Macrolophus pygmaeus (Heteroptera: Miridae) induces systemic resistance in tomato against Meloidogyne spp

Macrolophus pygmaeus (Heteroptera: Miridae) induces systemic resistance in tomato against Meloidogyne spp | 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 Article Macrolophus pygmaeus (Heteroptera: Miridae) induces systemic resistance in tomato against Meloidogyne spp Alejandro Expósito, Pablo Urbaneja-Bernat, Sara Boncompte, Aida Magdalena Fullana, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5181542/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The ability of Macrolophus pygmaeus to induce systemic resistance in susceptible and Mi1.2 resistant tomato against Meloidogyne spp. was evaluated in pot experiments. The susceptible cv. Roma and the resistant cv. Caramba were exposed to 15 M. pygmaeus nymphs per plant in mesh bags for 48h and then were inoculated with 1 second-stage juvenile (J2) of M. incognita or 3 J2 cm − 3 of soil of a mixed community of M. arenaria, M. hapla , and M. javanica . Tomato plants were maintained in a growth chamber during 40 days. Then the number of egg masses and eggs per plant were determined. In addition, the preference of the insect was estimated confronting nematode-infected vs. non-infected plants in a Y-tube olfactometer and in insect cages, where 10 females were released into each cage containing resistant or susceptible tomato plants. After 1, 2, 4, 24, 48 and 72h, the number of M. pygmaeus was counted as well as the offspring after 14 days. M. pygmaeus reduced the infectivity and reproduction by 37% and 53%, in the susceptible tomato inoculated with M. incognita and by 52% and 37% when inoculated with the nematode community but no effect was observed in the Mi1.2 resistant tomato irrespective of the nematode inoculum. The preference and the offspring of M. pygmaeus was not negatively affected by the nematode infection or the tomato cultivar. In conclusion, pre-induction of tomato plants with M. pygmaeus reduces RKN infectivity and reproduction in susceptible but not in Mi1.2 resistant tomato. Biological sciences/Zoology/Entomology Biological sciences/Plant sciences/Plant immunity Biological sciences/Plant sciences/Plant stress responses RKN biological control induced resistance tomato Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Meloidogyne spp. is the most challenging plant-parasitic nematode (PPN) genus affecting global plant production [ 1 ]. More than 100 species have been described, however, the root-knot nematodes (RKN) tropical species M. arenaria , M. incognita , and M. javanica are the most widespread and limiting for vegetable production in tropical and subtropical climates [ 2 ]. These species are obligate parasites, which reproduce parthenogenetically. The second stage juvenile (J2) penetrates the root near the elongation zone, moves intercellular and induce a hypertrophied and multinucleate giant cells (GC) in the vascular cylinder becoming a feeding site, supplying nutrients to the nematode. After that, the J2 undergoes three moults until it reaches the adult stage. The juveniles develop into females that lays eggs into a gelatinous matrix [ 3 ]. Because of that, plant roots become galled, interfering with the correct uptake of water and nutrients, which causes yellowing, wilting, and dwarfism in aboveground parts of the plant, leading plant death in severe attacks. In tomato, one of the main cash crops in the Mediterranean area, RKN can reduce crop yield until 62% and 72%, depending on the cropping season and crop duration [ 4 , 5 ]. Among sustainable nematode control methods, plant resistance stands out as one of the most effective, economically profitable and environmentally safe [ 6 , 4 , 7 ]. In tomato, the resistance against Meloidogyne is mediated by the Mi1.2 resistance gene and conferred resistance against M. arenaria, M. incognita and M. javanica [ 8 ], M. luci and M. ethiopica [ 9 ] but not to M. hapla [ 8 ] or M. enterolobii [ 10 ]. This gene induces a hypersensitive reaction (HR)-mediated cell death around the feeding site, involving an oxidative burst by reactive oxygen species (ROS) that prevents nematode infection and development [ 11 ]. Nevertheless, its effectiveness is reduced or lost after repeated cultivation due to the emergence of virulent populations [ 12 , 4 , 13 ]. An alternative strategy to reduce nematode building up populations is to induce resistance in susceptible germplasm by biotic or abiotic factors. This property can be used to drive apart the use of the same R-gene, reducing its selection pressure and the selection of virulent populations, if the defense mechanism differs from those expressed by the R-genes. Bacteria and fungi have previously described induced plant resistance to RKN by biotic elicitors [ 14 , 15 , 16 , 17 , 18 , 19 ]. Recently, the ability of other organisms, such as insects, to be used in biological pest control has been investigated. For instance, zoophytophagous mirid bugs such as Macrolophus pygmaeus and Nesiodiocoris tenuis (Heteroptera: Miridae), known predators of various aboveground pests, have been observed to trigger plant resistance against several key pests: the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), the South American tomato pinworm Tuta absoluta Meyrick (Lepidoptera: Gelichiidae), the Western flower thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), and the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). This resistance is induced when these mirid bugs engage in phytophagous behavior and puncture the plants [ 20 , 21 , 22 , 23 , 24 , 25 ]. However, the effect of punctured plants infected with RKN in susceptible and resistant germplasm remains unknown, and also the possibility that the induced resistance could be additive to the genetic resistance mediated by the Mi1.2 resistance gene, as reported for Trichoderma asperellum T-34 [ 19 ]. Moreover, PPN infection can modify the host preference and development of certain phytophagous insects and mites, affecting the performance of their development stages and offspring. For example, Pratylenchus penetrans enhances the production of leaf phenolics and glucosinolates in Brassica nigra , where the caterpillar Pieris rapae (Lepidoptera: Pieridae) larvae grew slower and produced fewer pupae compared to non-infested plants [ 26 ]. Likewise, root infection by the cyst nematode Heterodera schachtii reduces the population density of the aphid Brevicoryne brassicae (Hemiptera: Aphididae) and induces changes in the composition of glucosinolates in Brassica oleracea [ 27 ]. Moreover, Arabidopsis thaliana infected by H. schachtii enhances the attractiveness of the spider mite T. uricae and enhances its life history traits [ 28 ], whereas P. penetrans reduces its fertility on Phaseolus vulgaris [ 29 ]. The effect of RKN infection also can affect the aboveground phytophages. For example, the leaf miner T. absoluta prefers uninfected RKN tomato plants for its oviposition, and that root infection negatively affects its pupation process [ 30 ]. Interestingly, positive effects of RKN infection on leaf-chewing herbivores have also been observed. For example, Spodoptera exigua (Lepidoptera: Noctuidae) benefits from M. incognita infection, increasing its weight compared to non-infected plants, depending on the life cycle of the nematode [ 31 ]. Furthermore, M. incognita infection inhibits the root-produced defense chemical nicotine in response to foliar herbivory by Manduca sexta (Lepidoptera: Sphingidae) and Trichoplusia ni (Lepidoptera: Noctuidae), resulting in low-leaf nicotine levels in tobacco [ 32 ]. This study aims to investigate the potential of M. pygmaeus in enhancing plant resistance against root-knot nematodes (RKN) in susceptible and resistant tomato cultivars. Macrolophus pygmaeus was selected for its known safety profile for plants, contrasting with N. tenuis , which has been associated with adverse effects such as necrotic rings on stems and flowers, as well as fruit punctures [ 33 , 34 ]. Additionally, this research explores the insect's preference between RKN-infected and non-infected plants using a vertical Y-tube olfactometer and insect cages, providing insights into the ecological interactions between predator behaviour and plant defense mechanisms. MATERIALS AND METHODS Plant material, insects and nematodes The experiments were conducted at the Institute of Agrifood Research and Technology (IRTA) (Cabrils, Spain) during 2023. The susceptible tomato cv. Roma (Fitó Seeds) and the Mi1.2 resistant cv. Caramba (Seminis Seeds) were used in the experiments. Tomato plants were germinated in a seedling tray containing a mixture consisting of pit:perlite (95:5; v:v) and placed inside insect cages to avoid insect interferences and maintained in a glasshouse. Subsequently, the plants were transplanted into 200 cm³ pots filled with sterile river sand when they had developed four true leaves. They were allowed to establish roots for one week under controlled conditions in a growth chamber previous to perform the experiments. (25 ± 2ºC; RH% 70 ± 10%; 16:8 h L:D photoperiod). M. pygmaeus N4-5 nymphs of 4–7 days and mated females used in the experiments were supply by the SELMAR (Federació d’Agrupacions de Defensa Vegetal) mass rearing located at Santa Susanna (Barcelona, Spain). The RKN used in the experiments consisted in a M. incognita (Agropolis) population isolated from an experimental greenhouse [ 7 ] and a mixed community containing M. arenaria, M. hapla , and M. javanica (Community) isolated from grafted tomato in a commercial field production. Both strains were reproduced and maintained in the susceptible tomato cv. Durinta (Seminis Seeds) and J2 were obtained from nematode eggs produced in tomato roots and extracted by maceration in a 5% commercial bleach solution (40 g L − 1 NaOCl) [ 35 ] and placed in Baermann trays [ 36 ]. J2 hatched during the first 24 h were discarded, and subsequent ones were collected daily and stored at 9 ºC until use. The RKN species were identified using specific SCAR PCR for M. arenaria, M. incognita and M. javanica [ 37 ] and specific PCR tests for M. hapla , M. chitwoody and M. fallax [ 38 ]. Inducing resistance of tomato to RKN in pot experiments. After a week of transplanting, plants were covered with a 100 microns mesh bag of 20 x 5.1 x 27.9 cm, and 15 N4-5 nymphs of 4–7 days old of M. pygmaeus per plant were released inside the bag and fixed to the pot using an elastic rubber (Fig. 1 ). Forty-eight hours later, bags and the insects were removed, and plants were inoculated with 1 second-stage juvenile (J2) cm − 3 of soil of M. incogita (Agropolis) or 3 J2 cm − 3 of soil of a mixed community of M. arenaria, M. hapla , and M. javanica (Community) to increase the likelihood of all RKN species infect the plant. The J2 suspension was applied in two opposite holes, 2 cm deep and 2 cm apart from the plant, and covered with the soil. Plants not exposed to M. pygmaeus were included for comparison, and each combination of tomato cultivar-RKN-mirid exposure was repeated 12 times, and the experiment was conducted twice. Plants were watered and fertilized as needed during the experiments. Forty days after nematode inoculation (DANI), the aboveground part of the plants were removed, and the roots were carefully washed. The RKN infectivity was assessed by counting the egg masses after being stained with a 0.01% erioglaucine solution for 30 min [ 39 ]. RKN eggs were extracted from roots by maceration in a 10% commercial bleach solution (40g L − 1 NaOCl) [ 35 ] and then counted. The nematode fertility was expressed as the mean number of eggs per egg mass. The reproduction index (RI) was calculated as the percentage of the number of eggs produced in the resistant germplasm in relation to the number of eggs produced in the susceptible germplasm in the plants not exposed to M. pygmaeus . The level of resistance was categorized as highly resistant (RI < 1%), resistant (1% ≤ RI < 10%), moderately resistant (10% ≤ RI < 25%), slightly resistant (25% ≤ RI < 50%), or susceptible (RI ≥ 50%) [ 40 ]. Preference experiments Vertical Y-tube olfactometer The preference of 7-day-old females of M. pygmaeus for RKN infected or non-infected with 3 J2 cm − 3 of soil the susceptible tomato cv. Roma or the resistant cv. Caramba plants after 14 DANI were investigated in a Y-tube olfactometer (Nathura, ECIS, Bessanvido, Italy) consisting of a Y-shaped glass tube with an internal diameter of 3.5 cm and 17 cm long arms. Both arms were connected to an air pump in the upper part. The Y-tube was positioned vertically, producing controlled air that flowed from the arms to the bottom. The air was controlled using an anemometer (TESTO, Barcelona, Spain), at the ends of both pump tubes and maintained at 2.7 ± 0.1 m s-1. The airflow at the exit of the olfactometer was maintained at 0.20 ± 0.02 m s-1. At the beginning of the trial, a M. pygmaeus female was allowed to walk onto a mesh lid placed at the base of the olfactometer. After the female had walked off the lid, the lid was then removed, and the time was counted. Each individual was observed until it had either crossed a line drawn above the bottom third of the olfactometer arm or until a total of 5 min had elapsed, after which the insect was removed with a mouth aspirator and discarded. Sixty insects were tested per each treatment, and each individual was used only once. To minimize potential experimental biases from environmental variables or location-specific effects, the olfactometer was cleaned with 96% alcohol after every five insects tested. Additionally, the positions of the olfactometer arms were alternated between the two plants, and the orientation of the jars was rotated after every ten insects [ 41 , 42 ]. The trials were conducted at the same location, under uniform light conditions, and at the same time of the day (between 9:00 h and 16:00 h) to avoid circadian variations in the insect behaviour [ 43 ]. Each insect’s final choice was recorded using the method proposed by Du et al. [ 44 ], to prevent the inclusion of random choices resulting from the exploration of the arms by the insects. At the end of the experiment, the root infection by RKN was confirmed by staining the nematodes inside the roots in acid fuchsine [ 45 ]. Insect cages experiments Three experiments were carried out to assess the preference and the offspring of M. pygmaeus for non-infected or infected plants with 3 J2 cm − 3 of soil by M. incognita or the RKN community after 14 DANI. The susceptible tomato cv. Roma and the resistant cv. Caramba were germinated, transplanted, cultivated, and inoculated as previously described. After 14 DANI, one inoculated and one non-inoculated plant of the same cultivar were transferred to a 250 microns mesh cage of 30 x 30 x 30 cm and placed in a growth chamber (25ºC ± 2; 70% RH;16: 8 h L:D photoperiod). Then, 10 mated females of M. pygmaeus of, 7- days-old, were released into the cage. After, 1, 2, 4, 24, 48, and 72h, the number of M. pygmaeus females on and outside the plant was counted (Fig. 3 ). After that, the aboveground part of the plant was removed, cut into pieces, and transferred to 480 mL insect pots of 12cm diameter with a 100 micron mesh at the top to allow air circulation and placed in the growth chamber (Fig. 4 ). After 14 days, the number of nymphs produced in each plant was evaluated under a stereomicroscope. Each treatment was repeated 10 times. At the end of the experiment, RKN infection was confirmed by staining the roots in acid fuchsine [ 45 ]. Statistical analyses Data of nematode infectivity (egg masses), reproduction (eggs per plant), and fertility (eggs per egg mass) belonging to the inducing of tomato resistance experiments, and data of the number of insects per plant and period along with the insect offspring in the cage experiments were assessed for normality and homogeneity of variances. Data were compared using the Student-t test if no differences ( P > 0.05) between variances were observed or using the Welch test otherwise. Data were compared between experiments and pooled if there were not significant differences ( P > 0.05). Significant differences in the proportion of M. pygmaeus choosing a particular host plant in the olfactometer were tested using a two-sided binomial test. Females that did not make choice were discarded in the statistical analysis. Statistical analyses were performed using JMP 16.2.0 (SAS Institute inc.). RESULTS Inducing resistance of tomato to RKN in pot experiments. Both M. incognita (Agropolis) and the community of RKN used in the experiments overcome the resistance (RI > 50%) to the Mi1.2 resistance gene of the tomato cv. Caramba performing as susceptible in both experiments (Agropolis: 89% and 116%; Community: 52% and 63%) (Fig. 1 ). The infectivity and reproduction of M. incognita in the first experiment in the susceptible tomato cv. Roma exposed to M. pygmaeus was reduced ( P < 0.05) by 40 and 62% respectively. In addition, the fertility was reduced by 39% in plants infected with M. incognita and induced by M. pygmaeus . Concerning the resistant cv. Caramba, the infectivity of the induced plants was reduced by 50% in plants infected with M. incognita ( P 0.05), increasing its fecundity by 49% in plants exposed to M. pygmaeus . The number of eggs per plant and the fecundity were 42% and 57% lower ( P < 0.05) respectively, in the susceptible compared to the resistant germplasm in the plants exposed to M. pygmaeus , but not in plants not exposed to the insect (Fig. 1 ). In the second experiment, in the susceptible germplasm, the infectivity was reduced by 34%, and the reproduction by 43% in the induced plants, respectively (P 0.05). The number of eggs per plant in plants exposed to M. pygmaeus were 38% lower in the susceptible compared to the resistant germplasm, but not in the plants not exposed. Concerning nematode fecundity in both exposed and non-exposed plants were 44% and 33% lower respectively in the susceptible compared to the resistant germplasm (Fig. 1 ). In the susceptible plants of the first experiment infected with the nematode community strain, the number of eggs masses per plant in plants exposed to M. pygmaeus were 65% lower ( P 0.05), increasing its fecundity by 132% in the exposed plants ( P < 0.05). Regarding the resistant cv. Caramba, both eggs per plant and nematode fecundity increased by 196% and 220% ( P < 0.05) in plants exposed to M. pygmaeus compared to non-exposed plants. The number of egg masses and eggs per plant were 49 and 49% lower ( P 0.05). In the second experiment, both egg masses per plant and eggs per plant were reduced by 39% and 53% ( P 0.05). Finally, the number of egg masses per plant was 43% higher ( P 0.05) (Fig. 1 ). Vertical Y-tube olfactometer The olfactometer setup showed that 97, 87, 85 and 75% of the M. pygmaeus females used responded to the odors from tomato plants. However, their preference was not affected by the nematode infection irrespective of the tomato cultivar neither the RKN inoculum used in the experiment ( P > 0.05) (Fig. 2 ) Insect cages experiments In the experiments inoculated with M. incognita there were no differences between experiments ( P > 0.05), therefore data were pooled. The number of M. pygmaeus in RKN infected resistant tomato compared to non-infected were 2-fold and 1.7-fold higher after 1h and 48h of releasing the insects into the cages, respectively ( P 0.05) (Fig. 3 ). In addition, no differences in the offspring were recorded irrespective of the nematode inoculation or the tomato cultivar (P > 0.05) (Fig. 4 ). In the experiment inoculated with the nematode community, no differences in the number of M. pygmaeus choosing infected versus non-infected plants were recorded irrespective of the time and the tomato cultivar ( P > 0.05) (Fig. 3 ). Moreover, no significant differences in the offspring were recorded irrespective of the nematode inoculation or the tomato cultivar (P > 0.05) (Fig. 4 ). DISCUSSION The present study reveals M. pygmaeus ability to induce plant resistance against RKN, effectively reducing infectivity and nematode reproduction in susceptible tomato plants. However, in resistant tomato plants, only the initial pot experiment showed a reduced infectivity with M. incognita , while reproduction remained unaffected. Moreover, neither infectivity nor reproduction was impacted in the subsequent pot experiment. These findings underscore the influence of plant genetics on the phenotypic response to RKN when exposed to M. pygmaeus . The effect of plant feeding by M. pygmaeus has been previously reported to upregulate the genes related to the jasmonic acid (JA) pathway, increasing the concentration of 12-oxo-phytodienoic acid and jasmonic acid–isoleucine in the punctured leaves, affecting the performance of T. urticae and F. occidentalis in other leaves [ 24 ]. Besides, the JA-related genes were also upregulated in tomato leaves induced by the mirid bug N. tenuis [ 20 ]. For that, the JA pathway seems responsible for mediating the resistance to RKN. In addition, Wang et al. [ 46 ] demonstrated through a series of grafting experiments using mutants lacking the GLUTAMATE RECEPTOR-LIKE 3.5 or the RESPIRATORY BURST OXIDASE HOMOLOG 1 , key for ROS and JA accumulation in the upper stems and leaves that basal resistance of roots against RKN relies significantly on JA synthesis in shoots but not in roots. The JA is then transported from the shoots to the roots to help trigger defense responses. The exogenous application of JA and its derivatives, such as methyl jasmonate, have also been demonstrated to reduce nematode infection, probably by increasing toxic compounds to nematodes produced by roots such as hytoectosteroids, flavonoids and proteinase inhibitors [ 47 , 48 , 49 , 50 ]. Salicylic acid (SA) seems to be an important signaling compound associated with the hypersensitive reaction to prevent nematode establishment in the Mi -mediated resistance [ 51 ]. Furthermore, SA and JA could interact antagonistically depending on the combination of their respective concentrations and could be exploited by pathogens to enhance plant susceptibility [ 52 ]. For instance, the bacterium Pseudomonas syringae uses coronatine, a substance similar to jasmonate-isoleucine (JA-I1e) to repress the SA-mediated defense pathway [ 53 ]. Thus, a negative crosstalk between the SA and JA signaling pathways may occur leading to a deficiency to induce resistance in the Mi1.2 plants. Interestingly, Copper et al. [ 49 ] found a reduction in nematode infection when JA application was performed on susceptible tomato plants at 25ºC and 32ºC one day prior to inoculation and 7 days after, using avirulent nematode populations compared to non-treated plants. When Mi -resistant plants were used, no effect of the JA application at 25ºC or 32ºC was found, although the resistance had partially loosed at 32ºC due to high temperature. When virulent nematode populations were used, the JA application did not reduce the nematode infection in susceptible and resistant plants. Therefore, the interaction between the JA application, the (a)virulent status of the nematode, and the plant genetic background affects the plant response against the nematode. Thus, further studies related to the gene expression in susceptible and resistant plants, including grafting, must be explored to understand the potential interaction of susceptible scions induced by M. pygmaeus grafted onto resistant rootstocks and its interactions with Mi1.2 -virulent and avirulent RKN. Furthermore, the mechanisms related to defense induction by M. pygmaeus seem to differ from those mediated by the resistance conferred by the Mi1.2 gene and therefore alternating these strategies would be advisable to reduce nematode populations and avoid the selection of virulence to R genes. In our work, no significant differences were found between M. pygmaeus choice of nematode-infected or non-infected plants and the offspring produced, regardless of the plant background or the nematode used. The preference of M. pygmaeus for tomato plants is significantly influenced by the Herbivore Induced Plant Volatiles (HIPVs) emitted when the plants are infested by various pests, such as spider mites, aphids, whiteflies, and caterpillars [ 54 , 55 ]. Additionally, RKN infection is known to alter the volatile organic compounds (VOC) profile, potentially affecting the preference and development of both pests and their predators. Arce et al. [ 30 ] reported a strong suppression of 8 out of 33 compounds emitted by RKN infected plants, including α-terpinene, β-phellandrene β-caryophyllene, α-pinene, and α-humulene, affecting the oviposition and development of T. absoluta and thus, the biological control of T. absoluta with M. pygmaeus would be enhaced, since M. pygmaeus would not be affected by the nematode infection, as our results show. Moreover, the root infection of wheat plants by M. incognita reduced the feeding of Sitobion avenae (Hemiptera: Aphididae) 7 days after inoculation and interestingly, its aphid predator Harmonia axyridis (Coleoptera: Coccinellidae) preferred plants co-damaged by M. incognita and S. avenae from those only infested by the aphid [ 56 ]. In addition, the accumulation of other compounds in RKN-infected roots, such as α-tomatine, a steroidal glycoalkaloid related to herbivory defense, is associated with the nematode life cycle stage, increasing its concentration at the nematode reproduction stage [ 31 , 57 ]. In our study, as no prey were introduced in the plant, the response of the predator was not affected by the nematode infection. Those results are encouraging since the presence of the predator will not be affected by the nematode, ensuring the integrated pest management in these conditions. In conclusion, the induction of susceptible tomato plants with M. pygmaeus prior to planting significantly reduces RKN infectivity and reproduction. Given that the preference of M. pygmaeus is influenced by plant-emitted HIPVs, but not for those altered by RKN infection, the biological control of pests will not be affected and is proposed as a tool to include into integrated pest and nematode management. Declarations COMPETING INTERESTS The authors declare no competing interests. Author Contribution AE: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing – original draft, visualization, resources, supervision PUB: Conceptualization, methodology, validation, investigation, data curation, writing – review & editing, visualization SB: Conceptualization, methodology, validation, investigation, data curation, AMF: Validation, data curation, writing – review & editing, visualization AG: Validation, data curation, writing – review & editing, visualization FJS: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing – review & editing, visualization, resources, supervision, project administration and funding acquisition JR: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing – review & editing, visualization, supervision Acknowledgement UPC authors acknowledge the funding from the R+D+i project PID2021-129001OB-100 and AGL2017-89785-R, financed by MCIN and FEDER and Fondo Social Europeo (PRE2018-084265, Aïda Magdalena Fullana) and for the post-doctoral grant Funded by European Union-NextGenerationEU, Ministry of Universities and Recovery, Transformation and Resilience Plan, through a call from Universitat Politècnica de Catalunya (Grant Ref. 2022UPC-MSC-93765). The authors from IRTA were also funded by the CERCA Programme / Generalitat de Catalunya. The authors also want to thank the IRTA technicians Victor Muñoz, Pilar Hernández, and Silvia Rascon for their technical support during the experiments and to the doctoral student Luis Guillermo Montes for his technical support on the olfactometer setup. Data Availability The data supporting this study are available from the corresponding author upon reasonable request References Jones, J. T. et al. Top 10 plant-parasitic nematodes in molecular plant pathology. Mol. Plant. Pathol. 14 (9), 946–961. 10.1111/mpp.12057 (2013). Hallmann, J. & Meressa, B. H. Nematode parasites of vegetables in Plant parasitic nematodes in subtropical and tropical agriculture (ed. Sikora, R.A., Coyne, D., Hallman, J. & Timper, P.) 346–410 (CABI International, Wallingford, (2018). 10.1079/9781786391247.0346 . Abad, P. et al. feeding and development in Root-knot nematodes (ed. 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Weights in the balance: jasmonic acid and salicylic acid signaling in root-biotroph interactions. Mol. Plant-Microb Interac . 22 (7), 763–772. 10.1094/MPMI-22-7-0763 (2009). Zheng, X. Y. et al. Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell. Host Microbe . 11 (6), 587–596. 10.1016/j.chom.2012.04.014 (2012). Moayeri, H. R. S., Ashouri, A., Brødsgaard, H. F. & Enkegaard, A. Odour-mediated preference and prey preference of Macrolophus caliginosus between spider mites and green peach aphids. J. Appl. Entomol. 130 (9‐10), 504–508. 10.1111/j.1439-0418.2006.01094.x (2006). Lins, J. C. et al. Response of the zoophytophagous predators Macrolophus pygmaeus and Nesidiocoris tenuis to volatiles of uninfested plants and to plants infested by prey or conspecifics. BioControl . 59 , 707–718. 10.1007/s10526-014-9602-y (2014). Shi, J. H. et al. Volatiles and hormones mediated root-knot nematode induced wheat defense response to foliar herbivore aphid. Sci. Total Environ. 815 , 152840. 10.1016/j.scitotenv.2021.152840 (2022). Mbaluto, C. M., Ahmad, E. M., Fu, M. & Martínez-Medina, A. Dam, N. M. The impact of Spodoptera exigua herbivory on Meloidogyne incognita -induced root responses depends on the nematodes’ life cycle stages. AoB Plants . 12 (4), plaa029. 10.1093/jxb/erab370 (2020). van. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Dec, 2024 Reviews received at journal 19 Nov, 2024 Reviewers agreed at journal 07 Nov, 2024 Reviews received at journal 04 Nov, 2024 Reviews received at journal 28 Oct, 2024 Reviewers agreed at journal 24 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers invited by journal 21 Oct, 2024 Editor assigned by journal 20 Oct, 2024 Editor invited by journal 20 Oct, 2024 Submission checks completed at journal 16 Oct, 2024 First submitted to journal 30 Sep, 2024 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. 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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-5181542","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":386131204,"identity":"8fc38c85-3523-43f4-baac-28722b828cc6","order_by":0,"name":"Alejandro Expósito","email":"data:image/png;base64,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","orcid":"","institution":"Universitat Politècnica de Catalunya, BarcelonaTech (UPC)","correspondingAuthor":true,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Expósito","suffix":""},{"id":386131214,"identity":"223838a1-a68f-4a89-aaed-8d2339b56dcf","order_by":1,"name":"Pablo Urbaneja-Bernat","email":"","orcid":"","institution":"IRTA","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Urbaneja-Bernat","suffix":""},{"id":386131218,"identity":"b1e5da80-5e1a-4938-baeb-846d02016d57","order_by":2,"name":"Sara Boncompte","email":"","orcid":"","institution":"Universitat Politècnica de Catalunya, BarcelonaTech (UPC)","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Boncompte","suffix":""},{"id":386131227,"identity":"01e3421d-c36e-496f-864c-99658e0f7ef3","order_by":3,"name":"Aida Magdalena Fullana","email":"","orcid":"","institution":"Universitat Politècnica de Catalunya, BarcelonaTech (UPC)","correspondingAuthor":false,"prefix":"","firstName":"Aida","middleName":"Magdalena","lastName":"Fullana","suffix":""},{"id":386131228,"identity":"e650b307-0c78-4aff-867d-224c88fd6db7","order_by":4,"name":"Ariadna Giné","email":"","orcid":"","institution":"Universitat Politècnica de Catalunya, BarcelonaTech (UPC)","correspondingAuthor":false,"prefix":"","firstName":"Ariadna","middleName":"","lastName":"Giné","suffix":""},{"id":386131230,"identity":"9b8d7845-c0a6-4237-b639-0bcadead56ee","order_by":5,"name":"Francisco Javier Sorribas","email":"","orcid":"","institution":"Universitat Politècnica de Catalunya, BarcelonaTech (UPC)","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"Javier","lastName":"Sorribas","suffix":""},{"id":386131232,"identity":"46e6f7f3-54b2-4507-bd0e-6abc4528afa0","order_by":6,"name":"Jordi Riudavets","email":"","orcid":"","institution":"IRTA","correspondingAuthor":false,"prefix":"","firstName":"Jordi","middleName":"","lastName":"Riudavets","suffix":""}],"badges":[],"createdAt":"2024-09-30 14:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5181542/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5181542/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-90233-7","type":"published","date":"2025-03-04T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71251363,"identity":"4b4c9865-d8eb-4dfc-afab-e92b598e0e94","added_by":"auto","created_at":"2024-12-12 14:41:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1135033,"visible":true,"origin":"","legend":"\u003cp\u003eInfectivity (egg masses per plant), reproduction (eggs per plant), and fecundity (eggs per egg mass) (mean ± SE) were assessed on both susceptible tomato cv. Roma (S) and the \u003cem\u003eMi\u003c/em\u003e1.2 resistant cv. Caramba (R) in 200 cm³ pot experiments. Plants were either induced (with) or not (without) with 15 nymphs of \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e over a 48-hour period and subsequently inoculated with 1 J2 cm\u003csup\u003e-\u003c/sup\u003e³ of soil of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e (Agropolis strain) or 3 J2 cm\u003csup\u003e-\u003c/sup\u003e³ of soil containing a mixed community of \u003cem\u003eM. hapla\u003c/em\u003e, \u003cem\u003eM. arenaria\u003c/em\u003e, and \u003cem\u003eM. javanica\u003c/em\u003e (Community strain). Data followed by * are different according to the Student-t test or the Welch test (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5181542/v1/60b53fcf7071408bce3371f2.png"},{"id":71251367,"identity":"a0709629-850d-4604-9728-78909013a29e","added_by":"auto","created_at":"2024-12-12 14:41:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":524199,"visible":true,"origin":"","legend":"\u003cp\u003ePreference of \u003cem\u003eM. pygmaeus \u003c/em\u003efemales\u003cem\u003e \u003c/em\u003eto nematode infected or non-infected plants in a vertical Y-tube olfactometer, 14 days after nematode inoculation. The susceptible tomato cv. Roma (S) or the \u003cem\u003eMi\u003c/em\u003e1.2 resistant cv. Caramba (R) were inoculated with 3 J2 cm\u003csup\u003e-\u003c/sup\u003e³ of soil of \u003cem\u003eM. incognita \u003c/em\u003e(Agropolis strain) or a mixed community of \u003cem\u003eM. hapla\u003c/em\u003e, \u003cem\u003eM. arenaria\u003c/em\u003e and \u003cem\u003eM. javanica \u003c/em\u003e(Community strain). NC indicates the number of tested individuals that did not respond. Significant differences using a two side binomial test are marked with (*) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5181542/v1/7f27d597b4408ac2bdfba655.png"},{"id":71251769,"identity":"03d7f5e5-6d5a-401b-bb3c-05f003e61fd8","added_by":"auto","created_at":"2024-12-12 14:49:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":964925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMacrolophus. pygmaeus\u003c/em\u003e (mean ± SE) per plant counted in insect cages 1, 2, 4, 24, 48 and 72h\u0026nbsp; after releasing the insects in the susceptible cv. Roma (S) or the resistant tomato cv. Caramba (R), inoculated or not with 3 J2 cm\u003csup\u003e-\u003c/sup\u003e³ of soil \u0026nbsp;of \u003cem\u003eM. incognita \u003c/em\u003e(Agropolis strain) or a mixed community of \u003cem\u003eM. hapla\u003c/em\u003e, \u003cem\u003eM. arenaria\u003c/em\u003e and \u003cem\u003eM. javanica \u003c/em\u003e(Community strain) 14 days after nematode inoculation (DANI). Data followed by * are different according to the Student-t test or the Welch test (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5181542/v1/39a63baf697567ecd1d15898.png"},{"id":71251358,"identity":"7b4e7dd5-aade-43bc-80d7-274b922fbaaa","added_by":"auto","created_at":"2024-12-12 14:41:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":931981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMacrolophus. pygmaeus\u003c/em\u003e (mean ± SE) offspring nymphs per plant produced in separated boxes after 14 days of the last count in each plant of the susceptible cv. Roma (S) or the resistant tomato cv. Caramba (R), inoculated or not with 3 J2 cm\u003csup\u003e-\u003c/sup\u003e³ of soil \u0026nbsp;of \u003cem\u003eM. incognita \u003c/em\u003e(Agropolis strain) or a mixed community of \u003cem\u003eM. hapla\u003c/em\u003e, \u003cem\u003eM. arenaria\u003c/em\u003e and \u003cem\u003eM. javanica \u003c/em\u003e(Community strain). Data followed by * are different according to the Student-t test or the Welch test (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5181542/v1/02a178476a4eaba831ba96c5.png"},{"id":78191455,"identity":"48344520-3b93-49b5-8e5d-393ad6bd25c6","added_by":"auto","created_at":"2025-03-10 20:01:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4191428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5181542/v1/4b9ae903-9696-4beb-ab90-b850dfff8afa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Macrolophus pygmaeus (Heteroptera: Miridae) induces systemic resistance in tomato against Meloidogyne spp","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eMeloidogyne\u003c/em\u003e spp. is the most challenging plant-parasitic nematode (PPN) genus affecting global plant production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. More than 100 species have been described, however, the root-knot nematodes (RKN) tropical species \u003cem\u003eM. arenaria\u003c/em\u003e, \u003cem\u003eM. incognita\u003c/em\u003e, and \u003cem\u003eM. javanica\u003c/em\u003e are the most widespread and limiting for vegetable production in tropical and subtropical climates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These species are obligate parasites, which reproduce parthenogenetically. The second stage juvenile (J2) penetrates the root near the elongation zone, moves intercellular and induce a hypertrophied and multinucleate giant cells (GC) in the vascular cylinder becoming a feeding site, supplying nutrients to the nematode. After that, the J2 undergoes three moults until it reaches the adult stage. The juveniles develop into females that lays eggs into a gelatinous matrix [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Because of that, plant roots become galled, interfering with the correct uptake of water and nutrients, which causes yellowing, wilting, and dwarfism in aboveground parts of the plant, leading plant death in severe attacks. In tomato, one of the main cash crops in the Mediterranean area, RKN can reduce crop yield until 62% and 72%, depending on the cropping season and crop duration [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among sustainable nematode control methods, plant resistance stands out as one of the most effective, economically profitable and environmentally safe [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In tomato, the resistance against \u003cem\u003eMeloidogyne\u003c/em\u003e is mediated by the \u003cem\u003eMi1.2\u003c/em\u003e resistance gene and conferred resistance against \u003cem\u003eM. arenaria, M. incognita\u003c/em\u003e and \u003cem\u003eM. javanica\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eM. luci\u003c/em\u003e and \u003cem\u003eM. ethiopica\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] but not to \u003cem\u003eM. hapla\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] or \u003cem\u003eM. enterolobii\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This gene induces a hypersensitive reaction (HR)-mediated cell death around the feeding site, involving an oxidative burst by reactive oxygen species (ROS) that prevents nematode infection and development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nevertheless, its effectiveness is reduced or lost after repeated cultivation due to the emergence of virulent populations [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. An alternative strategy to reduce nematode building up populations is to induce resistance in susceptible germplasm by biotic or abiotic factors. This property can be used to drive apart the use of the same R-gene, reducing its selection pressure and the selection of virulent populations, if the defense mechanism differs from those expressed by the R-genes.\u003c/p\u003e \u003cp\u003eBacteria and fungi have previously described induced plant resistance to RKN by biotic elicitors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recently, the ability of other organisms, such as insects, to be used in biological pest control has been investigated. For instance, zoophytophagous mirid bugs such as \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e and \u003cem\u003eNesiodiocoris tenuis\u003c/em\u003e (Heteroptera: Miridae), known predators of various aboveground pests, have been observed to trigger plant resistance against several key pests: the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e Gennadius (Hemiptera: Aleyrodidae), the South American tomato pinworm \u003cem\u003eTuta absoluta\u003c/em\u003e Meyrick (Lepidoptera: Gelichiidae), the Western flower thrips \u003cem\u003eFrankliniella occidentalis\u003c/em\u003e Pergande (Thysanoptera: Thripidae), and the two-spotted spider mite \u003cem\u003eTetranychus urticae\u003c/em\u003e Koch (Acari: Tetranychidae). This resistance is induced when these mirid bugs engage in phytophagous behavior and puncture the plants [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the effect of punctured plants infected with RKN in susceptible and resistant germplasm remains unknown, and also the possibility that the induced resistance could be additive to the genetic resistance mediated by the \u003cem\u003eMi1.2\u003c/em\u003e resistance gene, as reported for \u003cem\u003eTrichoderma asperellum\u003c/em\u003e T-34 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, PPN infection can modify the host preference and development of certain phytophagous insects and mites, affecting the performance of their development stages and offspring. For example, \u003cem\u003ePratylenchus penetrans\u003c/em\u003e enhances the production of leaf phenolics and glucosinolates in \u003cem\u003eBrassica nigra\u003c/em\u003e, where the caterpillar \u003cem\u003ePieris rapae\u003c/em\u003e (Lepidoptera: Pieridae) larvae grew slower and produced fewer pupae compared to non-infested plants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Likewise, root infection by the cyst nematode \u003cem\u003eHeterodera schachtii\u003c/em\u003e reduces the population density of the aphid \u003cem\u003eBrevicoryne brassicae\u003c/em\u003e (Hemiptera: Aphididae) and induces changes in the composition of glucosinolates in \u003cem\u003eBrassica oleracea\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e infected by \u003cem\u003eH. schachtii\u003c/em\u003e enhances the attractiveness of the spider mite \u003cem\u003eT. uricae\u003c/em\u003e and enhances its life history traits [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], whereas \u003cem\u003eP. penetrans\u003c/em\u003e reduces its fertility on \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The effect of RKN infection also can affect the aboveground phytophages. For example, the leaf miner \u003cem\u003eT. absoluta\u003c/em\u003e prefers uninfected RKN tomato plants for its oviposition, and that root infection negatively affects its pupation process [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Interestingly, positive effects of RKN infection on leaf-chewing herbivores have also been observed. For example, \u003cem\u003eSpodoptera exigua\u003c/em\u003e (Lepidoptera: Noctuidae) benefits from \u003cem\u003eM. incognita\u003c/em\u003e infection, increasing its weight compared to non-infected plants, depending on the life cycle of the nematode [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, \u003cem\u003eM. incognita\u003c/em\u003e infection inhibits the root-produced defense chemical nicotine in response to foliar herbivory by \u003cem\u003eManduca sexta\u003c/em\u003e (Lepidoptera: Sphingidae) and \u003cem\u003eTrichoplusia ni\u003c/em\u003e (Lepidoptera: Noctuidae), resulting in low-leaf nicotine levels in tobacco [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to investigate the potential of \u003cem\u003eM. pygmaeus\u003c/em\u003e in enhancing plant resistance against root-knot nematodes (RKN) in susceptible and resistant tomato cultivars. \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e was selected for its known safety profile for plants, contrasting with \u003cem\u003eN. tenuis\u003c/em\u003e, which has been associated with adverse effects such as necrotic rings on stems and flowers, as well as fruit punctures [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, this research explores the insect's preference between RKN-infected and non-infected plants using a vertical Y-tube olfactometer and insect cages, providing insights into the ecological interactions between predator behaviour and plant defense mechanisms.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material, insects and nematodes\u003c/h2\u003e \u003cp\u003eThe experiments were conducted at the Institute of Agrifood Research and Technology (IRTA) (Cabrils, Spain) during 2023. The susceptible tomato cv. Roma (Fit\u0026oacute; Seeds) and the \u003cem\u003eMi1.2\u003c/em\u003e resistant cv. Caramba (Seminis Seeds) were used in the experiments. Tomato plants were germinated in a seedling tray containing a mixture consisting of pit:perlite (95:5; v:v) and placed inside insect cages to avoid insect interferences and maintained in a glasshouse. Subsequently, the plants were transplanted into 200 cm\u0026sup3; pots filled with sterile river sand when they had developed four true leaves. They were allowed to establish roots for one week under controlled conditions in a growth chamber previous to perform the experiments. (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C; RH% 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10%; 16:8 h L:D photoperiod).\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. pygmaeus\u003c/em\u003e N4-5 nymphs of 4\u0026ndash;7 days and mated females used in the experiments were supply by the SELMAR (Federaci\u0026oacute; d\u0026rsquo;Agrupacions de Defensa Vegetal) mass rearing located at Santa Susanna (Barcelona, Spain).\u003c/p\u003e \u003cp\u003eThe RKN used in the experiments consisted in a \u003cem\u003eM. incognita\u003c/em\u003e (Agropolis) population isolated from an experimental greenhouse [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and a mixed community containing \u003cem\u003eM. arenaria, M. hapla\u003c/em\u003e, and \u003cem\u003eM. javanica\u003c/em\u003e (Community) isolated from grafted tomato in a commercial field production. Both strains were reproduced and maintained in the susceptible tomato cv. Durinta (Seminis Seeds) and J2 were obtained from nematode eggs produced in tomato roots and extracted by maceration in a 5% commercial bleach solution (40 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOCl) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and placed in Baermann trays [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. J2 hatched during the first 24 h were discarded, and subsequent ones were collected daily and stored at 9 \u0026ordm;C until use. The RKN species were identified using specific SCAR PCR for \u003cem\u003eM. arenaria, M. incognita\u003c/em\u003e and \u003cem\u003eM. javanica\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and specific PCR tests for \u003cem\u003eM. hapla\u003c/em\u003e, \u003cem\u003eM. chitwoody\u003c/em\u003e and \u003cem\u003eM. fallax\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eInducing resistance of tomato to RKN in pot experiments.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAfter a week of transplanting, plants were covered with a 100 microns mesh bag of 20 x 5.1 x 27.9 cm, and 15 N4-5 nymphs of 4\u0026ndash;7 days old of \u003cem\u003eM. pygmaeus\u003c/em\u003e per plant were released inside the bag and fixed to the pot using an elastic rubber (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Forty-eight hours later, bags and the insects were removed, and plants were inoculated with 1 second-stage juvenile (J2) cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of soil of \u003cem\u003eM. incogita\u003c/em\u003e (Agropolis) or 3 J2 cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of soil of a mixed community of \u003cem\u003eM. arenaria, M. hapla\u003c/em\u003e, and \u003cem\u003eM. javanica\u003c/em\u003e (Community) to increase the likelihood of all RKN species infect the plant. The J2 suspension was applied in two opposite holes, 2 cm deep and 2 cm apart from the plant, and covered with the soil. Plants not exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e were included for comparison, and each combination of tomato cultivar-RKN-mirid exposure was repeated 12 times, and the experiment was conducted twice. Plants were watered and fertilized as needed during the experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eForty days after nematode inoculation (DANI), the aboveground part of the plants were removed, and the roots were carefully washed. The RKN infectivity was assessed by counting the egg masses after being stained with a 0.01% erioglaucine solution for 30 min [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. RKN eggs were extracted from roots by maceration in a 10% commercial bleach solution (40g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOCl) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and then counted. The nematode fertility was expressed as the mean number of eggs per egg mass. The reproduction index (RI) was calculated as the percentage of the number of eggs produced in the resistant germplasm in relation to the number of eggs produced in the susceptible germplasm in the plants not exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e. The level of resistance was categorized as highly resistant (RI\u0026thinsp;\u0026lt;\u0026thinsp;1%), resistant (1% \u0026le; RI\u0026thinsp;\u0026lt;\u0026thinsp;10%), moderately resistant (10% \u0026le; RI\u0026thinsp;\u0026lt;\u0026thinsp;25%), slightly resistant (25% \u0026le; RI\u0026thinsp;\u0026lt;\u0026thinsp;50%), or susceptible (RI\u0026thinsp;\u0026ge;\u0026thinsp;50%) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreference experiments\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVertical Y-tube olfactometer\u003c/h2\u003e \u003cp\u003eThe preference of 7-day-old females of \u003cem\u003eM. pygmaeus\u003c/em\u003e for RKN infected or non-infected with 3 J2 cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of soil the susceptible tomato cv. Roma or the resistant cv. Caramba plants after 14 DANI were investigated in a Y-tube olfactometer (Nathura, ECIS, Bessanvido, Italy) consisting of a Y-shaped glass tube with an internal diameter of 3.5 cm and 17 cm long arms. Both arms were connected to an air pump in the upper part. The Y-tube was positioned vertically, producing controlled air that flowed from the arms to the bottom. The air was controlled using an anemometer (TESTO, Barcelona, Spain), at the ends of both pump tubes and maintained at 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 m s-1. The airflow at the exit of the olfactometer was maintained at 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 m s-1. At the beginning of the trial, a \u003cem\u003eM. pygmaeus\u003c/em\u003e female was allowed to walk onto a mesh lid placed at the base of the olfactometer. After the female had walked off the lid, the lid was then removed, and the time was counted. Each individual was observed until it had either crossed a line drawn above the bottom third of the olfactometer arm or until a total of 5 min had elapsed, after which the insect was removed with a mouth aspirator and discarded. Sixty insects were tested per each treatment, and each individual was used only once. To minimize potential experimental biases from environmental variables or location-specific effects, the olfactometer was cleaned with 96% alcohol after every five insects tested. Additionally, the positions of the olfactometer arms were alternated between the two plants, and the orientation of the jars was rotated after every ten insects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The trials were conducted at the same location, under uniform light conditions, and at the same time of the day (between 9:00 h and 16:00 h) to avoid circadian variations in the insect behaviour [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Each insect\u0026rsquo;s final choice was recorded using the method proposed by Du et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], to prevent the inclusion of random choices resulting from the exploration of the arms by the insects. At the end of the experiment, the root infection by RKN was confirmed by staining the nematodes inside the roots in acid fuchsine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInsect cages experiments \u003c/h3\u003e\n\u003cp\u003eThree experiments were carried out to assess the preference and the offspring of \u003cem\u003eM. pygmaeus\u003c/em\u003e for non-infected or infected plants with 3 J2 cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of soil by \u003cem\u003eM. incognita\u003c/em\u003e or the RKN community after 14 DANI. The susceptible tomato cv. Roma and the resistant cv. Caramba were germinated, transplanted, cultivated, and inoculated as previously described. After 14 DANI, one inoculated and one non-inoculated plant of the same cultivar were transferred to a 250 microns mesh cage of 30 x 30 x 30 cm and placed in a growth chamber (25\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2; 70% RH;16: 8 h L:D photoperiod). Then, 10 mated females of \u003cem\u003eM. pygmaeus\u003c/em\u003e of, 7- days-old, were released into the cage. After, 1, 2, 4, 24, 48, and 72h, the number of \u003cem\u003eM. pygmaeus\u003c/em\u003e females on and outside the plant was counted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After that, the aboveground part of the plant was removed, cut into pieces, and transferred to 480 mL insect pots of 12cm diameter with a 100 micron mesh at the top to allow air circulation and placed in the growth chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After 14 days, the number of nymphs produced in each plant was evaluated under a stereomicroscope. Each treatment was repeated 10 times. At the end of the experiment, RKN infection was confirmed by staining the roots in acid fuchsine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eData of nematode infectivity (egg masses), reproduction (eggs per plant), and fertility (eggs per egg mass) belonging to the inducing of tomato resistance experiments, and data of the number of insects per plant and period along with the insect offspring in the cage experiments were assessed for normality and homogeneity of variances. Data were compared using the Student-t test if no differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between variances were observed or using the Welch test otherwise. Data were compared between experiments and pooled if there were not significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Significant differences in the proportion of \u003cem\u003eM. pygmaeus\u003c/em\u003e choosing a particular host plant in the olfactometer were tested using a two-sided binomial test. Females that did not make choice were discarded in the statistical analysis. Statistical analyses were performed using JMP 16.2.0 (SAS Institute inc.).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cem\u003eInducing resistance of tomato to RKN in pot experiments.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBoth \u003cem\u003eM. incognita\u003c/em\u003e (Agropolis) and the community of RKN used in the experiments overcome the resistance (RI\u0026thinsp;\u0026gt;\u0026thinsp;50%) to the \u003cem\u003eMi1.2\u003c/em\u003e resistance gene of the tomato cv. Caramba performing as susceptible in both experiments (Agropolis: 89% and 116%; Community: 52% and 63%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The infectivity and reproduction of \u003cem\u003eM. incognita\u003c/em\u003e in the first experiment in the susceptible tomato cv. Roma exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e was reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by 40 and 62% respectively. In addition, the fertility was reduced by 39% in plants infected with \u003cem\u003eM. incognita\u003c/em\u003e and induced by \u003cem\u003eM. pygmaeus\u003c/em\u003e. Concerning the resistant cv. Caramba, the infectivity of the induced plants was reduced by 50% in plants infected with \u003cem\u003eM. incognita\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not the nematode reproduction (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), increasing its fecundity by 49% in plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e. The number of eggs per plant and the fecundity were 42% and 57% lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively, in the susceptible compared to the resistant germplasm in the plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e, but not in plants not exposed to the insect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the second experiment, in the susceptible germplasm, the infectivity was reduced by 34%, and the reproduction by 43% in the induced plants, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, in the case of resistant plants, no differences were found (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The number of eggs per plant in plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e were 38% lower in the susceptible compared to the resistant germplasm, but not in the plants not exposed. Concerning nematode fecundity in both exposed and non-exposed plants were 44% and 33% lower respectively in the susceptible compared to the resistant germplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the susceptible plants of the first experiment infected with the nematode community strain, the number of eggs masses per plant in plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e were 65% lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in relation to non-exposed plants, but the reproduction was not affected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), increasing its fecundity by 132% in the exposed plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Regarding the resistant cv. Caramba, both eggs per plant and nematode fecundity increased by 196% and 220% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e compared to non-exposed plants. The number of egg masses and eggs per plant were 49 and 49% lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively, in the susceptible plants induced by the insect compared to the resistant, but not in plants not exposed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the second experiment, both egg masses per plant and eggs per plant were reduced by 39% and 53% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively in the susceptible plants exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e, but not in the resistant germplasm (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Finally, the number of egg masses per plant was 43% higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the susceptible compared to the resistant plants non-exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e, but not in the exposed ones (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eVertical Y-tube olfactometer\u003c/h3\u003e\n\u003cp\u003eThe olfactometer setup showed that 97, 87, 85 and 75% of the \u003cem\u003eM. pygmaeus\u003c/em\u003e females used responded to the odors from tomato plants. However, their preference was not affected by the nematode infection irrespective of the tomato cultivar neither the RKN inoculum used in the experiment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInsect cages experiments\u003c/h3\u003e\n\u003cp\u003eIn the experiments inoculated with \u003cem\u003eM. incognita\u003c/em\u003e there were no differences between experiments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), therefore data were pooled. The number of \u003cem\u003eM. pygmaeus\u003c/em\u003e in RKN infected resistant tomato compared to non-infected were 2-fold and 1.7-fold higher after 1h and 48h of releasing the insects into the cages, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but no differences were recorded in the susceptible (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, no differences in the offspring were recorded irrespective of the nematode inoculation or the tomato cultivar (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the experiment inoculated with the nematode community, no differences in the number of \u003cem\u003eM. pygmaeus\u003c/em\u003e choosing infected versus non-infected plants were recorded irrespective of the time and the tomato cultivar (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, no significant differences in the offspring were recorded irrespective of the nematode inoculation or the tomato cultivar (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study reveals \u003cem\u003eM. pygmaeus\u003c/em\u003e ability to induce plant resistance against RKN, effectively reducing infectivity and nematode reproduction in susceptible tomato plants. However, in resistant tomato plants, only the initial pot experiment showed a reduced infectivity with \u003cem\u003eM. incognita\u003c/em\u003e, while reproduction remained unaffected. Moreover, neither infectivity nor reproduction was impacted in the subsequent pot experiment. These findings underscore the influence of plant genetics on the phenotypic response to RKN when exposed to \u003cem\u003eM. pygmaeus\u003c/em\u003e. The effect of plant feeding by \u003cem\u003eM. pygmaeus\u003c/em\u003e has been previously reported to upregulate the genes related to the jasmonic acid (JA) pathway, increasing the concentration of 12-oxo-phytodienoic acid and jasmonic acid\u0026ndash;isoleucine in the punctured leaves, affecting the performance of \u003cem\u003eT. urticae\u003c/em\u003e and \u003cem\u003eF. occidentalis\u003c/em\u003e in other leaves [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Besides, the JA-related genes were also upregulated in tomato leaves induced by the mirid bug \u003cem\u003eN. tenuis\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For that, the JA pathway seems responsible for mediating the resistance to RKN. In addition, Wang et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] demonstrated through a series of grafting experiments using mutants lacking the \u003cem\u003eGLUTAMATE RECEPTOR-LIKE\u003c/em\u003e 3.5 or the \u003cem\u003eRESPIRATORY BURST OXIDASE HOMOLOG 1\u003c/em\u003e, key for ROS and JA accumulation in the upper stems and leaves that basal resistance of roots against RKN relies significantly on JA synthesis in shoots but not in roots. The JA is then transported from the shoots to the roots to help trigger defense responses. The exogenous application of JA and its derivatives, such as methyl jasmonate, have also been demonstrated to reduce nematode infection, probably by increasing toxic compounds to nematodes produced by roots such as hytoectosteroids, flavonoids and proteinase inhibitors [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSalicylic acid (SA) seems to be an important signaling compound associated with the hypersensitive reaction to prevent nematode establishment in the \u003cem\u003eMi\u003c/em\u003e-mediated resistance [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Furthermore, SA and JA could interact antagonistically depending on the combination of their respective concentrations and could be exploited by pathogens to enhance plant susceptibility [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. For instance, the bacterium \u003cem\u003ePseudomonas syringae\u003c/em\u003e uses coronatine, a substance similar to jasmonate-isoleucine (JA-I1e) to repress the SA-mediated defense pathway [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Thus, a negative crosstalk between the SA and JA signaling pathways may occur leading to a deficiency to induce resistance in the \u003cem\u003eMi1.2\u003c/em\u003e plants. Interestingly, Copper et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] found a reduction in nematode infection when JA application was performed on susceptible tomato plants at 25\u0026ordm;C and 32\u0026ordm;C one day prior to inoculation and 7 days after, using avirulent nematode populations compared to non-treated plants. When \u003cem\u003eMi\u003c/em\u003e-resistant plants were used, no effect of the JA application at 25\u0026ordm;C or 32\u0026ordm;C was found, although the resistance had partially loosed at 32\u0026ordm;C due to high temperature. When virulent nematode populations were used, the JA application did not reduce the nematode infection in susceptible and resistant plants. Therefore, the interaction between the JA application, the (a)virulent status of the nematode, and the plant genetic background affects the plant response against the nematode. Thus, further studies related to the gene expression in susceptible and resistant plants, including grafting, must be explored to understand the potential interaction of susceptible scions induced by \u003cem\u003eM. pygmaeus\u003c/em\u003e grafted onto resistant rootstocks and its interactions with \u003cem\u003eMi1.2\u003c/em\u003e-virulent and avirulent RKN. Furthermore, the mechanisms related to defense induction by \u003cem\u003eM. pygmaeus\u003c/em\u003e seem to differ from those mediated by the resistance conferred by the \u003cem\u003eMi1.2\u003c/em\u003e gene and therefore alternating these strategies would be advisable to reduce nematode populations and avoid the selection of virulence to R genes.\u003c/p\u003e \u003cp\u003eIn our work, no significant differences were found between \u003cem\u003eM. pygmaeus\u003c/em\u003e choice of nematode-infected or non-infected plants and the offspring produced, regardless of the plant background or the nematode used. The preference of \u003cem\u003eM. pygmaeus\u003c/em\u003e for tomato plants is significantly influenced by the Herbivore Induced Plant Volatiles (HIPVs) emitted when the plants are infested by various pests, such as spider mites, aphids, whiteflies, and caterpillars [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, RKN infection is known to alter the volatile organic compounds (VOC) profile, potentially affecting the preference and development of both pests and their predators. Arce et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] reported a strong suppression of 8 out of 33 compounds emitted by RKN infected plants, including α-terpinene, β-phellandrene β-caryophyllene, α-pinene, and α-humulene, affecting the oviposition and development of \u003cem\u003eT. absoluta\u003c/em\u003e and thus, the biological control of \u003cem\u003eT. absoluta\u003c/em\u003e with \u003cem\u003eM. pygmaeus\u003c/em\u003e would be enhaced, since \u003cem\u003eM. pygmaeus\u003c/em\u003e would not be affected by the nematode infection, as our results show. Moreover, the root infection of wheat plants by \u003cem\u003eM. incognita\u003c/em\u003e reduced the feeding of \u003cem\u003eSitobion avenae\u003c/em\u003e (Hemiptera: Aphididae) 7 days after inoculation and interestingly, its aphid predator \u003cem\u003eHarmonia axyridis\u003c/em\u003e (Coleoptera: Coccinellidae) preferred plants co-damaged by \u003cem\u003eM. incognita\u003c/em\u003e and \u003cem\u003eS. avenae\u003c/em\u003e from those only infested by the aphid [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In addition, the accumulation of other compounds in RKN-infected roots, such as α-tomatine, a steroidal glycoalkaloid related to herbivory defense, is associated with the nematode life cycle stage, increasing its concentration at the nematode reproduction stage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In our study, as no prey were introduced in the plant, the response of the predator was not affected by the nematode infection. Those results are encouraging since the presence of the predator will not be affected by the nematode, ensuring the integrated pest management in these conditions.\u003c/p\u003e \u003cp\u003eIn conclusion, the induction of susceptible tomato plants with \u003cem\u003eM. pygmaeus\u003c/em\u003e prior to planting significantly reduces RKN infectivity and reproduction. Given that the preference of \u003cem\u003eM. pygmaeus\u003c/em\u003e is influenced by plant-emitted HIPVs, but not for those altered by RKN infection, the biological control of pests will not be affected and is proposed as a tool to include into integrated pest and nematode management.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAE: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing \u0026ndash; original draft, visualization, resources, supervision PUB: Conceptualization, methodology, validation, investigation, data curation, writing \u0026ndash; review \u0026amp; editing, visualization SB: Conceptualization, methodology, validation, investigation, data curation, AMF: Validation, data curation, writing \u0026ndash; review \u0026amp; editing, visualization AG: Validation, data curation, writing \u0026ndash; review \u0026amp; editing, visualization FJS: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing \u0026ndash; review \u0026amp; editing, visualization, resources, supervision, project administration and funding acquisition JR: Conceptualization, methodology, validation, investigation, formal analysis, data curation, writing \u0026ndash; review \u0026amp; editing, visualization, supervision\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eUPC authors acknowledge the funding from the R+D+i project PID2021-129001OB-100 and AGL2017-89785-R, financed by MCIN and FEDER and Fondo Social Europeo (PRE2018-084265, A\u0026iuml;da Magdalena Fullana) and for the post-doctoral grant Funded by European Union-NextGenerationEU, Ministry of Universities and Recovery, Transformation and Resilience Plan, through a call from Universitat Polit\u0026egrave;cnica de Catalunya (Grant Ref. 2022UPC-MSC-93765). The authors from IRTA were also funded by the CERCA Programme / Generalitat de Catalunya. The authors also want to thank the IRTA technicians Victor Mu\u0026ntilde;oz, Pilar Hern\u0026aacute;ndez, and Silvia Rascon for their technical support during the experiments and to the doctoral student Luis Guillermo Montes for his technical support on the olfactometer setup.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting this study are available from the corresponding author upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJones, J. T. et al. Top 10 plant-parasitic nematodes in molecular plant pathology. \u003cem\u003eMol. Plant. 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The impact of \u003cem\u003eSpodoptera exigua\u003c/em\u003e herbivory on \u003cem\u003eMeloidogyne incognita\u003c/em\u003e-induced root responses depends on the nematodes\u0026rsquo; life cycle stages. \u003cem\u003eAoB Plants\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e (4), plaa029. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/erab370\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erab370\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020). van.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RKN, biological control, induced resistance, tomato","lastPublishedDoi":"10.21203/rs.3.rs-5181542/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5181542/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ability of \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e to induce systemic resistance in susceptible and \u003cem\u003eMi1.2\u003c/em\u003e resistant tomato against \u003cem\u003eMeloidogyne\u003c/em\u003e spp. was evaluated in pot experiments. The susceptible cv. Roma and the resistant cv. Caramba were exposed to 15 \u003cem\u003eM. pygmaeus\u003c/em\u003e nymphs per plant in mesh bags for 48h and then were inoculated with 1 second-stage juvenile (J2) of \u003cem\u003eM. incognita\u003c/em\u003e or 3 J2 cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of soil of a mixed community of \u003cem\u003eM. arenaria, M. hapla\u003c/em\u003e, and \u003cem\u003eM. javanica\u003c/em\u003e. Tomato plants were maintained in a growth chamber during 40 days. Then the number of egg masses and eggs per plant were determined. In addition, the preference of the insect was estimated confronting nematode-infected vs. non-infected plants in a Y-tube olfactometer and in insect cages, where 10 females were released into each cage containing resistant or susceptible tomato plants. After 1, 2, 4, 24, 48 and 72h, the number of \u003cem\u003eM. pygmaeus\u003c/em\u003e was counted as well as the offspring after 14 days. \u003cem\u003eM. pygmaeus\u003c/em\u003e reduced the infectivity and reproduction by 37% and 53%, in the susceptible tomato inoculated with \u003cem\u003eM. incognita\u003c/em\u003e and by 52% and 37% when inoculated with the nematode community but no effect was observed in the \u003cem\u003eMi1.2\u003c/em\u003e resistant tomato irrespective of the nematode inoculum. The preference and the offspring of \u003cem\u003eM. pygmaeus\u003c/em\u003e was not negatively affected by the nematode infection or the tomato cultivar. In conclusion, pre-induction of tomato plants with \u003cem\u003eM. pygmaeus\u003c/em\u003e reduces RKN infectivity and reproduction in susceptible but not in \u003cem\u003eMi1.2\u003c/em\u003e resistant tomato.\u003c/p\u003e","manuscriptTitle":"Macrolophus pygmaeus (Heteroptera: Miridae) induces systemic resistance in tomato against Meloidogyne spp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 14:41:20","doi":"10.21203/rs.3.rs-5181542/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-04T10:57:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-19T08:04:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5190297890974775796725193867686617818","date":"2024-11-07T05:40:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-04T19:44:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-28T14:20:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47471462776869140674528755303122437567","date":"2024-10-24T06:44:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5204269099832007951761352553863990700","date":"2024-10-24T01:45:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-22T01:12:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-21T00:35:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-20T09:20:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-16T08:45:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-30T14:11:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"21e1c2ad-704a-4ea4-b1a0-f4e8dfa23a5e","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41140215,"name":"Biological sciences/Zoology/Entomology"},{"id":41140216,"name":"Biological sciences/Plant sciences/Plant immunity"},{"id":41140217,"name":"Biological sciences/Plant sciences/Plant stress responses"}],"tags":[],"updatedAt":"2025-03-10T20:01:20+00:00","versionOfRecord":{"articleIdentity":"rs-5181542","link":"https://doi.org/10.1038/s41598-025-90233-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-04 15:58:17","publishedOnDateReadable":"March 4th, 2025"},"versionCreatedAt":"2024-12-12 14:41:20","video":"","vorDoi":"10.1038/s41598-025-90233-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-90233-7","workflowStages":[]},"version":"v1","identity":"rs-5181542","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5181542","identity":"rs-5181542","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}