall'Oglio Tomazini","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"Dall'Oglio","lastName":"Tomazini","suffix":""},{"id":607076039,"identity":"373199a1-f2a5-4c8c-9d1f-96c124da41ba","order_by":3,"name":"Erika Seguchi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"","lastName":"Seguchi","suffix":""},{"id":607076040,"identity":"23cdaf87-35bc-41a8-98bd-a2ff2b7de551","order_by":4,"name":"Milena Yasmin da Silva Souza","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"Yasmin da Silva","lastName":"Souza","suffix":""},{"id":607076041,"identity":"3cfd3712-6ba1-43f5-91ce-bb8a04344337","order_by":5,"name":"Cláudio Marcelo Gonçalves de Oliveira","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cláudio","middleName":"Marcelo Gonçalves","lastName":"de Oliveira","suffix":""},{"id":607076042,"identity":"28cdc819-f4ae-4aed-b51b-d26bcfa3ad02","order_by":6,"name":"José Eduardo Marcondes de Almeida","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Eduardo Marcondes","lastName":"de Almeida","suffix":""}],"badges":[],"createdAt":"2026-01-29 13:20:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8732041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8732041/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104976265,"identity":"0f72c426-7641-4fc9-85e2-a27abe40efbf","added_by":"auto","created_at":"2026-03-19 12:12:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94580,"visible":true,"origin":"","legend":"\u003cp\u003eTomato plants 20 days after the application of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e(\u003cem\u003eM.a.\u003c/em\u003e) and inoculation with \u003cem\u003eMeloidogyne incognita \u003c/em\u003e(\u003cem\u003eM.i.\u003c/em\u003e) (Experiment 1).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8732041/v1/1054b1fa4a97a56c2cd510de.jpg"},{"id":104976250,"identity":"155e6db5-d72a-4d3d-b468-b2ef3e225462","added_by":"auto","created_at":"2026-03-19 12:12:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":298087,"visible":true,"origin":"","legend":"\u003cp\u003eTomato roots 60 days after treatment with \u003cem\u003eMetarhizium anisopliae\u003c/em\u003eand inoculation with \u003cem\u003eMeloidogyne incognita \u003c/em\u003e(Experiment 2).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8732041/v1/fc67f023e0877429334c53fc.jpg"},{"id":104976255,"identity":"07e2a651-a402-49b7-9547-5b5f1bfc2d5d","added_by":"auto","created_at":"2026-03-19 12:12:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49208,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on \u003cem\u003ein vitro\u003c/em\u003e hatching of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e second-stage juveniles over 7-day period.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8732041/v1/59606725974f92ac464f5cd8.jpg"},{"id":104976260,"identity":"e0a27219-39a4-49ad-85aa-8b7594ed77c2","added_by":"auto","created_at":"2026-03-19 12:12:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43404,"visible":true,"origin":"","legend":"\u003cp\u003eFinal nematode population (Pf) and number of nematodes per gram of root in tomato plants inoculated with \u003cem\u003eMeloidogyne incognita\u003c/em\u003e treated with \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e or water (control), evaluated 55 days after inoculation.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8732041/v1/c4f8481875e343615e3a8cb1.jpg"},{"id":105035351,"identity":"597df290-82d2-4dec-89df-0f314939675c","added_by":"auto","created_at":"2026-03-20 07:25:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1019281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8732041/v1/97570152-22d8-42e3-9056-ab4f82673891.pdf"}],"financialInterests":"","formattedTitle":"Metarhizium anisopliae reduces Meloidogyne incognita reproduction and promotes tomato plant growth","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe Brazilian agricultural sector has faced, in recent decades, an increasing demand for sustainability and efficiency, driven by environmental pressures, climate change, global food needs, and rising costs and restrictions associated with pesticide use. Within this scenario, biotic factors especially soilborne pathogens and pests stand out as major constraints to crop productivity, causing economic losses and compromising yield stability (Fontes and Valadares-Inglis \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these pathogens, plant parasitic nematodes are considered one of the most damaging threats to tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) production, a crop of high economic and social importance in Brazil and worldwide (Agrios \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pinheiro et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lopes and Ferraz \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is widely recognized that root-knot nematodes (\u003cem\u003eMeloidogyne\u003c/em\u003e spp.) can severely reduce tomato yield, and in many cases their impact becomes devastating when infestations reach high levels. In Brazil, \u003cem\u003eMeloidogyne incognita\u003c/em\u003e (Kofoid and White, 1919) Chitwood (1949), known as the Southern root-knot nematode, is widespread and frequently reported as one of the most critical soil pathogens affecting tomatoes and other high value crops (Pinheiro et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Oliveira and Rosa \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lopes and Ferraz \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Historically, the management of root-knot nematodes has relied on synthetic nematicides and traditional agronomic practices such as crop rotation, the use of nematode free seedlings, and when available resistant or less susceptible cultivars (Agrios \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Oliveira and Rosa \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, increasing environmental restrictions, high input costs, and risks to human health and non-target organisms have driven the search for more sustainable alternatives, among which biological control with antagonistic or growth promoting microorganisms has gained prominence (Fontes and Valadares-Inglis \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch on the interaction between \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Metschnikoff) Sorokin and plant parasitic nematodes has advanced substantially in recent decades. Early studies demonstrated the direct nematicidal action of the fungus, including its ability to reduce \u003cem\u003eMeloidogyne\u003c/em\u003e spp. in sugarcane crop (Rossi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), as well as its capacity to suppress \u003cem\u003eM. javanica\u003c/em\u003e populations in soils enriched with organic residues (Abdollahi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, there are reports of \u003cem\u003eM. anisopliae\u003c/em\u003e isolates obtained from suppressive soils being pathogenic to juveniles of other nematode species, such as \u003cem\u003eHeterodera avenae\u003c/em\u003e, reinforcing the versatility of this entomopathogenic species as a bionematicide (Ghayedi and Abdollahi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Complementary evidence from Brazilian agriculture indicates that \u003cem\u003eM. anisopliae\u003c/em\u003e is effective in reducing \u003cem\u003eM. incognita\u003c/em\u003e infestation in banana, coffee, and cotton crops (Oliveira et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Paes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Beyond its biocontrol activity, \u003cem\u003eM. anisopliae\u003c/em\u003e has also been recognized as a plant growth promoting microorganism, enhancing physiological performance and root development in several hosts, including tomato, maize, and \u003cem\u003eArabidopsis\u003c/em\u003e (Sasan and Bidochka \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Siqueira et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gonz\u0026aacute;lez-P\u0026eacute;rez et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite this substantial progress, no prior study has clearly demonstrated the simultaneous expression of these two functions, nematicidal activity and plant growth promotion. Considering the magnitude of losses caused by root-knot nematodes in solanaceous crops (Oliveira and Rosa \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pinheiro et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lopes and Ferraz \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the development of microbial agents capable of integrating pest suppression and plant stimulation aligns with the current demands of sustainable agriculture and consolidates the novelty and scientific relevance of the present study.\u003c/p\u003e \u003cp\u003eDespite these advances, there is still a lack of studies clearly documenting the dual function of \u003cem\u003eM. anisopliae.\u003c/em\u003e This gap represents an important research opportunity to investigate and demonstrate, for the first time, that a single isolate of \u003cem\u003eM. anisopliae\u003c/em\u003e can reduce \u003cem\u003eM. incognita\u003c/em\u003e infestation while also enhancing plant vigor. Therefore, the present study aimed to evaluate the impact of \u003cem\u003eM. anisopliae\u003c/em\u003e on plant growth and nematode suppression in tomato plants inoculated with \u003cem\u003eM. incognita\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e and greenhouse conditions.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eThree greenhouse experiments were conducted under controlled conditions at the Biological Institute, in the city of Campinas, SP, Brazil (22 \u0026ordm;54\u0026rsquo;S,47\u0026ordm;00\u0026rsquo;W and altitude of 707 m) at spring, 2025. The average temperature during the experiments was 34\u0026thinsp;\u0026plusmn;\u0026thinsp;3 \u0026ordm;C. The plots consisted of 800 cm\u0026sup3; plastic pots (15 cm height, 4.75 cm upper radius, and 3.5 cm lower radius) filled with 750 cm\u0026sup3; of autoclaved commercial substrate Tropstrato Florestal\u0026reg; (pH of 6.0; composed of pine bark, vermiculite and single superphosphate) and tomato hybrid \u0026lsquo;Sophia\u0026rsquo; (Sakata Seed Sudamerica) was used in both experiments. The seeds were first sown in seedling trays, and after 15 days the seedlings were transplanted to the pots, with one seedling per pot and 0.5g of a slow-release fertigation fertilizer (NPK 13-40-13) was applied. The experiments were conducted in a completely randomized design.\u003c/p\u003e \u003cp\u003eNematode inoculum\u003c/p\u003e \u003cp\u003eTo preserve nematode virulence, the isolate was periodically inoculated on different host plants. Annual identification of \u003cem\u003eM. incognita\u003c/em\u003e was confirmed using perineal pattern morphology (Kleynhans \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, Jepson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) and isoenzyme electrophoresis (esterase profile), identifying the isolate as \u003cem\u003eM. incognita\u003c/em\u003e race 3 (Alfenas and Brune \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Nematode inoculum consisted of eggs and second-stage juveniles (J2) extracted from infected roots using a modified Coolen and D\u0026rsquo;Herde (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) protocol. Roots were blended in a 0.5% NaOCl solution for one minute, and the suspension was filtered through a series of sieves (60-200-500 mesh; 0.250-0.074-0.025 mm). The resulting suspension was used for inoculation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. anisopliae\u003c/em\u003e isolate\u003c/p\u003e \u003cp\u003eThe isolate of \u003cem\u003eM. anisopliae\u003c/em\u003e was obtained from the fungal collection maintained at the Biological Control Reference Laboratory Unit/CAPSA of Biological Institute. The isolate was freshly cultured and exhibited spore viability above 80%. The fungal isolate was preserved thorough lyophilization and reactivated on Petri dishes containing PDA (Potato dextrose agar) supplemented with an antibiotic used to prevent contamination. Once sporulation was observed, conidia were scraped from the agar surface and suspended in sterile Milli-Q water containing 0.01% of Tween 80. This suspension was then inoculated onto autoclaved rice incubated under controlled conditions (25 \u0026ordm;C; 12:12 h light-dark photoperiod).\u003c/p\u003e \u003cp\u003eAfter approximately seven days of incubation, once intense sporulation was evident, conidia were collected from the rice substrate through a dry separation method. The colonized rice was transferred to a 20-mesh sieve (0.25 mm aperture) and shaken by hand for about three minutes. This procedure allowed the conidia to detach from the rice grains and pass through the sieve openings by gravity. The released conidia were collected in a clean plastic container placed beneath the sieve. Conidial density was then quantified using a Neubauer hemocytometer under a light microscope (Leica DMLS), and the suspension was adjusted to 1 \u0026times; 10⁸ viable conidia mL⁻\u0026sup1;. Each plant received 30 mL of the suspension, applied in the soil near the seedlings.\u003c/p\u003e \u003cp\u003eExperiment 1 \u0026ndash; Greenhouse test of a single application of \u003cem\u003eM. anisopliae\u003c/em\u003e isolate on tomato plants infested with \u003cem\u003eM. incognita\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe first experiment consisted of 4 treatments: one non-inoculated control, one inoculated with \u003cem\u003eM. incognita\u003c/em\u003e (Mi), one treated with \u003cem\u003eM. anisopliae\u003c/em\u003e isolate (Ma) and one inoculated with \u003cem\u003eM. incognita\u003c/em\u003e and treated with \u003cem\u003eM. anisopliae\u003c/em\u003e (Mi\u0026thinsp;+\u0026thinsp;Ma), each with seven replicates. The inoculation of \u003cem\u003eM. incognita\u003c/em\u003e was performed in the same day of transplanting, at sowing by 5,000 eggs and J2 nematodes into two 2 cm deep holes near the seedlings. Plants were maintained in the greenhouse throughout the tomato cycle and irrigated daily. At 60 days after transplanting (DAT), the following variables were evaluated: final nematode population in the roots (Fp); reproduction factor (Rf\u0026thinsp;=\u0026thinsp;Fp/initial population); nematode suppression efficiency compared to control (E%), calculated according to the Abbott\u0026rsquo;s formula, using the expression: E% =[(C - T)/C] \u0026times; 100, where C represents the mean value observed in the control treatment and T the mean value in the treated group; root fresh mass (RFM); shoot dry (SDM) and fresh mass (SFM); and shoot height (SH). Nematodes were extracted from the roots using the same method as for inoculum preparation, whereas the nematode counts were performed under a light microscope (Leica DMLS) using Peters counting slides with two 1-mL sub samples.\u003c/p\u003e \u003cp\u003eExperiment 2 \u0026ndash; Greenhouse test of two applications of \u003cem\u003eM. anisopliae\u003c/em\u003e isolate on tomato plants infested with \u003cem\u003eM. incognita\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe second experiment was conducted under the same period, inoculation of \u003cem\u003eM. incognita\u003c/em\u003e and greenhouse conditions as experiment 1. This experiment included 5 treatments: one non-inoculated control, one inoculated with \u003cem\u003eM. incognita\u003c/em\u003e, one treated with \u003cem\u003eM. anisopliae\u003c/em\u003e isolate, one treated with twice application of \u003cem\u003eM. anisopliae\u003c/em\u003e (Ma2) and one inoculated with \u003cem\u003eM. incognita\u003c/em\u003e and treated with \u003cem\u003eM. anisopliae\u003c/em\u003e, with eight replicates per treatment. The evaluations were carried out at 60 DAT and included the same variables.\u003c/p\u003e \u003cp\u003eExperiment 3 - Effects of \u003cem\u003eM. anisopliae\u003c/em\u003e on second-stage juveniles hatching of \u003cem\u003eM. incognita\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions followed by a greenhouse assay\u003c/p\u003e \u003cp\u003eExperiment 3 was conducted in two sequential stages to evaluate the effects of \u003cem\u003eM. anisopliae\u003c/em\u003e on the hatching of second-stage juveniles (J2) of \u003cem\u003eM. incognita\u003c/em\u003e and the persistence of its nematicidal activity over time.\u003c/p\u003e \u003cp\u003eThe stage 1 consisted of an \u003cem\u003ein vitro\u003c/em\u003e assay, using the modified Baermann funnel (Baermann \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1917\u003c/span\u003e) method for a shallow container (Whitehead and Hemming \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1965\u003c/span\u003e) to assess J2 hatching over a 7-day period.\u003c/p\u003e \u003cp\u003eEgg masses treated with distilled water (control) were compared with egg masses treated with \u003cem\u003eM. anisopliae\u003c/em\u003e, using the same fungal concentration adopted in Experiments 1 and 2. Six egg masses extracted from symptomatic tomato plants and stained with Phloxine B to facilitate visualization, were carefully placed in each container, with absorbent paper, with six replicates per treatment. Each container received 5 mL of either distilled water or fungal suspension and was maintained in a BOD incubator at 25\u0026deg;C. J2 hatching was evaluated at 3, 5, and 7 days after treatment using a stereomicroscope. At each assessment time, the liquid from the bottom of the container was collected, and the number of hatched J2 was counted.\u003c/p\u003e \u003cp\u003eThe Stage 2 consisted of a bioassay in tomato plants, in which all material from each container, containing remaining eggs and hatched J2, was transferred to tomato seedlings transplanted under the same conditions described for Experiments 1 and 2. This bioassay was designed to confirm whether \u003cem\u003eM. anisopliae\u003c/em\u003e exerts a direct effect on egg hatching and to determine whether this effect persists during the period in which the tomato plants were maintained under greenhouse conditions. After 55 days of inoculation, tomato roots were processed to determine the final nematode population (Pf) and the number of nematodes/g⁻\u0026sup1; root, following the same root nematode extraction method described for Experiments 1 and 2. Nematode quantification at this stage was performed using an optical microscope with the aid of a Peters counting slide.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the Sisvar software (Ferreira \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and treatment means compared by the Tukey test at 5% of significance. When necessary, data were log-transformed [log (x\u0026thinsp;+\u0026thinsp;1)] to meet the assumptions of normality, which were verified using the Shapiro-Wilk test.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eSignificant differences among treatments were observed for most of the evaluated variables in both experiments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In Experiment 1, tomato plants treated with \u003cem\u003eM. anisopliae\u003c/em\u003e exhibited the greatest increases in agronomic variables, clearly indicating that the fungus stimulated plant development. Application of \u003cem\u003eM. anisopliae\u003c/em\u003e resulted in the tallest plants (76.1 cm) and the highest shoot fresh mass (44.0 g), values statistically similar to those obtained in the combined Ma\u0026thinsp;+\u0026thinsp;Mi treatment (77.0 cm and 46.7 g), and both superior to the control and to plants inoculated only with \u003cem\u003eM. incognita\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant differences were detected for shoot dry mass. Root fresh mass followed the same trend, with \u003cem\u003eM. anisopliae\u003c/em\u003e producing the highest root mass (27.3 g), followed by Ma\u0026thinsp;+\u0026thinsp;Mi (24.5 g), both significantly greater than the control (14.5 g). Plants inoculated only with \u003cem\u003eM. incognita\u003c/em\u003e exhibited reduced root mass (16.2 g) relative to the fungal treatments. Regarding nematode parameters, \u003cem\u003eM. incognita\u003c/em\u003e alone reached a final population of 21,107 specimens per root system, resulting in the highest reproduction factor (4.2). When \u003cem\u003eM. anisopliae\u003c/em\u003e was applied to infested plants, nematode reproduction decreased, with the Ma\u0026thinsp;+\u0026thinsp;Mi treatment reducing the final population to 9,189 and the reproduction factor to 1.8, corresponding to 56% efficiency compared with the nematode-only treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on tomato plant growth and \u003cem\u003eMeloidogyne incognita\u003c/em\u003e reproduction in greenhouse experiments evaluated 60 days after inoculation. SH\u0026thinsp;=\u0026thinsp;shoot height; SFM\u0026thinsp;=\u0026thinsp;shoot fresh mass; SDM\u0026thinsp;=\u0026thinsp;shoot dry mass; RFM\u0026thinsp;=\u0026thinsp;root fresh mass; Fp\u0026thinsp;=\u0026thinsp;final nematode population; Rf\u0026thinsp;=\u0026thinsp;reproduction factor; E% = control efficacy.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eEXPERIMENT 1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSH (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSFM (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSDM (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRFM (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eE%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.8 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.1 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.5 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. incognita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.7 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.2 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.6 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.2 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21,107 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. anisopliae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.1 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.0 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.4 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMa\u0026thinsp;+\u0026thinsp;Mi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.0 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.7 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.5 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9,189 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEXPERIMENT 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSH (cm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eSFM (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSDM (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eRFM (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eFp\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eRf\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eE%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.2 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.4 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.4 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. incognita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.0 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.7 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.7 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.6 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26,429 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. anisopliae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.5 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.7 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.4 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.3 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. anisopliae\u003c/em\u003e 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.7 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.1 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.9 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.9 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMa\u0026thinsp;+\u0026thinsp;Mi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.4 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.8 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.1 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11,746 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.3 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans followed by different lower case in column differ from each other by Tukey test at 0.05 significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Experiment 2, \u003cem\u003eM. anisopliae\u003c/em\u003e again stimulated plant growth, reinforcing its beneficial effect on agronomic variables. The highest shoot heights were observed in plants receiving either one (93.5 cm) or two (90.7 cm) fungal applications, which also resulted in the greatest shoot fresh and dry mass accumulation. Importantly, applying \u003cem\u003eM. anisopliae\u003c/em\u003e twice at transplanting and again 30 days later did not differ significantly from a single application for any agronomic variable, indicating that one application was sufficient to promote plant development. Although root fresh mass did not differ significantly among treatments, the fungal applications tended to produce higher values than the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs in Experiment 2, the highest nematode reproduction occurred in plants inoculated with \u003cem\u003eM. incognita\u003c/em\u003e alone, reaching a final population of 26,429 and a reproduction factor of 5.2. The application of \u003cem\u003eM. anisopliae\u003c/em\u003e to infested plants significantly reduced nematode multiplication, lowering the final population to 11,746 and the reproduction factor to 2.3, resulting in 55% efficiency relative to the nematode-only treatment. Overall, \u003cem\u003eM. anisopliae\u003c/em\u003e consistently reduced \u003cem\u003eM. incognita\u003c/em\u003e reproduction and stimulated tomato plant development across both experiments.\u003c/p\u003e \u003cp\u003eFinally, the Experiment 3, in the \u003cem\u003ein vitro\u003c/em\u003e assay (Stage 1), the hatching of second-stage juveniles of \u003cem\u003eM. incognita\u003c/em\u003e increased progressively over time in both treatments. However, egg masses treated with \u003cem\u003eM. anisopliae\u003c/em\u003e showed consistently lower numbers of hatched juveniles compared with the control throughout the evaluation period. At 3 days after incubation, the control treatment presented 1,234 J2, whereas the treatment with \u003cem\u003eM. anisopliae\u003c/em\u003e showed 405 J2. At 5 days, the number of hatched juveniles increased to 2,176 in the control and to 534 in the \u003cem\u003eM. anisopliae\u003c/em\u003e treatment. At the final evaluation, 7 days after incubation, the control reached 2,371 hatched J2, while the \u003cem\u003eM. anisopliae\u003c/em\u003e treatment resulted in 613 J2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the biotest conducted in tomato plants (Stage 2), significant differences were observed between treatments for both nematode population variables evaluated at 55 days after inoculation. Plants inoculated with control treatment showed a Pf of 25,964 and 5,753 nematodes/g⁻\u0026sup1; of root. In contrast, plants inoculated with the nematode treated with \u003cem\u003eM. anisopliae\u003c/em\u003e presented lower values, with a Pf of 4,977 and 693 nematodes/g⁻\u0026sup1; of root (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results of this study demonstrate that the application of \u003cem\u003eM. anisopliae\u003c/em\u003e simultaneously promoted tomato plant growth and reduced the reproduction of \u003cem\u003eM. incognita\u003c/em\u003e. The increases observed in plant height, shoot fresh mass, and root fresh mass clearly indicate a biostimulant effect on plant development. These findings corroborate previous studies showing the capacity of \u003cem\u003eMetarhizium\u003c/em\u003e spp. to act as plant growth promoters. Siqueira et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that the fungus produces biochemical compounds capable of modulating plant metabolism, enhancing endophytic colonization, and stimulating tomato growth. Similarly, Sasan and Bidochka (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) demonstrated that \u003cem\u003eM. robertsii\u003c/em\u003e can colonize plant roots and promote greater root development, improving nutrient acquisition. Although no molecular, biochemical, or colonization assays were performed in the present study, the stimulatory effect was clearly verified, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFurther support for this biostimulant role comes from Gonz\u0026aacute;lez et al. (2022), who demonstrated that multiple \u003cem\u003eM. anisopliae\u003c/em\u003e strains significantly enhanced fresh weight and chlorophyll content in \u003cem\u003eArabidopsis\u003c/em\u003e within seven days and stimulated primary root elongation through volatile organic compounds such as β-caryophyllene and o-cymene. These effects were also confirmed in soil-grown tomato, and maize plants. Consistent with these findings, our results showed that \u003cem\u003eM. anisopliae\u003c/em\u003e improved several agronomic variables in tomato and effectively reduced nematode reproduction, reinforcing its dual activity as both a plant growth promoter and a biological control agent.\u003c/p\u003e \u003cp\u003eIn the present study, the \u003cem\u003eM. anisopliae\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eM. incognita\u003c/em\u003e treatment significantly reduced the nematode reproduction factor in both experiments, with efficiencies of 55% and 56%. This reduction confirms the potential of the fungus as a biological control agent against plant parasitic nematodes. Similar effects have been documented by Abdollahi (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who observed suppression of \u003cem\u003eM. javanica\u003c/em\u003e in soil amended with organic material colonized by \u003cem\u003eM. anisopliae\u003c/em\u003e, and by Ghayedi and Abdollahi (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), who reported high pathogenicity of isolates from suppressive soils against juveniles of \u003cem\u003eHeterodera avenae\u003c/em\u003e. The present findings also align with Paes et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who demonstrated that multiple \u003cem\u003eM. anisopliae\u003c/em\u003e isolates significantly reduced \u003cem\u003eM. incognita\u003c/em\u003e reproduction in cotton, indicating that the nematicidal potential of the fungus is consistent across different cropping systems. In perennial crops, Oliveira et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found the fungus to be effective against \u003cem\u003eM. incognita\u003c/em\u003e in banana and coffee, and Rossi et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) also reported nematicidal activity of microbial agents containing \u003cem\u003eM. anisopliae\u003c/em\u003e in sugarcane. Collectively, these studies suggest that the suppressive effect of \u003cem\u003eM. anisopliae\u003c/em\u003e is robust and not strongly dependent on the host plant, underscoring its potential applicability in diverse agricultural systems.\u003c/p\u003e \u003cp\u003eAn additional finding of practical relevance is that a second application of \u003cem\u003eM. anisopliae\u003c/em\u003e performed 30 days after transplanting did not enhance plant growth or reduce nematode reproduction beyond the effects obtained with a single application. In both experiments, one application at transplanting was sufficient to get biostimulant and nematicidal responses. Although authors have demonstrated the efficacy of a single application of \u003cem\u003eM. anisopliae\u003c/em\u003e against plant parasitic nematodes (Oliveira et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Paes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), none have compared single versus repeated applications. Therefore, our observation that a second application provides no additional benefit represents a contribution. This finding reinforces a more economical and operationally efficient strategy for growers, reducing labor and input use without compromising efficacy.\u003c/p\u003e \u003cp\u003eThe results obtained in Experiment 3 are consistent with previous reports describing the inhibitory effect of \u003cem\u003eM. anisopliae\u003c/em\u003e on \u003cem\u003eM. incognita\u003c/em\u003e egg hatching under \u003cem\u003ein vitro\u003c/em\u003e conditions. In the present work, egg masses treated with \u003cem\u003eM. anisopliae\u003c/em\u003e showed a reduction in the number of hatched second-stage juveniles throughout the 7-day evaluation period compared with the control, indicating a direct ovistatic or ovicidal effect of the fungus. Similar responses were reported by Youssef et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who demonstrated that culture filtrates and spore suspensions of \u003cem\u003eM. anisopliae\u003c/em\u003e significantly inhibited \u003cem\u003eM. incognita\u003c/em\u003e egg hatching \u003cem\u003ein vitro\u003c/em\u003e, with inhibition levels exceeding 70% depending on concentration and exposure time. Although methodological differences exist between the studies, particularly regarding exposure period and data expression, both sets of results converge in demonstrating that \u003cem\u003eM. anisopliae\u003c/em\u003e negatively affects egg hatching in a time-dependent manner. Moreover, the reduced hatching observed in the present \u003cem\u003ein vitro\u003c/em\u003e assay was reflected in the subsequent biotest on tomato plants, in which lower final nematode population (Pf) and nematode density per gram of root were recorded, reinforcing the biological relevance of early hatching inhibition and ovicidal effect, corroborating the nematicidal potential of \u003cem\u003eM. anisopliae\u003c/em\u003e reported in previous studies.\u003c/p\u003e \u003cp\u003eAlthough several biological nematicides are commercially available, mainly products based on \u003cem\u003ePurpureocillium lilacinum, Pochonia chlamydosporia\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e species (MAPA 2025), there are no registered nematicides formulated with \u003cem\u003eM. anisopliae\u003c/em\u003e. Still marketed exclusively as an entomopathogenic fungus, its use against plant parasitic nematodes has not yet been incorporated. The present study reinforces its potential, showing that \u003cem\u003eM. anisopliae\u003c/em\u003e not only suppresses nematodes but also stimulates plant growth. This dual functionality underscores \u003cem\u003eM. anisopliae\u003c/em\u003e as a promising candidate to advance beyond current biological options and may encourage future efforts toward its registration for nematode management.\u003c/p\u003e \u003cp\u003eTaken together, the results of this work confirmed the multifunctional role of \u003cem\u003eM. anisopliae\u003c/em\u003e in the plant nematode system: the fungus reduced \u003cem\u003eM. incognita\u003c/em\u003e reproduction while simultaneously stimulating plant agronomic development. These findings reinforce the relevance of \u003cem\u003eM. anisopliae\u003c/em\u003e as a viable bioinput and the potential integration into nematode management programs as a sustainable strategy for both plant growth promotion and nematode control in tomato cultivation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis work demonstrates that \u003cem\u003eM. anisopliae\u003c/em\u003e can simultaneously promote tomato growth and suppress \u003cem\u003eM. incognita\u003c/em\u003e under greenhouse conditions. Treated plants showed increased height and biomass, confirming the fungus\u0026rsquo;s biostimulant effect, while nematode reproduction was reduced by 55 and 56% in inoculated plants with nematode. Furthermore, a single or double applications performed similarly, indicating that one application at transplanting is sufficient.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eABDOLLAHI M (2018) Application of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e against \u003cem\u003eMeloidogyne javanica\u003c/em\u003e in soil amended with oak debris. Int J Agricultural Biosystems Eng 12(2):35\u0026ndash;41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAGRIOS GN (2005) Plant pathology. Elsevier, Boston, p 921\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eALFENAS AC, BRUNE W (2006) Eletroforese em gel de amido. In: ALFENAS AC (ed) Eletroforese e marcadores bioqu\u0026iacute;micos em plantas e microrganismos. 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Boletim T\u0026eacute;cnico n. 3. pp. 233\u0026ndash;246\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOLIVEIRA CMG et al (2021) Efficiency of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e in the control of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e in banana and coffee crops. Coffee Sci 16:e161957\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePAES BSJ, ARA\u0026Uacute;JO HR, OLIVEIRA CMG, ALMEIDA JEM (2025) Efficacy of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and \u003cem\u003ePurpureocillium lilacinum\u003c/em\u003e isolates in the control of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e race 3 in cotton. Pesquisa Agropecu\u0026aacute;ria Trop. e83171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePINHEIRO JB, PEREIRA RB, SUINAGA FA (2014) \u003cem\u003eManejo de nematoides na cultura do tomate\u003c/em\u003e. Circular T\u0026eacute;cnica 132. Bras\u0026iacute;lia, DF: Embrapa Hortali\u0026ccedil;as. 12 p\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eROSSI CE et al (2006) Efeito de inseticidas fitoqu\u0026iacute;micos e microbianos em nematoides da cana-de-a\u0026ccedil;\u0026uacute;car. STAB \u0026ndash; A\u0026ccedil;\u0026uacute;car \u0026Aacute;lcool e Subprodutos 24(3):6\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSASAN RK, BIDOCHKA MJ (2012) The insect-pathogenic fungus \u003cem\u003eMetarhizium robertsii\u003c/em\u003e (Clavicipitaceae) is also an endophyte that stimulates plant root development. Am J Bot 99(1):101\u0026ndash;107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSIQUEIRA ACO et al (2020) Multi-trait biochemical features of \u003cem\u003eMetarhizium\u003c/em\u003e species and their activities that stimulate the growth of tomato plants. Front Sustainable Food Syst 4:137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fsufs.2020.00137\u003c/span\u003e\u003cspan address=\"10.3389/fsufs.2020.00137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHITEHEAD AG, HEMMING JR (1965) A comparison of some quantitative methods of extracting small vermiform nematodes from soil. Ann Appl Biol 55:25\u0026ndash;38\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYOUSSEF MMA, EL-NAGDI WMA, LOTFY DEM (2020) Evaluation of the fungal activity of \u003cem\u003eBeauveria bassiana\u003c/em\u003e, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and \u003cem\u003ePaecilomyces lilacinus\u003c/em\u003e as biocontrol agents against root-knot nematode, \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, on cowpea. \u003cem\u003eBulletin of the National Research Centre\u003c/em\u003e. 44: 112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s42269-020-00367-z\u003c/span\u003e\u003cspan address=\"10.1186/s42269-020-00367-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Southern root-knot nematode, Solanum lycopersicum L., bionematicidal, biostimulant","lastPublishedDoi":"10.21203/rs.3.rs-8732041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8732041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRoot-knot nematodes, especially \u003cem\u003eMeloidogyne incognita\u003c/em\u003e, cause substantial yield losses in tomato production and remain one of the most challenging soilborne pathogens in Brazil. The entomopathogenic fungus \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e has been recognized for both its nematicidal activity and its capacity to promote plant growth, although these two functions have rarely been demonstrated simultaneously in the same study. This work evaluated the effectiveness of \u003cem\u003eM. anisopliae\u003c/em\u003e in stimulating tomato growth and reducing \u003cem\u003eM. incognita\u003c/em\u003e reproduction under \u003cem\u003ein vitro\u003c/em\u003e and greenhouse conditions. Three experiments were conducted using tomato hybrid \u0026lsquo;Sophia\u0026rsquo;, with treatments including fungal application, nematode inoculation, and their combination. In both experiments, \u003cem\u003eM. anisopliae\u003c/em\u003e significantly increased plant height, shoot fresh mass, and root fresh mass compared with untreated controls. The effect of \u003cem\u003eM. anisopliae\u003c/em\u003e was also directly evaluated on egg masses under \u003cem\u003ein vitro\u003c/em\u003e conditions to verify the hatching rate of juveniles. When applied to nematode infested plants, the fungus reduced the nematode multiplication by 55 and 56%, lowering the reproduction factor from 4.2 and 5.2 (nematode-only) to 1.8 and 2.3. Single and double applications of \u003cem\u003eM. anisopliae\u003c/em\u003e produced similar agronomic responses, indicating that one application was sufficient for growth promotion. Overall, the results demonstrate that \u003cem\u003eM. anisopliae\u003c/em\u003e acts as both a plant growth promoter and an effective biological control agent against \u003cem\u003eM. incognita\u003c/em\u003e, bringing greater prominence to the management of plant-parasitic nematodes, particularly root-knot nematodes in tomato.\u003c/p\u003e","manuscriptTitle":"Metarhizium anisopliae reduces Meloidogyne incognita reproduction and promotes tomato plant growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 12:11:48","doi":"10.21203/rs.3.rs-8732041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-03-17T11:53:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T16:34:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2026-02-02T13:32:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-31T18:25:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2026-01-30T10:36:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8bcc2a6e-0539-41ae-8bc7-218dbc17ad00","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T12:11:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 12:11:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8732041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8732041","identity":"rs-8732041","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}