Assessing the residual effects of the “defeated” tomato Mi-1.2 gene against a Meloidogyne enterolobii (guava race) population via comparative assays with contrasting near-isogenic lines

Assessing the residual effects of the “defeated” tomato Mi-1.2 gene against a Meloidogyne enterolobii (guava race) population via comparative assays with contrasting near-isogenic lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessing the residual effects of the “defeated” tomato Mi-1.2 gene against a Meloidogyne enterolobii (guava race) population via comparative assays with contrasting near-isogenic lines Dwillian F. CUNHA, Thávio J. B. PINTO, Jadir B. PINHEIRO, Giovani O. SILVA, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5364816/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Meloidogyne enterolobii represents a major threat to the global tomato ( Solanum lycopersicum L.) production due to its ability to “break-down” the resistance conferred by the dominant Mi -1.2 gene. However, a subgroup of “defeated” resistance genes in various pathosystems exhibits residual effects characterized by an enduring interference in quantitative levels of disease expression induced by novel virulent pathogens. Thus far, residual effects of the “defeated” Mi -1.2 gene to M. enterolobii have not been properly investigated. Herein, two comparative assays using contrasting near-isogenic lines (NILs) for presence/absence of the Mi -1.2 locus were carried out using a guava race population of M. enterolobii . Seedlings of two pairs of contrasting NILs ‘Nemadoro’ (homozygous dominant; Mi -1.2/ Mi -1.2) / ‘Rio Grande’ (homozygous recessive, mi -1.2/ mi -1.2) and ‘Del Rey’ ( Mi -1.2/ Mi -1.2) / ‘Calipso’ ( mi -1.2/ mi -1.2) were inoculated with ≈ 2,000 M. enterolobii eggs. The homozygous dominant ( Mi -1.2/ Mi -1.2) NILs displayed values for the quantitative parameter NEGR (number of eggs + occasional J2 per gram of root tissue) similar or even superior to their corresponding recessive ( mi -1.2/ mi -1.2) NILs. A slight positive impact of the resistance gene in the reproduction factor (RF) value was observed only for one pair of contrasting NILs (‘Del Rey’ / ‘Calipso’), which was restricted to one bioassay. The employment of NILs in our bioassays allowed us to hypothesize that the Mi -1.2 gene, although extremely effective against populations of at least 13 Meloidogyne species, does not confer significant residual effects against M. enterolobii race from guava. root-knot nematode defeated resistance gene resistance Solanum lycopersicum Figures Figure 1 Figure 2 Introduction Root-knot nematodes (RKNs) of the genus Meloidogyne are obligate endoparasites able to invade and parasitize the root systems of a wide range of plants, encompassing more than 3,000 host species (Sikandar et al., 2020). The tomato ( Solanum lycopersicum L.) crop is one of the major global hosts of Meloidogyne species, being severely affected by this group of pathogens mainly across tropical and subtropical areas (Tariq-Khan et al., 2017 ). In Brazil, nationwide surveys indicated M. incognita Kofoid & White, 1919; Chitwood, 1949, M. javanica Treub, 1885; Chitwood, 1949, and M. arenaria Neal, 1889; Chitwood, 1949 as the main species associated with the tomato crop (Pinheiro et al., 2014 ). However, populations of M. enterolobii Yang & Eisenback, 1983 were also detected across distinct vegetable production areas in the country (Pinheiro et al., 2015 ). Meloidogyne enterolobii represents a major threat to tomato production due to its recent geographical expansion, wide host range, and especially due to its ability to “break-down” the resistance conferred by the dominant Mi -1.2 gene (Gabriel et al., 2020 ; Dareus et al., 2021 ). In addition, M. enterolobii populations are able to infect other sources of resistance in different host species (Pinheiro et al., 2015 ; Koutsovoulos et al., 2019 ; Pinto et al., 2023 ), including the N gene in Capsicum annuum L., the Rk gene in Vigna unguiculata (L.), the Mir 1 gene in Glycine max (L.) Merril, the Mh gene in S. tuberosum L., and the Mi -1 gene in Gossypium hirsutum L., and in sources of resistance detected in Ipomoea batatas (L.) Lam (Collett et al., 2019 ; Schwarz, 2023 ). More recently, a new M . enterolobii race was detected parasitizing cotton and soybean crops in Brazil (Verssiani et al., 2023 ), expanding the virulence profile of this pathogen. Various resistance genes to RKN species have been reported in tomato relatives of the genus Solanum (section Lycopersicon ) (Williamson 1998 ; Jaiteh et al., 2012 ; Wang et al., 2013 ; Okorley et al., 2018 ). However, the Mi -1.2 gene remains as the major resistance factor employed in tomato breeding thus far (Williamson 1998 ; Gabriel et al., 2020 ; Gabriel et al., 2022 ). This dominant gene was introgressed from the wild tomato relative S. peruvianum L. (Bailey, 1941 ; Gilbert & McGuire, 1956 ) and it is phenotypic manifestation is characterized by a very fast hypersensitive reaction, which encompasses the cells around the feeding sites of avirulent Meloidogyne populations (Gabriel et al., 2024b ). The genomic region on tomato chromosome 6 where the Mi -1-2 gene is located contains a cluster of different NBS-LRR type of resistance genes (designated Mi -1.1 to Mi -1.7) that exhibit high levels of structural similarity (Milligan et al., 1998 ). However, additional studies established that Mi -1.2 gene is the only one encoding a functional RKN resistance gene (Williamson 1998 ; Milligan et al., 1998 ; Seah et al., 2007 ). This source of resistance has proven effective against the three most important RKN species: M. javanica , M. arenaria , and M. incognita (Roberts, 1992 ; Williamson, 1998 ). More recently, the Mi -1.2 gene was reported to be effective against populations of 13 Meloidogyne species (Gabriel et al., 2020 ). Nevertheless, the resistance conferred by the Mi -1.2 gene was surpassed when challenged against a virulent M. javanica isolate (Gabriel et al., 2022 ) as well as to populations of M. hapla Chitwood, 1949 and M. enterolobii (guava and cotton races) from Brazil (Gabriel et al., 2020 ; Verssiani et al., 2023 ). This virulent phenotype of M. enterolobii guava and cotton races in relation to the Mi -1.2 gene represents a serious threat to tomato cultivation in tropical and subtropical regions. Across distinct pathosystems, “defeated” resistance genes (i.e. the ones that are no longer effective against a pathogen variant, biotype, or race) can still have positive “residual” or “ghost effects” on reducing the damages caused by a novel virulent population (Van Der Plank, 1984 ; Damann Jr, 1987 ). These residual effects may manifest as a quantitative interference in a subset of parameters related to the interactions with the virulent pathogens (Nass et al., 1981 ; Singh et al., 2021 ). From the breeding standpoint, these effects might result in a rate-reducing phenotype (Tomczyńska et al., 2014 ) or correspond to a quantitative trait locus – QTL (Wang et al., 2001 ). However, the potential residual effects of the Mi -1.2 gene on M. enterolobii populations have not been properly investigated thus far in tomatoes. The employment of near-isogenic lines (NILs) is an outstanding tool to isolate the effect of specific genes/loci, such as Mi -1.2 in tomato plants (Boiteux et al., 1995 ). NILs are sister lines obtained after repeated generations of backcrossing that share an overall identical genome, except for one specific genomic region/locus for which the selection was applied (Michelmore et al., 1991 ; Wenzel & Foroughi-Wehr, 1994 ). Therefore, the employment of comparative analyses with NILs can either eliminates or minimizes the genetic “noise” caused by unrelated background variability that occurs when the comparison in done with lines/cultivars contrasting not only for the gene/locus in study. In this context, the objective of the present study was to estimate potential residual effects of the Mi -1.2 gene on parameters of the interaction tomato– M. enterolobii by employing comparative inoculation assays with contrasting NILs for presence/absence of this resistant locus. Materials and methods Production of contrasting near isogenic lines (NILs) of tomato for the locus M i - 1.2 – The Mi -1.2 gene was introgressed into the susceptible cultivars ‘Rio Grande’ (homozygous recessive, mi -1.2/ mi -1.2) and ‘Calipso’ ( mi -1.2/ mi -1.2) via a series of crosses and backcrosses. Pollen was collected from parental plants carrying the Mi -1.2 and transferred to (previously emasculated) flowers of ‘Rio Grande’ and ‘Calipso’ plants. The resulting F 1 hybrids were selfed to obtain recombinant F 2 populations, which were subsequently screened for the resistance phenotype using a M. incognita population (data not shown). The selected plants were backcrossed with ‘Rio Grande’ and ‘Calipso’ up to the sixth backcross generation (BC6), selfing every generation to screen for resistance to M. incognita . After six backcross generations and progeny testing, the resulting cultivars ‘Nemadoro’ (homozygous dominant; Mi -1.2/ Mi -1.2) and ‘Del Rey’ ( Mi -1.2/ Mi -1.2) were considered as near-isogenic lines (NILs) of Rio Grande’ and ‘Calipso’, respectively. inoculation bioassays employing contrasting tomato NILs for the locus M i - 1.2 – Meloidogyne enterolobii inoculum was obtained from a pure culture multiplied by periodic subculturing on plants of the tomato cultivar ‘Santa Cruz’. This RKN population was maintained in a greenhouse (25–30 o C), being previously identified by esterase and SCAR phenotyping (Gabriel et al., 2020 ). Two bioassays were carried out under greenhouse conditions at Embrapa Vegetables Crops (Brasilia–DF, Brazil) in two distinct periods: November/2022 to January/2023 and February/2024 to April/2024. The average air temperature in the first assay was 30.1 ℃ (39.2 ℃ maximum and 16.1 ℃ minimum), while in the second experiment the average air temperature was 27.2 ℃ (31.7 ℃ maximum and 15.5 ℃ minimum). The bioassays were conducted in completely randomized design with four treatments (two pairs contrasting NILs) and six replicates (Tables 1 & 2 ). Table 1 Mean values of gall index (GI) and egg mass index (EMI) induced by Meloidogyne enterolobii (guava race) on the root systems of contrasting tomato ( Solanum lycopersicum ) near isogenic lines (NILs) for the presence/absence of the Mi -1.2 locus. Evaluation was carried out under greenhouse conditions at 45 days after inoculation NILs GI 1* EMI 2* Mi -1.2 gene Experiment I Experiment II Experiment I Experiment II ‘Nemadoro’ 3 Present homozygous 4.5 5.0 3.5 4.0 ‘Rio Grande’ 3 Absent homozygous 4.3 3.0 4.0 2.0 ‘Del Rey’ 4 Present homozygous 5.0 3.0 4.3 2.3 ‘Calipso’ 4 Absent homozygous 4.5 5.0 4.0 3.3 ‘Rutgers’ (control) Absent homozygous 3.5 5.0 5.0 4.0 Average 4.3 4.2 4.1 3.1 CV (%) 5 14.2 7.7 12.0 7.7 *Means (n = 6) were not significantly different across pairs of NILs according to the Scott–Knott test ( P < 0.05). 1 GI and 2 EMI: grades 1–5, according to Taylor and Sasser ( 1978 ). The pairs of contrasting NILs were ‘Nemadoro’ 3 and ‘Rio Grande’ 3 and ‘Del Rey’ 4 and ‘Calipso’ 4 . The cultivar ‘Rutgers’ was employed as susceptible control. CV (%) 5 = coefficient of variation. Table 2 Number of eggs + occasional second stage juveniles per gram of roots ( NEGR ) and nematode reproduction factor ( RF ) of Meloidogyne enterolobii on contrasting near isogenic lines (NILs) of tomato ( Solanum lycopersicum ) for the presence/absence of the Mi -1.2 locus. Evaluation was carried out under greenhouse conditions at 45 days after inoculation NILs NEGR 1* RF 2* /NIL reaction Mi -1.2 gene Experiment I Experiment II Experiment I Experiment II ‘Nemadoro’ 3 Present homozygous 1193 b 14783 a 15.83 c / S 38.38 a / S ‘Rio Grande’ 3 Absent homozygous 623 b 5049 b 11.59 c / S 13.00 c / S ‘Del Rey’ 4 Present homozygous 2610 a 4946 b 47.19 b / S 11.00 c / S ‘Calipso’ 4 Absent homozygous 957 b 2290 b 11.66 c / S 21.75 b / S ‘Rutgers’ (control) Absent homozygous 3608 b 13130 a 70.63 a / S 43.50 a / S Average 1798 8040 31.38 26.18 CV (%) 5 33.8 35.7 42.7 12.9 *The data were transformed to √x + 0.5 for analysis but are presented without transformation. Means (n = 6) followed by different letters lowercase in the columns is significantly different, according to the Scott–Knott test ( P < 0.05). 1 Eggs + occasional second stage juveniles per gram of roots (NEGR) = final population / fresh weight of root (g), 2 RF = final population/2000 eggs + J2 of M. enterolobii ; 3 Reaction of inoculated plants, RF > 1 = suitable host (S) and RF < 1 = poor host (P) and RF = 0 immune (I) (Oostenbrink, 1966 ). CV = coefficient of variation. The pairs of NILs were ‘Nemadoro’ 3 versus ‘Rio Grande’ 3 and ‘Dell Rey’ 4 versus ‘Calipso’ 4 . The cultivar ‘Rutgers’ was employed as susceptible control. **NIL Reaction: S (susceptible). A distinct set of plants of the NIL ‘Nemadoro’ ( Mi -1.2/ Mi -1.2) and ‘Heinz 1706’ was inoculated with an Mi -1.2-avirulent population of M. incognita race 1 aiming to verify if the resistance reaction conferred by this gene could be affected environmental conditions similar to that of the experiments with M. enterolobii . The inoculum of the M. incognita race 1 population was obtained also from a pure culture multiplied by periodic subculturing on plants of the tomato cultivar ‘Santa Cruz’. This RKN population was maintained under greenhouse conditions (25–30 o C), being previously identified through morphological features as well as through esterase phenotyping I2 (Rm: 0.39 and 0.42). This M. incognita population was identified as race 1 based upon its ability to reproduce in pepper (‘Califonia Wonder’), watermelon (‘Charleston Gray’) and tomato (‘Rutgers’) and by its inability to infect cotton (‘Deltapine’), tobacco (‘NC95’) and peanut (‘Florunner’) (Hartman & Sasser, 1985). The bioassay with M. incognita race 1 inoculum was conducted in February/2024 to April/2024. The experiment was carried out in a completely randomized design with two treatments (‘Nemadoro’ and ‘Heinz 1706’) and six replicates. The average air temperature in the assay was 27.2 ℃ (31.7 ℃ maximum and 15.5 ℃ minimum). Inoculation procedures – Eggs + occasional J2 juveniles of the M. enterolobii and M. incognita race 1 populations were extracted from the infected roots, according to Hussey and Barker ( 1973 ) modified by Bonetti and Ferraz ( 1981 ). The roots were washed with tap water, and nematode eggs in the egg masses were extracted using 0.5% sodium hypochlorite. Roots were cut into 1–2 cm-long pieces, mixed with sodium hypochlorite, shaken for 30 seconds, washed with tap water, and collected using a 500-mesh screen. The total number of eggs per milliliter was quantified under a light microscope (Nikon Eclipse 80i model) using a nematode-counting slide (Peter’s slide). Twenty days after sowing, the tomato seedlings were transplanted to 2000 cm 3 plastic pots with a diameter of 12 cm (filled with sterilized soil and sand in a 2:1 ratio). Plants were subsequently inoculated with 2,000 eggs + occasional J2 by placing 2 mL of the suspension into three holes with 2 cm deep around the plants. The tomato cultivar ‘Rutgers’ was employed as susceptible control in the assays with M. enterolobii population and, as previously mentioned, the cultivar ‘Heinz 1706’ was employed as susceptible control in the assay with the M. incognita race 1 population. Molecular genotyping of individual plants for Confirmation of the presence/Absence of the M i - 1.2 locus – The genomic DNA was extracted from each evaluated plant in order to confirm the presence/absence of the Mi -1.2 gene/locus. For nucleic acid purification of young apical leaves, a modified 2X CTAB buffer (pH = 8.0) and organic solvents methodology was employed (Boiteux et al., 1999 ). The DNA pellet was carefully washed with 70% ethanol. The micro centrifuge tubes were then placed in an incubator at 37°C for 20 minutes and the solution resuspended in 100 µL of Tris-EDTA buffer + RNase. The purified samples were vortexed and stored in a freezer (-20°C). PCR was performed with the primer pair ‘Mi23F’ (5’–TGG AAA AAT GTT GAA TTT CTT TTG–3’) and ‘Mi23R’ (5’–GCA TAC TAT ATG GCT TGT TTA CCC–3’), considered the most reliable codominant molecular marker for detecting contrasting alleles at the Mi -1.2 gene/locus (Seah et al., 2007 ; Bhavana et al., 2019 ). The final volume of the mixture was 25 µL (23 µL master mix + 2 µL DNA). The master mix consisted of 11.32 µL milliQ water, 2.5 µL of 10× Taq polymerase buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl), 5.7 µL of dNTPs (0.5 mM, Invitrogen), 0.5 µL of each primer (0.5 mM), 2.3 µL of MgCl 2 (25 mM), and 0.18 µL of Taq DNA polymerase (5 U/µL, Invitrogen). Amplifications were performed with the following parameters: an initial denaturation step at 94°C for 2 minutes; 40 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 1 minute, extension at 72°C for 1.5 minutes; and a final extension step at 68°C for 10 minutes. Bromophenol blue (5 µL) was added to each sample, and the amplified DNA fragments were separated by electrophoresis in TBE buffer (40 mM Tris-borate, 1 mM EDTA) on a 1.7% agarose gel supplemented with ethidium bromide, at 80 V for approximately 4 to 5 hours. The gels were photographed under UV light (Fig. 1 ). The 1kb Plus DNA Ladder (Invitrogen) was used to locate the amplification profile. Sample processing and statistical analysis – At sixty days after inoculation, the roots were carefully removed from the soil, rinsed with tap water, and dried with a paper towel. The roots were then stained with floxin B (15 mg/L water) (Daykin and Hussey 1985 ) to facilitate the visualization of the nematode's external egg masses, which were quantified with the help of a magnifying glass, assessing the gall and egg mass indices. The following qualitative parameters were evaluated: gall index ( GI ) and egg mass index ( EMI ) (Taylor and Sasser, 1978 ). The quantitative parameter NEGR (number of eggs + occasional J2 juveniles per gram of root) was also estimated by counting nematode structures in a Peter’s counting chamber under an optical microscope (Nikon Eclipse 80i model). The quantitative parameter nematode reproduction factor– RF = (final population/initial population) was also calculated (Oostenbrink, 1966 ). Individual plants were rated according to their reaction to the nematode as immune ( RF = 0 ), resistant/poor host ( RF 1 ), with. Data were transformed √x + 05 for statistical analysis, that is, normality test, analysis of variance, and Scott–Knott test ( P < 0.05). The data were back-transformed to display the original values. Results The contrasting NILs for the Mi -1.2 locus reacted differently to M. enterolobii as inferred from the qualitative parameters of gall index ( GI ) and egg mass index ( EMI ) (Table 1 ) and by the quantitative parameters NEGR and RF (Table 2 ). The homozygous dominant ( Mi -1.2/ Mi -1.2) NILs displayed values for the parameter NEGR similar or even superior to their corresponding recessive ( mi -1.2/ mi -1.2) NILs ( Table 2 ). All the contrasting NILs showed RF > 1.0 ( Table 2 ). Therefore, they can be categorized as susceptible/suitable hosts of M. enterolobii (Oostenbrink, 1966 ). In the present study, the susceptible standard (cultivar ‘Rutgers’) displayed the highest RF in the bioassays I and II (70.63 and 43.50, respectively), indicating proper experimental conditions for M. enterolobii development and reproduction (Table 2 ). Furthermore, ‘Rutgers’ displayed significantly higher M. enterolobii reproduction even in comparison with the two susceptible NILs ‘Rio Grande’ ( mi -1.2/ mi -1.2) and ‘Calipso’ ( mi -1.2/ mi -1.2) (Table 2 ). The inoculation assay with the M. incognita race 1 population displayed GI and EMI values equal to 1 and RF value of 0.1 for the NIL ‘Nemadoro’ (homozygous, Mi -1.2/ Mi -1.2) (Fig. 2 ), indicating the effectiveness of the resistance gene against this avirulent population under similar environmental conditions of the assays carried out with M. enterolobii . The susceptible standard (cultivar ‘Heinz 1706’) displayed GI, EMI, and RF values of 5.0, 4.18, 44.20, respectively, indicating the inoculum viability of the M. incognita race 1 population. The PCR analyses with the specific primer pair Mi23F/Mi23R using as template DNA extracted from each individual plant of the contrasting NILs under evaluation (Fig. 1 ), yielded only amplicons of 380 bp in ‘Nemadoro’ and ‘Del Rey’, confirming that the dominant allele is fixed in these NILs (i.e. all plants displaying the dominant Mi -1.2 gene in homozygous condition), whereas PCR assays with their corresponding contrasting NILs (‘Rio Grande’ and ‘Calipso’) yielded only amplicons of 430 bp, confirming their homozygous configuration for the recessive allele ( mi -1.2/ mi -1.2). The NIL ‘Nemadoro’ showed RFs of 15.83 and 38.38 in bioassays I and II, respectively, whereas its contrasting NIL (‘Rio Grande’), showed RFs of 11.59 and 13.00 in bioassays I and II, respectively. Likewise, the cultivar ‘Del Rey’ (homozygous Mi -1.2/ Mi -1.2) exhibited a higher RF (47.19) in comparison to its corresponding NIL ‘Calipso’, with displayed FR of 11.66 in bioassay I. However, a slight positive impact of the resistance gene in the reproduction factor (RF) value was observed in bioassay II for ‘Del Rey’ (RF = 11.00) in relation to ‘Calipso’ (RF = 21.75). The GI and EMI are auxiliary semi-quantitative/qualitative parameters that can help estimate plant resistance to Meloidogyne infections. Generally, a correlation exists between these indices and RF. Herein, most GI and EMI were correlated with RF. For example, the NILs ‘Rio Grande’ and ‘Del Rey’ displayed lower GI and EMI values in bioassay II (2.0 and 2.3, respectively), and their FR values were 13.00 and 11.00, respectively (Tables 1 & 2 ). Therefore, the auxiliary parameters GI and EMI were also in agreement with the observation that the Mi -1.2 gene does not influence M. enterolobii infection. Discussion From the tomato breeding standpoint, the Mi -1.2 is a unique gene able to control resistant reactions to populations of 13 Meloidogyne species (Gabriel et al., 2020 ) and to three species of Hempiteran insects, including the aphid Macrosiphum euphorbiae , the whiteflies of the Bemisia tabaci complex, and the psyllid Bactericerca cockerelli (Rossi et al., 1998 ; Vos et al., 1998 ; Nombela et al. 2003 ; Kaloshian & Teixeira, 2019 ). Even though the Mi -1.2 might display negative effects on the beneficial biocontrol agent, Orius insidiosus (Pallipparambil et al. 2015 ), its ability to control a wide array of pests and pathogens makes this gene a central component in integrated pest management in the tomato crop. In this context, it is important to assess if this gene might have some residual effects on virulent RKN species and populations that are able to ‘break-down’ this resistance such as M. enterolobii (Castagnone-Sereno, 2012 ; Pinheiro et al., 2014 ; Gabriel et al., 2020 ; Philbrick et al., 2020 ; Gabriel et al., 2024a ). To address this question, we evaluated the reaction of contrasting NILs with very similar genetic background but differing for presence/absence of the Mi -1.2-carrying locus. The molecular-marker analyses in association with the simultaneous bioassays with contrasting pairs of NILs reinforce the notion that the Mi -1.2 gene does not confer significant residual effects on this M. enterolobii (guava race) population. Our assays with the contrasting pair of NILs ‘Nemadoro’ (homozygous Mi -1.2/ Mi -1.2) and ‘Rio Grande’ (homozygous mi -1.2/ mi -1.2), showed similar RF values across the two bioassays. These results were also consistent with the reactions of one alternative pair of contrasting NILs (‘Del Rey’ and ‘Calipso’). The NIL ‘Del Rey’ (homozygous Mi -1.2/ Mi -1.2) exhibited either similar or even a higher RF in comparison to the corresponding NIL ‘Calipso’ (homozygous mi -1.2/ mi -1.2) in the first bioassay, even though a slight (but statistically significant) positive impact of the resistance gene in RF value was observed only in bioassay II. Our results are not in agreement with previous investigations involving the comparison of the susceptible cultivar ‘Rutgers’ (homozygous mi -1.2/ mi -1.2) with the cultivars ‘Nemadoro’ and ‘Tospodoro’ (both homozygous for the Mi -1.2 gene). ‘Rutgers’ displayed significantly higher RF, suggesting a residual effect of this gene against distinct M. enterolobii populations (Pinheiro et al., 2022 ; Verssiani et al., 2023 ; Pinto et al., 2024 ). Even before these studies, Brito et al. ( 2007 ) also detected a putative significant residual effect (approximately 80%) of the cultivar ‘Sanibel’ (carrying the Mi -1.2 gene) to a group of isolates of M. mayaguensis (= M. enterolobii ) when comparing with ‘Rutgers’. In addition, data presented by Gabriel et al. ( 2020 ) indicated lower levels of susceptibility in the hybrid ‘Debora Plus’ (heterozygous Mi -1.2/ mi -1.2) when compared to the cultivar ‘Santa Clara’ (homozygous mi -1.2/ mi -1.2) in response to a Brazilian population of M. enterolobii from guava. These contradictory results can be explained either by distinct virulence profiles or by the variable levels of aggressiveness observed across isolates of the cotton and guava races of M . enterolobii (Verssiani et al., 2023 ). However, it is important to point out that all these comparative studies were carried out with cultivars/accessions displaying different genetic backgrounds. In the present study, the susceptible standard (cultivar ‘Rutgers’) displayed significantly higher levels susceptibility to M. enterolobii even when compared to the two susceptible NILs ‘Rio Grande’ ( mi -1.2/ mi -1.2) and ‘Calipso’ ( mi -1.2/ mi -1.2). This reinforces the observation that comparison between genetically unrelated accessions with distinct backgrounds, even in the absence of the Mi -1.2, can induce misleading interpretations of the putative residual effects of this gene. Therefore, we cannot exclude in these previous reports, the potential contribution of other genes, in addition to Mi -1.2, that might influence the levels of resistance/susceptibility to this pathogen, making difficult to assess with confidence the putative residual effects of this gene. In this regard, our employment of contrasting NILs in the present study either eliminates or minimizes the genetic “noise” caused by unrelated background variability when the comparison in done with lines/cultivars contrasting not only for the gene/locus under evaluation. In our study, we could provide a stronger test of this ‘residual effect hypothesis’ since we carry out a comparative analysis with pairs of NILs, sharing similar genetic background: ‘Nemadoro’ ( Mi -1.2/ Mi -1.2) versus its NIL ‘Rio Grande’ ( mi -1.2/ mi -1.2) as well as ‘Del Rey’ (homozygous Mi -1.2/ Mi -1.2) and its corresponding NIL ‘Calipso’ (homozygous mi -1.2/ mi -1.2). With if few exceptions, no significant differences were observed across the contrasting NILs for a subset of quantitative parameters in both bioassays, providing evidence that Mi -1.2 gene do not display any residual effect against M. enterolobii. Herein, we can exclude the potential negative impacts of the environmental conditions established on the efficient expression Mi -1.2 gene as indicated by it highly resistant reaction of the NIL ‘Nemadoro’ against a well-known avirulent population of M. incognita race 1. As expected, M. incognita race 1 was unable to overcome the resistance conferred by the Mi -1.2 gene (see Gabriel et al., 2020 ). The cultivar ‘Nemadoro’ displayed very low GI, IMO, and RF values. This result allowed us to confirm that environmental components were not able to interfere with the Mi -1.2 gene expression under our experimental conditions The confirmation of the complete ‘break-down’ of the Mi -1.2 gene with no significant residual effect against M. enterolobii will intensify the need to search for novel sources of genetic resistance effective against this RKN species. However, searches for sources of resistance to M. enterolobii have not been very successful thus far. Besides the Mi -1.2, nine distinct genes of resistance to Meloidogyne species have been identified in accessions of Solanum ( Lycopersicon ) (Williamson 1998 ). However, four of these genes ( Mi -3, Mi -5, Mi -9, and Mi -HT) were found to be ineffective against M. enterolobii populations (Williamson 1998 ; El-Sappah et al., 2019 ). A collection of 101 commercial and wild tomatoes was screened in a search resistance/tolerance to M. enterolobii (guava race) population, but no useful sources were detected (Silva et al. 2019 ). One additional problem related to M. enterolobii management is the fact that this RKN is able to break-down a wide array of resistance sources of different host species (Pinheiro et al., 2015 ; Koutsovoulos et al., 2019 ; Pinto et al., 2023 ), including the N gene in Capsicum annuum , the Rk gene in Vigna unguiculata , the Mir 1 gene in Glycine max , the Mh gene in S. tuberosum , and the Mi -1 gene in Gossypium hirsutum , and in sources carrying Mh gene in Ipomoea batatas (Fery et al., 1998 ; Thies & Fery, 2000 ; Williamson & Roberts, 2009 ; Castagnone-Sereno, 2012 ; Quenouille et al., 2013 ; Verssiani et al., 2023 ; Collett et al., 2019 ; Schwarz, 2023 ). This feature of M. enterolobii populations limits the crop rotation alternatives. Alternative measures for the genetic management of M. enterolobii could be implemented, including grafting tomato cultivars onto resistant rootstocks (Louws et al., 2010 ; Schwarz et al., 2010 ; Baidya et al., 2017 ; Pinheiro et al., 2022 ) as well as biotech-based approaches (Claverie et al., 2011 ; Zhuo et al., 2017 ; El-Sappah et al., 2019 ; Manivannan et al., 2020 ). The continuous investigation into the genetic and molecular mechanisms by which M. enterolobii circumvents resistance will be also a crucial piece of information (Sato et al., 2012 ; Szitenberg et al., 2017 ; Koutsovoulos et al., 2019 ). In summary, the evaluation of contrasting NILs for the Mi- 1.2 locus indicated that all are susceptible to M. enterolobii , displaying similar responses to all parameters (including RF). Therefore, Mi -1.2 gene, although extremely effective against at least 13 Meloidogyne species, is not able of imposing any significant interference with the infection process of M. enterolobii in tomato. Therefore, search for effective sources of resistance to this pathogen can be considered as a major tomato breeding priority. Declarations Funding The authors acknowledge EMBRAPA Hortaliças for the support in all steps of this research the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001 – for providing scholarships to Dwillian F. Cunha and Thávio J. B. Pinto and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for fellowships and for partially funding the research project. Author information Authors and Affiliations University of Brasília (UnB), Institute of Biological Sciences, Department of Plant Pathology, Brasília–DF, 70910-900, Brazil Dwillian Firmiano Cunha, Thávio Júnior Barbosa Pinto, Juvenil Enrique Cares & Leonardo Silva Boiteux Embrapa Vegetable Crops (Hortaliças), Brasília–DF, 70359-970, Brazil Jadir Borges Pinheiro, Maria Esther de Noronha Fonseca & Leonardo Silva Boiteux Embrapa Hortaliças, Estação Experimental Canoinhas, Canoinhas–SC, 89460-000, Brazil Giovani Olegário da Silva Centro Universitário ICESP, Brasília–DF, 71961-540, Brazil Felipe Santos Rafael & Leandro Alves Santos Contributions Dwillian F. Cunha; Thávio J.B. Pinto; Felipe S. Rafael; Leandro A. Santos: Investigation, Formal analysis. Leonardo S. Boiteux; Jadir B. Pinheiro; Juvenil E. Cares: Conceptualization, Writing - original draft, Supervision, Funding acquisition, Project administration. Thávio J.B. Pinto; Dwillian F. Cunha; Maria Esther N. Fonseca; Giovani O. 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B.","lastName":"PINTO","suffix":""},{"id":410779691,"identity":"e28e39cf-b574-48d9-9482-725ef3c7a5c1","order_by":2,"name":"Jadir B. PINHEIRO","email":"","orcid":"","institution":"Embrapa Vegetable Crops (Hortaliças)","correspondingAuthor":false,"prefix":"","firstName":"Jadir","middleName":"B.","lastName":"PINHEIRO","suffix":""},{"id":410779692,"identity":"663955e3-1de5-4054-9f66-d11647ded33f","order_by":3,"name":"Giovani O. SILVA","email":"","orcid":"","institution":"Embrapa Hortaliças, Estação Experimental Canoinhas","correspondingAuthor":false,"prefix":"","firstName":"Giovani","middleName":"O.","lastName":"SILVA","suffix":""},{"id":410779693,"identity":"a4d5ad64-b0d1-450e-8677-dd3523b8ec17","order_by":4,"name":"Felipe S. RAFAEL","email":"","orcid":"","institution":"Centro Universitário ICESP","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"S.","lastName":"RAFAEL","suffix":""},{"id":410779694,"identity":"689333cf-8ae3-4d87-9204-7790693e9f1b","order_by":5,"name":"Leandro A. SANTOS","email":"","orcid":"","institution":"Centro Universitário ICESP","correspondingAuthor":false,"prefix":"","firstName":"Leandro","middleName":"A.","lastName":"SANTOS","suffix":""},{"id":410779695,"identity":"e549ca93-a17f-458d-ae3c-d7d22c5c2623","order_by":6,"name":"Maria Esther N. FONSECA","email":"","orcid":"","institution":"Embrapa Vegetable Crops (Hortaliças)","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Esther N.","lastName":"FONSECA","suffix":""},{"id":410779696,"identity":"03b4a1ad-71ff-48b8-80cf-6423435902e5","order_by":7,"name":"Juvenil E. CARES","email":"","orcid":"","institution":"University of Brasília (UnB), Brasília–DF","correspondingAuthor":false,"prefix":"","firstName":"Juvenil","middleName":"E.","lastName":"CARES","suffix":""},{"id":410779697,"identity":"b368c98e-0ef8-4e8c-b707-45c1ce544875","order_by":8,"name":"Leonardo S. BOITEUX","email":"","orcid":"","institution":"University of Brasília (UnB), Brasília–DF","correspondingAuthor":false,"prefix":"","firstName":"Leonardo","middleName":"S.","lastName":"BOITEUX","suffix":""}],"badges":[],"createdAt":"2024-10-31 04:53:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5364816/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5364816/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75486387,"identity":"43da35ca-851b-4959-a8db-094118d0a9ff","added_by":"auto","created_at":"2025-02-05 06:33:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55920,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis (1.7%) displaying the PCR amplicon profiles of the codominant marker closely linked to the \u003cem\u003eMeloidogyne\u003c/em\u003e resistance gene \u003cem\u003eMi\u003c/em\u003e-1.2 in tomato chromosome 6. PCR assays were performed using the specific primer pair \u003cem\u003eMi\u003c/em\u003e23F/\u003cem\u003eMi\u003c/em\u003e23R essentially as described by Bhavana \u003cem\u003eet al.\u003c/em\u003e(2019). This gel shows the allelic configuration of the \u003cem\u003eMi\u003c/em\u003e-1.2 locus using DNA template from contrasting near-isogenic lines (NILs) evaluated for their reaction to a \u003cem\u003eM. enterolobii\u003c/em\u003e population: ‘Nemadoro’ (\u003cstrong\u003eNE\u003c/strong\u003e) and Del Rey (\u003cstrong\u003eDR\u003c/strong\u003e) which are homozygous for the resistant locus (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and ‘Rio Grande’ (\u003cstrong\u003eRG\u003c/strong\u003e) and Calypso (\u003cstrong\u003eCA\u003c/strong\u003e), which are homozygous for the susceptible locus (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2). \u003cstrong\u003eTest. R =\u003c/strong\u003eHomozygous (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003e Mi\u003c/em\u003e-1.2) resistant control and \u003cstrong\u003eTest. S =\u003c/strong\u003eHomozygous (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003e mi\u003c/em\u003e-1.2) susceptible control. \u003cstrong\u003eMM\u003c/strong\u003e = Molecular marker 1kb plus DNA Ladder (Invitrogen)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5364816/v1/d29dd07533ccace8a5730af5.jpg"},{"id":75486388,"identity":"b30d75df-9285-4705-b679-9b9780ac978f","added_by":"auto","created_at":"2025-02-05 06:33:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":368402,"visible":true,"origin":"","legend":"\u003cp\u003eControl assay conducted under greenhouse conditions aiming to verify the efficiency of the \u003cem\u003eMi\u003c/em\u003e-1.2 gene to an avirulent \u003cem\u003eMeloidogyne\u003c/em\u003e population under the experimental conditions established in the present study. Reaction with a distinct set of plants of the NIL ‘Nemadoro’ (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and ‘Heinz 1706’ at 60 days after inoculation with an avirulent population of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e race 1. On the left, no conspicuous galls were displayed, indicating the efficiency of the resistance gene, while on the right, numerous galls were observed\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5364816/v1/c342039da22867997ee160c7.jpg"},{"id":75487619,"identity":"bc9ce3fa-6b64-4b2a-b866-082927e98a9d","added_by":"auto","created_at":"2025-02-05 06:41:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5364816/v1/ae0a0eb1-b337-4d26-b4fd-535fa48cc03a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessing the residual effects of the “defeated” tomato Mi-1.2 gene against a Meloidogyne enterolobii (guava race) population via comparative assays with contrasting near-isogenic lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRoot-knot nematodes (RKNs) of the genus \u003cem\u003eMeloidogyne\u003c/em\u003e are obligate endoparasites able to invade and parasitize the root systems of a wide range of plants, encompassing more than 3,000 host species (Sikandar et al., 2020). The tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) crop is one of the major global hosts of \u003cem\u003eMeloidogyne\u003c/em\u003e species, being severely affected by this group of pathogens mainly across tropical and subtropical areas (Tariq-Khan et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In Brazil, nationwide surveys indicated \u003cem\u003eM. incognita\u003c/em\u003e Kofoid \u0026amp; White, 1919; Chitwood, 1949, \u003cem\u003eM. javanica\u003c/em\u003e Treub, 1885; Chitwood, 1949, and \u003cem\u003eM. arenaria\u003c/em\u003e Neal, 1889; Chitwood, 1949 as the main species associated with the tomato crop (Pinheiro et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, populations of \u003cem\u003eM. enterolobii\u003c/em\u003e Yang \u0026amp; Eisenback, 1983 were also detected across distinct vegetable production areas in the country (Pinheiro et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e represents a major threat to tomato production due to its recent geographical expansion, wide host range, and especially due to its ability to \u0026ldquo;break-down\u0026rdquo; the resistance conferred by the dominant \u003cem\u003eMi\u003c/em\u003e-1.2 gene (Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dareus et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, \u003cem\u003eM. enterolobii\u003c/em\u003e populations are able to infect other sources of resistance in different host species (Pinheiro et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Koutsovoulos et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pinto et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), including the \u003cem\u003eN\u003c/em\u003e gene in \u003cem\u003eCapsicum annuum\u003c/em\u003e L., the \u003cem\u003eRk\u003c/em\u003e gene in \u003cem\u003eVigna unguiculata\u003c/em\u003e (L.), the \u003cem\u003eMir\u003c/em\u003e1 gene in \u003cem\u003eGlycine max\u003c/em\u003e (L.) Merril, the \u003cem\u003eMh\u003c/em\u003e gene in \u003cem\u003eS. tuberosum\u003c/em\u003e L., and the \u003cem\u003eMi\u003c/em\u003e-1 gene in \u003cem\u003eGossypium hirsutum\u003c/em\u003e L., and in sources of resistance detected in \u003cem\u003eIpomoea batatas\u003c/em\u003e (L.) Lam (Collett et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schwarz, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). More recently, a new \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eenterolobii\u003c/em\u003e race was detected parasitizing cotton and soybean crops in Brazil (Verssiani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), expanding the virulence profile of this pathogen.\u003c/p\u003e \u003cp\u003eVarious resistance genes to RKN species have been reported in tomato relatives of the genus \u003cem\u003eSolanum\u003c/em\u003e (section \u003cem\u003eLycopersicon\u003c/em\u003e) (Williamson \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Jaiteh et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Okorley et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the \u003cem\u003eMi\u003c/em\u003e-1.2 gene remains as the major resistance factor employed in tomato breeding thus far (Williamson \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gabriel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This dominant gene was introgressed from the wild tomato relative \u003cem\u003eS. peruvianum\u003c/em\u003e L. (Bailey, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1941\u003c/span\u003e; Gilbert \u0026amp; McGuire, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1956\u003c/span\u003e) and it is phenotypic manifestation is characterized by a very fast hypersensitive reaction, which encompasses the cells around the feeding sites of avirulent \u003cem\u003eMeloidogyne\u003c/em\u003e populations (Gabriel et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). The genomic region on tomato chromosome 6 where the \u003cem\u003eMi\u003c/em\u003e-1-2 gene is located contains a cluster of different NBS-LRR type of resistance genes (designated \u003cem\u003eMi\u003c/em\u003e-1.1 to \u003cem\u003eMi\u003c/em\u003e-1.7) that exhibit high levels of structural similarity (Milligan et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). However, additional studies established that \u003cem\u003eMi\u003c/em\u003e-1.2 gene is the only one encoding a functional RKN resistance gene (Williamson \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Milligan et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Seah et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This source of resistance has proven effective against the three most important RKN species: \u003cem\u003eM. javanica\u003c/em\u003e, \u003cem\u003eM. arenaria\u003c/em\u003e, and \u003cem\u003eM. incognita\u003c/em\u003e (Roberts, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Williamson, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). More recently, the \u003cem\u003eMi\u003c/em\u003e-1.2 gene was reported to be effective against populations of 13 \u003cem\u003eMeloidogyne\u003c/em\u003e species (Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, the resistance conferred by the \u003cem\u003eMi\u003c/em\u003e-1.2 gene was surpassed when challenged against a virulent \u003cem\u003eM. javanica\u003c/em\u003e isolate (Gabriel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) as well as to populations of \u003cem\u003eM. hapla\u003c/em\u003e Chitwood, 1949 and \u003cem\u003eM. enterolobii\u003c/em\u003e (guava and cotton races) from Brazil (Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Verssiani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This virulent phenotype of \u003cem\u003eM. enterolobii\u003c/em\u003e guava and cotton races in relation to the \u003cem\u003eMi\u003c/em\u003e-1.2 gene represents a serious threat to tomato cultivation in tropical and subtropical regions.\u003c/p\u003e \u003cp\u003eAcross distinct pathosystems, \u0026ldquo;defeated\u0026rdquo; resistance genes (i.e. the ones that are no longer effective against a pathogen variant, biotype, or race) can still have positive \u0026ldquo;residual\u0026rdquo; or \u0026ldquo;ghost effects\u0026rdquo; on reducing the damages caused by a novel virulent population (Van Der Plank, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Damann Jr, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). These residual effects may manifest as a quantitative interference in a subset of parameters related to the interactions with the virulent pathogens (Nass et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). From the breeding standpoint, these effects might result in a rate-reducing phenotype (Tomczyńska et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) or correspond to a quantitative trait locus \u0026ndash; QTL (Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, the potential residual effects of the \u003cem\u003eMi\u003c/em\u003e-1.2 gene on \u003cem\u003eM. enterolobii\u003c/em\u003e populations have not been properly investigated thus far in tomatoes.\u003c/p\u003e \u003cp\u003eThe employment of near-isogenic lines (NILs) is an outstanding tool to isolate the effect of specific genes/loci, such as \u003cem\u003eMi\u003c/em\u003e-1.2 in tomato plants (Boiteux et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). NILs are sister lines obtained after repeated generations of backcrossing that share an overall identical genome, except for one specific genomic region/locus for which the selection was applied (Michelmore et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Wenzel \u0026amp; Foroughi-Wehr, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Therefore, the employment of comparative analyses with NILs can either eliminates or minimizes the genetic \u0026ldquo;noise\u0026rdquo; caused by unrelated background variability that occurs when the comparison in done with lines/cultivars contrasting not only for the gene/locus in study. In this context, the objective of the present study was to estimate potential residual effects of the \u003cem\u003eMi\u003c/em\u003e-1.2 gene on parameters of the interaction tomato\u0026ndash;\u003cem\u003eM. enterolobii\u003c/em\u003e by employing comparative inoculation assays with contrasting NILs for presence/absence of this resistant locus.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eProduction of contrasting near isogenic lines (NILs) of tomato for the locus\u003c/span\u003e \u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003eM\u003c/span\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003e-\u003c/span\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e1.2\u003c/span\u003e \u0026ndash; The \u003cem\u003eMi\u003c/em\u003e-1.2 gene was introgressed into the susceptible cultivars \u0026lsquo;Rio Grande\u0026rsquo; (homozygous recessive, \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) and \u0026lsquo;Calipso\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) via a series of crosses and backcrosses. Pollen was collected from parental plants carrying the \u003cem\u003eMi\u003c/em\u003e-1.2 and transferred to (previously emasculated) flowers of \u0026lsquo;Rio Grande\u0026rsquo; and \u0026lsquo;Calipso\u0026rsquo; plants. The resulting F\u003csub\u003e1\u003c/sub\u003e hybrids were selfed to obtain recombinant F\u003csub\u003e2\u003c/sub\u003e populations, which were subsequently screened for the resistance phenotype using a \u003cem\u003eM. incognita\u003c/em\u003e population (data not shown). The selected plants were backcrossed with \u0026lsquo;Rio Grande\u0026rsquo; and \u0026lsquo;Calipso\u0026rsquo; up to the sixth backcross generation (BC6), selfing every generation to screen for resistance to \u003cem\u003eM. incognita\u003c/em\u003e. After six backcross generations and progeny testing, the resulting cultivars \u0026lsquo;Nemadoro\u0026rsquo; (homozygous dominant; \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and \u0026lsquo;Del Rey\u0026rsquo; (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) were considered as near-isogenic lines (NILs) of Rio Grande\u0026rsquo; and \u0026lsquo;Calipso\u0026rsquo;, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003einoculation bioassays\u003c/span\u003e \u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eemploying contrasting tomato NILs for the locus\u003c/span\u003e \u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003eM\u003c/span\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003e-\u003c/span\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e1.2\u003c/span\u003e \u003cstrong\u003e\u0026ndash;\u003c/strong\u003e \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e inoculum was obtained from a pure culture multiplied by periodic subculturing on plants of the tomato cultivar \u0026lsquo;Santa Cruz\u0026rsquo;. This RKN population was maintained in a greenhouse (25\u0026ndash;30 \u003csup\u003eo\u003c/sup\u003eC), being previously identified by esterase and SCAR phenotyping (Gabriel et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Two bioassays were carried out under greenhouse conditions at Embrapa Vegetables Crops (Brasilia\u0026ndash;DF, Brazil) in two distinct periods: November/2022 to January/2023 and February/2024 to April/2024. The average air temperature in the first assay was 30.1 ℃ (39.2 ℃ maximum and 16.1 ℃ minimum), while in the second experiment the average air temperature was 27.2 ℃ (31.7 ℃ maximum and 15.5 ℃ minimum). The bioassays were conducted in completely randomized design with four treatments (two pairs contrasting NILs) and six replicates (Tables \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean values of gall index (GI) and egg mass index (EMI) induced by \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e (guava race) on the root systems of contrasting tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) near isogenic lines (NILs) for the presence/absence of the \u003cem\u003eMi\u003c/em\u003e-1.2 locus. Evaluation was carried out under greenhouse conditions at 45 days after inoculation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003eNILs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 11.7167%;\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 20.309%;\"\u003e\n \u003cp\u003eGI\u003csup\u003e1*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 20.2222%;\"\u003e\n \u003cp\u003eEMI\u003csup\u003e2*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003e\u003cem\u003eMi\u003c/em\u003e-1.2 gene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003eExperiment I\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003eExperiment II\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003eExperiment I\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003eExperiment II\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003e\u0026lsquo;Nemadoro\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003ePresent homozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003e\u0026lsquo;Rio Grande\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003e\u0026lsquo;Del Rey\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003ePresent homozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003e\u0026lsquo;Calipso\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003eAbsent homozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003e\u0026lsquo;Rutgers\u0026rsquo; (control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\n \u003cp\u003eAbsent homozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003eAverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.3696%;\"\u003e\n \u003cp\u003eCV (%)\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.7167%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.0677%;\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.9809%;\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 10.2413%;\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 64.4853%;\"\u003e*Means (n\u0026thinsp;=\u0026thinsp;6) were not significantly different across pairs of NILs according to the Scott\u0026ndash;Knott test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003csup\u003e1\u003c/sup\u003eGI and \u003csup\u003e2\u003c/sup\u003eEMI: grades 1\u0026ndash;5, according to Taylor and Sasser (\u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e). The pairs of contrasting NILs were \u0026lsquo;Nemadoro\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e and \u0026lsquo;Rio Grande\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e and \u0026lsquo;Del Rey\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e and \u0026lsquo;Calipso\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e. The cultivar \u0026lsquo;Rutgers\u0026rsquo; was employed as susceptible control. CV (%)\u003csup\u003e5\u003c/sup\u003e = coefficient of variation.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of eggs\u0026thinsp;+\u0026thinsp;occasional second stage juveniles per gram of roots (\u003cstrong\u003eNEGR\u003c/strong\u003e) and nematode reproduction factor (\u003cstrong\u003eRF\u003c/strong\u003e) of \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e on contrasting near isogenic lines (NILs) of tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) for the presence/absence of the \u003cem\u003eMi\u003c/em\u003e-1.2 locus. Evaluation was carried out under greenhouse conditions at 45 days after inoculation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003eNILs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.0546%;\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 20.8893%;\"\u003e\n \u003cp\u003eNEGR\u003csup\u003e1*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 20.8893%;\"\u003e\n \u003cp\u003eRF\u003csup\u003e2*\u003c/sup\u003e/NIL reaction\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003e\u003cem\u003eMi\u003c/em\u003e-1.2 gene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003eExperiment I\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003eExperiment II\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003eExperiment I\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003eExperiment II\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003e\u0026lsquo;Nemadoro\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e1193 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e14783 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e15.83 c / S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e38.38 a / S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003e\u0026lsquo;Rio Grande\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e623 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e5049 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e11.59 c / S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e13.00 c / S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003e\u0026lsquo;Del Rey\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e2610 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e4946 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e47.19 b / S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e11.00 c / S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003e\u0026lsquo;Calipso\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e957 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e2290 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e11.66 c / S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e21.75 b / S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003e\u0026lsquo;Rutgers\u0026rsquo; (control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ehomozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e3608 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e13130 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e70.63 a / S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e43.50 a / S\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003eAverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e1798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e8040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e31.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e26.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 11.7015%;\"\u003e\n \u003cp\u003eCV (%)\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.0546%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e33.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.3146%;\"\u003e\n \u003cp\u003e42.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 10.5747%;\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 64.4015%;\"\u003e*The data were transformed to \u0026radic;x\u0026thinsp;+\u0026thinsp;0.5 for analysis but are presented without transformation. Means (n\u0026thinsp;=\u0026thinsp;6) followed by different letters lowercase in the columns is significantly different, according to the Scott\u0026ndash;Knott test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003csup\u003e1\u003c/sup\u003eEggs + occasional second stage juveniles per gram of roots (NEGR)\u0026thinsp;=\u0026thinsp;final population / fresh weight of root (g), \u003csup\u003e2\u003c/sup\u003eRF = final population/2000 eggs\u0026thinsp;+\u0026thinsp;J2 of \u003cem\u003eM. enterolobii\u003c/em\u003e; \u003csup\u003e3\u003c/sup\u003eReaction of inoculated plants, RF\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026thinsp;=\u0026thinsp;suitable host (S) and RF\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026thinsp;=\u0026thinsp;poor host (P) and RF\u0026thinsp;=\u0026thinsp;0 immune (I) (Oostenbrink, \u003cspan class=\"CitationRef\"\u003e1966\u003c/span\u003e). CV\u0026thinsp;=\u0026thinsp;coefficient of variation. The pairs of NILs were \u0026lsquo;Nemadoro\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e versus \u0026lsquo;Rio Grande\u0026rsquo;\u003csup\u003e3\u003c/sup\u003e and \u0026lsquo;Dell Rey\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e versus \u0026lsquo;Calipso\u0026rsquo;\u003csup\u003e4\u003c/sup\u003e. The cultivar \u0026lsquo;Rutgers\u0026rsquo; was employed as susceptible control. **NIL Reaction: S (susceptible).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eA distinct set of plants of the NIL \u0026lsquo;Nemadoro\u0026rsquo; (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and \u0026lsquo;Heinz 1706\u0026rsquo; was inoculated with an \u003cem\u003eMi\u003c/em\u003e-1.2-avirulent population of \u003cem\u003eM. incognita\u003c/em\u003e race 1 aiming to verify if the resistance reaction conferred by this gene could be affected environmental conditions similar to that of the experiments with \u003cem\u003eM. enterolobii\u003c/em\u003e. The inoculum of the \u003cem\u003eM. incognita\u003c/em\u003e race 1 population was obtained also from a pure culture multiplied by periodic subculturing on plants of the tomato cultivar \u0026lsquo;Santa Cruz\u0026rsquo;. This RKN population was maintained under greenhouse conditions (25\u0026ndash;30 \u003csup\u003eo\u003c/sup\u003eC), being previously identified through morphological features as well as through esterase phenotyping I2 (Rm: 0.39 and 0.42). This \u003cem\u003eM. incognita\u003c/em\u003e population was identified as race 1 based upon its ability to reproduce in pepper (\u0026lsquo;Califonia Wonder\u0026rsquo;), watermelon (\u0026lsquo;Charleston Gray\u0026rsquo;) and tomato (\u0026lsquo;Rutgers\u0026rsquo;) and by its inability to infect cotton (\u0026lsquo;Deltapine\u0026rsquo;), tobacco (\u0026lsquo;NC95\u0026rsquo;) and peanut (\u0026lsquo;Florunner\u0026rsquo;) (Hartman \u0026amp; Sasser, 1985). The bioassay with \u003cem\u003eM. incognita\u003c/em\u003e race 1 inoculum was conducted in February/2024 to April/2024. The experiment was carried out in a completely randomized design with two treatments (\u0026lsquo;Nemadoro\u0026rsquo; and \u0026lsquo;Heinz 1706\u0026rsquo;) and six replicates. The average air temperature in the assay was 27.2 ℃ (31.7 ℃ maximum and 15.5 ℃ minimum).\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eInoculation procedures\u003c/span\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e Eggs\u0026thinsp;+\u0026thinsp;occasional J2 juveniles of the \u003cem\u003eM. enterolobii\u003c/em\u003e and \u003cem\u003eM. incognita\u003c/em\u003e race 1 populations were extracted from the infected roots, according to Hussey and Barker (\u003cspan class=\"CitationRef\"\u003e1973\u003c/span\u003e) modified by Bonetti and Ferraz (\u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e). The roots were washed with tap water, and nematode eggs in the egg masses were extracted using 0.5% sodium hypochlorite. Roots were cut into 1\u0026ndash;2 cm-long pieces, mixed with sodium hypochlorite, shaken for 30 seconds, washed with tap water, and collected using a 500-mesh screen. The total number of eggs per milliliter was quantified under a light microscope (Nikon Eclipse 80i model) using a nematode-counting slide (Peter\u0026rsquo;s slide). Twenty days after sowing, the tomato seedlings were transplanted to 2000 cm\u003csup\u003e3\u003c/sup\u003e plastic pots with a diameter of 12 cm (filled with sterilized soil and sand in a 2:1 ratio). Plants were subsequently inoculated with 2,000 eggs\u0026thinsp;+\u0026thinsp;occasional J2 by placing 2 mL of the suspension into three holes with 2 cm deep around the plants. The tomato cultivar \u0026lsquo;Rutgers\u0026rsquo; was employed as susceptible control in the assays with \u003cem\u003eM. enterolobii\u003c/em\u003e population and, as previously mentioned, the cultivar \u0026lsquo;Heinz 1706\u0026rsquo; was employed as susceptible control in the assay with the \u003cem\u003eM. incognita\u003c/em\u003e race 1 population.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eMolecular genotyping of individual plants for Confirmation of the presence/Absence of the\u003c/span\u003e \u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003eM\u003c/span\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cspan type=\"BoldItalicSmallCaps\" class=\"BoldItalicSmallCaps\" name=\"Emphasis\"\u003e-\u003c/span\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003e1.2 locus\u003c/span\u003e \u003cstrong\u003e\u0026ndash;\u003c/strong\u003e The genomic DNA was extracted from each evaluated plant in order to confirm the presence/absence of the \u003cem\u003eMi\u003c/em\u003e-1.2 gene/locus. For nucleic acid purification of young apical leaves, a modified 2X CTAB buffer (pH\u0026thinsp;=\u0026thinsp;8.0) and organic solvents methodology was employed (Boiteux et al., \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). The DNA pellet was carefully washed with 70% ethanol. The micro centrifuge tubes were then placed in an incubator at 37\u0026deg;C for 20 minutes and the solution resuspended in 100 \u0026micro;L of Tris-EDTA buffer\u0026thinsp;+\u0026thinsp;RNase. The purified samples were vortexed and stored in a freezer (-20\u0026deg;C). PCR was performed with the primer pair \u0026lsquo;Mi23F\u0026rsquo; (5\u0026rsquo;\u0026ndash;TGG AAA AAT GTT GAA TTT CTT TTG\u0026ndash;3\u0026rsquo;) and \u0026lsquo;Mi23R\u0026rsquo; (5\u0026rsquo;\u0026ndash;GCA TAC TAT ATG GCT TGT TTA CCC\u0026ndash;3\u0026rsquo;), considered the most reliable codominant molecular marker for detecting contrasting alleles at the \u003cem\u003eMi\u003c/em\u003e-1.2 gene/locus (Seah et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Bhavana et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The final volume of the mixture was 25 \u0026micro;L (23 \u0026micro;L master mix\u0026thinsp;+\u0026thinsp;2 \u0026micro;L DNA). The master mix consisted of 11.32 \u0026micro;L milliQ water, 2.5 \u0026micro;L of 10\u0026times; \u003cem\u003eTaq\u003c/em\u003e polymerase buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl), 5.7 \u0026micro;L of dNTPs (0.5 mM, Invitrogen), 0.5 \u0026micro;L of each primer (0.5 mM), 2.3 \u0026micro;L of MgCl\u003csub\u003e2\u003c/sub\u003e (25 mM), and 0.18 \u0026micro;L of \u003cem\u003eTaq\u003c/em\u003e DNA polymerase (5 U/\u0026micro;L, Invitrogen). Amplifications were performed with the following parameters: an initial denaturation step at 94\u0026deg;C for 2 minutes; 40 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 59\u0026deg;C for 1 minute, extension at 72\u0026deg;C for 1.5 minutes; and a final extension step at 68\u0026deg;C for 10 minutes. Bromophenol blue (5 \u0026micro;L) was added to each sample, and the amplified DNA fragments were separated by electrophoresis in TBE buffer (40 mM Tris-borate, 1 mM EDTA) on a 1.7% agarose gel supplemented with ethidium bromide, at 80 V for approximately 4 to 5 hours. The gels were photographed under UV light (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The 1kb Plus DNA Ladder (Invitrogen) was used to locate the amplification profile.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003eSample processing and statistical analysis\u003c/span\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e At sixty days after inoculation, the roots were carefully removed from the soil, rinsed with tap water, and dried with a paper towel. The roots were then stained with floxin B (15 mg/L water) (Daykin and Hussey \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e) to facilitate the visualization of the nematode\u0026apos;s external egg masses, which were quantified with the help of a magnifying glass, assessing the gall and egg mass indices. The following qualitative parameters were evaluated: gall index (\u003cstrong\u003eGI\u003c/strong\u003e) and egg mass index (\u003cstrong\u003eEMI\u003c/strong\u003e) (Taylor and Sasser, \u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e). The quantitative parameter \u003cstrong\u003eNEGR\u003c/strong\u003e (number of eggs\u0026thinsp;+\u0026thinsp;occasional J2 juveniles per gram of root) was also estimated by counting nematode structures in a Peter\u0026rsquo;s counting chamber under an optical microscope (Nikon Eclipse 80i model). The quantitative parameter nematode reproduction factor\u0026ndash;\u003cstrong\u003eRF\u003c/strong\u003e = (final population/initial population) was also calculated (Oostenbrink, \u003cspan class=\"CitationRef\"\u003e1966\u003c/span\u003e). Individual plants were rated according to their reaction to the nematode as immune (\u003cstrong\u003eRF\u0026thinsp;=\u0026thinsp;0\u003c/strong\u003e), resistant/poor host (\u003cstrong\u003eRF\u0026thinsp;\u0026lt;\u0026thinsp;1\u003c/strong\u003e) or susceptible/suitable host (\u003cstrong\u003eRF\u0026thinsp;\u0026gt;\u0026thinsp;1\u003c/strong\u003e), with. Data were transformed \u0026radic;x\u0026thinsp;+\u0026thinsp;05 for statistical analysis, that is, normality test, analysis of variance, and Scott\u0026ndash;Knott test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The data were back-transformed to display the original values.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe contrasting NILs for the \u003cem\u003eMi\u003c/em\u003e-1.2 locus reacted differently to \u003cem\u003eM. enterolobii\u003c/em\u003e as inferred from the qualitative parameters of gall index (\u003cb\u003eGI\u003c/b\u003e) and egg mass index (\u003cb\u003eEMI\u003c/b\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and by the quantitative parameters \u003cb\u003eNEGR\u003c/b\u003e and \u003cb\u003eRF\u003c/b\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The homozygous dominant (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) NILs displayed values for the parameter \u003cb\u003eNEGR\u003c/b\u003e similar or even superior to their corresponding recessive (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) NILs \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All the contrasting NILs showed \u003cb\u003eRF\u0026thinsp;\u0026gt;\u0026thinsp;1.0 (\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, they can be categorized as susceptible/suitable hosts of \u003cem\u003eM. enterolobii\u003c/em\u003e (Oostenbrink, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). In the present study, the susceptible standard (cultivar \u0026lsquo;Rutgers\u0026rsquo;) displayed the highest RF in the bioassays I and II (70.63 and 43.50, respectively), indicating proper experimental conditions for \u003cem\u003eM. enterolobii\u003c/em\u003e development and reproduction (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, \u0026lsquo;Rutgers\u0026rsquo; displayed significantly higher \u003cem\u003eM. enterolobii\u003c/em\u003e reproduction even in comparison with the two susceptible NILs \u0026lsquo;Rio Grande\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) and \u0026lsquo;Calipso\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe inoculation assay with the \u003cem\u003eM. incognita\u003c/em\u003e race 1 population displayed GI and EMI values equal to 1 and RF value of 0.1 for the NIL \u0026lsquo;Nemadoro\u0026rsquo; (homozygous, \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating the effectiveness of the resistance gene against this avirulent population under similar environmental conditions of the assays carried out with \u003cem\u003eM. enterolobii\u003c/em\u003e. The susceptible standard (cultivar \u0026lsquo;Heinz 1706\u0026rsquo;) displayed GI, EMI, and RF values of 5.0, 4.18, 44.20, respectively, indicating the inoculum viability of the \u003cem\u003eM. incognita\u003c/em\u003e race 1 population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PCR analyses with the specific primer pair Mi23F/Mi23R using as template DNA extracted from each individual plant of the contrasting NILs under evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), yielded only amplicons of 380 bp in \u0026lsquo;Nemadoro\u0026rsquo; and \u0026lsquo;Del Rey\u0026rsquo;, confirming that the dominant allele is fixed in these NILs (i.e. all plants displaying the dominant \u003cem\u003eMi\u003c/em\u003e-1.2 gene in homozygous condition), whereas PCR assays with their corresponding contrasting NILs (\u0026lsquo;Rio Grande\u0026rsquo; and \u0026lsquo;Calipso\u0026rsquo;) yielded only amplicons of 430 bp, confirming their homozygous configuration for the recessive allele (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2).\u003c/p\u003e \u003cp\u003eThe NIL \u0026lsquo;Nemadoro\u0026rsquo; showed RFs of 15.83 and 38.38 in bioassays I and II, respectively, whereas its contrasting NIL (\u0026lsquo;Rio Grande\u0026rsquo;), showed RFs of 11.59 and 13.00 in bioassays I and II, respectively. Likewise, the cultivar \u0026lsquo;Del Rey\u0026rsquo; (homozygous \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) exhibited a higher RF (47.19) in comparison to its corresponding NIL \u0026lsquo;Calipso\u0026rsquo;, with displayed FR of 11.66 in bioassay I. However, a slight positive impact of the resistance gene in the reproduction factor (RF) value was observed in bioassay II for \u0026lsquo;Del Rey\u0026rsquo; (RF\u0026thinsp;=\u0026thinsp;11.00) in relation to \u0026lsquo;Calipso\u0026rsquo; (RF\u0026thinsp;=\u0026thinsp;21.75).\u003c/p\u003e \u003cp\u003eThe GI and EMI are auxiliary semi-quantitative/qualitative parameters that can help estimate plant resistance to \u003cem\u003eMeloidogyne\u003c/em\u003e infections. Generally, a correlation exists between these indices and RF. Herein, most GI and EMI were correlated with RF. For example, the NILs \u0026lsquo;Rio Grande\u0026rsquo; and \u0026lsquo;Del Rey\u0026rsquo; displayed lower GI and EMI values in bioassay II (2.0 and 2.3, respectively), and their FR values were 13.00 and 11.00, respectively (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the auxiliary parameters GI and EMI were also in agreement with the observation that the \u003cem\u003eMi\u003c/em\u003e-1.2 gene does not influence \u003cem\u003eM. enterolobii\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFrom the tomato breeding standpoint, the \u003cem\u003eMi\u003c/em\u003e-1.2 is a unique gene able to control resistant reactions to populations of 13 \u003cem\u003eMeloidogyne\u003c/em\u003e species (Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and to three species of Hempiteran insects, including the aphid \u003cem\u003eMacrosiphum euphorbiae\u003c/em\u003e, the whiteflies of the \u003cem\u003eBemisia tabaci\u003c/em\u003e complex, and the psyllid \u003cem\u003eBactericerca cockerelli\u003c/em\u003e (Rossi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Vos et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Nombela et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kaloshian \u0026amp; Teixeira, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Even though the \u003cem\u003eMi\u003c/em\u003e-1.2 might display negative effects on the beneficial biocontrol agent, \u003cem\u003eOrius insidiosus\u003c/em\u003e (Pallipparambil et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), its ability to control a wide array of pests and pathogens makes this gene a central component in integrated pest management in the tomato crop. In this context, it is important to assess if this gene might have some residual effects on virulent RKN species and populations that are able to \u0026lsquo;break-down\u0026rsquo; this resistance such as \u003cem\u003eM. enterolobii\u003c/em\u003e (Castagnone-Sereno, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pinheiro et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Philbrick et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gabriel et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). To address this question, we evaluated the reaction of contrasting NILs with very similar genetic background but differing for presence/absence of the \u003cem\u003eMi\u003c/em\u003e-1.2-carrying locus.\u003c/p\u003e \u003cp\u003eThe molecular-marker analyses in association with the simultaneous bioassays with contrasting pairs of NILs reinforce the notion that the \u003cem\u003eMi\u003c/em\u003e-1.2 gene does not confer significant residual effects on this \u003cem\u003eM. enterolobii\u003c/em\u003e (guava race) population. Our assays with the contrasting pair of NILs \u0026lsquo;Nemadoro\u0026rsquo; (homozygous \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and \u0026lsquo;Rio Grande\u0026rsquo; (homozygous \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2), showed similar RF values across the two bioassays. These results were also consistent with the reactions of one alternative pair of contrasting NILs (\u0026lsquo;Del Rey\u0026rsquo; and \u0026lsquo;Calipso\u0026rsquo;). The NIL \u0026lsquo;Del Rey\u0026rsquo; (homozygous \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) exhibited either similar or even a higher RF in comparison to the corresponding NIL \u0026lsquo;Calipso\u0026rsquo; (homozygous \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) in the first bioassay, even though a slight (but statistically significant) positive impact of the resistance gene in RF value was observed only in bioassay II.\u003c/p\u003e \u003cp\u003eOur results are not in agreement with previous investigations involving the comparison of the susceptible cultivar \u0026lsquo;Rutgers\u0026rsquo; (homozygous \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) with the cultivars \u0026lsquo;Nemadoro\u0026rsquo; and \u0026lsquo;Tospodoro\u0026rsquo; (both homozygous for the \u003cem\u003eMi\u003c/em\u003e-1.2 gene). \u0026lsquo;Rutgers\u0026rsquo; displayed significantly higher RF, suggesting a residual effect of this gene against distinct \u003cem\u003eM. enterolobii\u003c/em\u003e populations (Pinheiro et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Verssiani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pinto et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Even before these studies, Brito et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) also detected a putative significant residual effect (approximately 80%) of the cultivar \u0026lsquo;Sanibel\u0026rsquo; (carrying the \u003cem\u003eMi\u003c/em\u003e-1.2 gene) to a group of isolates of \u003cem\u003eM. mayaguensis\u003c/em\u003e (=\u0026thinsp;\u003cem\u003eM. enterolobii\u003c/em\u003e) when comparing with \u0026lsquo;Rutgers\u0026rsquo;. In addition, data presented by Gabriel et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) indicated lower levels of susceptibility in the hybrid \u0026lsquo;Debora Plus\u0026rsquo; (heterozygous \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) when compared to the cultivar \u0026lsquo;Santa Clara\u0026rsquo; (homozygous \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) in response to a Brazilian population of \u003cem\u003eM. enterolobii\u003c/em\u003e from guava. These contradictory results can be explained either by distinct virulence profiles or by the variable levels of aggressiveness observed across isolates of the cotton and guava races of \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eenterolobii\u003c/em\u003e (Verssiani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, it is important to point out that all these comparative studies were carried out with cultivars/accessions displaying different genetic backgrounds. In the present study, the susceptible standard (cultivar \u0026lsquo;Rutgers\u0026rsquo;) displayed significantly higher levels susceptibility to \u003cem\u003eM. enterolobii\u003c/em\u003e even when compared to the two susceptible NILs \u0026lsquo;Rio Grande\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) and \u0026lsquo;Calipso\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2). This reinforces the observation that comparison between genetically unrelated accessions with distinct backgrounds, even in the absence of the \u003cem\u003eMi\u003c/em\u003e-1.2, can induce misleading interpretations of the putative residual effects of this gene. Therefore, we cannot exclude in these previous reports, the potential contribution of other genes, in addition to \u003cem\u003eMi\u003c/em\u003e-1.2, that might influence the levels of resistance/susceptibility to this pathogen, making difficult to assess with confidence the putative residual effects of this gene.\u003c/p\u003e \u003cp\u003eIn this regard, our employment of contrasting NILs in the present study either eliminates or minimizes the genetic \u0026ldquo;noise\u0026rdquo; caused by unrelated background variability when the comparison in done with lines/cultivars contrasting not only for the gene/locus under evaluation. In our study, we could provide a stronger test of this \u0026lsquo;residual effect hypothesis\u0026rsquo; since we carry out a comparative analysis with pairs of NILs, sharing similar genetic background: \u0026lsquo;Nemadoro\u0026rsquo; (\u003cem\u003eMi\u003c/em\u003e-1.2/ \u003cem\u003eMi\u003c/em\u003e-1.2) versus its NIL \u0026lsquo;Rio Grande\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) as well as \u0026lsquo;Del Rey\u0026rsquo; (homozygous \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) and its corresponding NIL \u0026lsquo;Calipso\u0026rsquo; (homozygous \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2). With if few exceptions, no significant differences were observed across the contrasting NILs for a subset of quantitative parameters in both bioassays, providing evidence that \u003cem\u003eMi\u003c/em\u003e-1.2 gene do not display any residual effect against \u003cem\u003eM. enterolobii.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eHerein, we can exclude the potential negative impacts of the environmental conditions established on the efficient expression \u003cem\u003eMi\u003c/em\u003e-1.2 gene as indicated by it highly resistant reaction of the NIL \u0026lsquo;Nemadoro\u0026rsquo; against a well-known avirulent population of \u003cem\u003eM. incognita\u003c/em\u003e race 1. As expected, \u003cem\u003eM. incognita\u003c/em\u003e race 1 was unable to overcome the resistance conferred by the \u003cem\u003eMi\u003c/em\u003e-1.2 gene (see Gabriel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The cultivar \u0026lsquo;Nemadoro\u0026rsquo; displayed very low GI, IMO, and RF values. This result allowed us to confirm that environmental components were not able to interfere with the \u003cem\u003eMi\u003c/em\u003e-1.2 gene expression under our experimental conditions\u003c/p\u003e \u003cp\u003eThe confirmation of the complete \u0026lsquo;break-down\u0026rsquo; of the \u003cem\u003eMi\u003c/em\u003e-1.2 gene with no significant residual effect against \u003cem\u003eM. enterolobii\u003c/em\u003e will intensify the need to search for novel sources of genetic resistance effective against this RKN species. However, searches for sources of resistance to \u003cem\u003eM. enterolobii\u003c/em\u003e have not been very successful thus far. Besides the \u003cem\u003eMi\u003c/em\u003e-1.2, nine distinct genes of resistance to \u003cem\u003eMeloidogyne\u003c/em\u003e species have been identified in accessions of \u003cem\u003eSolanum\u003c/em\u003e (\u003cem\u003eLycopersicon\u003c/em\u003e) (Williamson \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). However, four of these genes (\u003cem\u003eMi\u003c/em\u003e-3, \u003cem\u003eMi\u003c/em\u003e-5, \u003cem\u003eMi\u003c/em\u003e-9, and \u003cem\u003eMi\u003c/em\u003e-HT) were found to be ineffective against \u003cem\u003eM. enterolobii\u003c/em\u003e populations (Williamson \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; El-Sappah et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A collection of 101 commercial and wild tomatoes was screened in a search resistance/tolerance to \u003cem\u003eM. enterolobii\u003c/em\u003e (guava race) population, but no useful sources were detected (Silva et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne additional problem related to \u003cem\u003eM. enterolobii\u003c/em\u003e management is the fact that this RKN is able to break-down a wide array of resistance sources of different host species (Pinheiro et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Koutsovoulos et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pinto et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), including the \u003cem\u003eN\u003c/em\u003e gene in \u003cem\u003eCapsicum annuum\u003c/em\u003e, the \u003cem\u003eRk\u003c/em\u003e gene in \u003cem\u003eVigna unguiculata\u003c/em\u003e, the \u003cem\u003eMir\u003c/em\u003e1 gene in \u003cem\u003eGlycine max\u003c/em\u003e, the \u003cem\u003eMh\u003c/em\u003e gene in \u003cem\u003eS. tuberosum\u003c/em\u003e, and the \u003cem\u003eMi\u003c/em\u003e-1 gene in \u003cem\u003eGossypium hirsutum\u003c/em\u003e, and in sources carrying \u003cem\u003eMh\u003c/em\u003e gene in \u003cem\u003eIpomoea batatas\u003c/em\u003e (Fery et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Thies \u0026amp; Fery, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Williamson \u0026amp; Roberts, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Castagnone-Sereno, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Quenouille et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Verssiani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Collett et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schwarz, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This feature of \u003cem\u003eM. enterolobii\u003c/em\u003e populations limits the crop rotation alternatives.\u003c/p\u003e \u003cp\u003eAlternative measures for the genetic management of \u003cem\u003eM. enterolobii\u003c/em\u003e could be implemented, including grafting tomato cultivars onto resistant rootstocks (Louws et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Schwarz et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Baidya et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pinheiro et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) as well as biotech-based approaches (Claverie et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhuo et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; El-Sappah et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Manivannan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The continuous investigation into the genetic and molecular mechanisms by which \u003cem\u003eM. enterolobii\u003c/em\u003e circumvents resistance will be also a crucial piece of information (Sato et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Szitenberg et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Koutsovoulos et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, the evaluation of contrasting NILs for the \u003cem\u003eMi-\u003c/em\u003e1.2 locus indicated that all are susceptible to \u003cem\u003eM. enterolobii\u003c/em\u003e, displaying similar responses to all parameters (including RF). Therefore, \u003cem\u003eMi\u003c/em\u003e-1.2 gene, although extremely effective against at least 13 \u003cem\u003eMeloidogyne\u003c/em\u003e species, is not able of imposing any significant interference with the infection process of \u003cem\u003eM. enterolobii\u003c/em\u003e in tomato. Therefore, search for effective sources of resistance to this pathogen can be considered as a major tomato breeding priority.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge EMBRAPA Hortali\u0026ccedil;as for the support in all steps of this research\u003cbr\u003e\u0026nbsp;the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) \u0026ndash; Finance Code 001\u003cbr\u003e\u0026nbsp;\u0026ndash; for providing scholarships to Dwillian F. Cunha and Th\u0026aacute;vio J. B. Pinto and the Conselho Nacional de Desenvolvimento\u003cbr\u003e\u0026nbsp;Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) for fellowships and for partially funding the research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUniversity of Bras\u0026iacute;lia (UnB), Institute of Biological Sciences, Department of Plant Pathology, Bras\u0026iacute;lia\u0026ndash;DF, 70910-900, Brazil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDwillian Firmiano Cunha, Th\u0026aacute;vio J\u0026uacute;nior Barbosa Pinto, Juvenil Enrique Cares \u0026amp; Leonardo Silva Boiteux\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmbrapa Vegetable Crops (Hortali\u0026ccedil;as), Bras\u0026iacute;lia\u0026ndash;DF, 70359-970, Brazil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJadir Borges Pinheiro, Maria Esther de Noronha Fonseca \u0026amp; Leonardo Silva Boiteux\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmbrapa Hortali\u0026ccedil;as, Esta\u0026ccedil;\u0026atilde;o Experimental Canoinhas, Canoinhas\u0026ndash;SC, 89460-000, Brazil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiovani Oleg\u0026aacute;rio da Silva\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCentro Universit\u0026aacute;rio ICESP, Bras\u0026iacute;lia\u0026ndash;DF, 71961-540, Brazil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFelipe Santos Rafael \u0026amp; Leandro Alves Santos\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDwillian F. Cunha; Th\u0026aacute;vio J.B. Pinto; Felipe S. Rafael; Leandro A. Santos:\u0026nbsp;\u003c/strong\u003eInvestigation, Formal analysis.\u0026nbsp;\u003cstrong\u003eLeonardo S. Boiteux;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eJadir B. Pinheiro; Juvenil E. Cares:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing - original draft, Supervision, Funding acquisition, Project administration.\u0026nbsp;\u003cstrong\u003eTh\u0026aacute;vio J.B. Pinto; Dwillian F. Cunha; Maria Esther N. Fonseca; Giovani O. Silva\u003c/strong\u003e\u003cstrong\u003e; Jadir B. Pinheiro; Leonardo S. Boiteux; Juvenil E. Cares:\u0026nbsp;\u003c/strong\u003eMethodology,Data curation, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to \u003cu\u003eDwillian F. Cunha\u003c/u\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data supporting the findings of this study are available in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaidya, S., Timila, R.D., Bahadur, K.C.R., Manandhar, H.K., \u0026amp; Manandhar, C. (2017). Management of root-knot nematode on tomato through grafting rootstock of \u003cem\u003eSolanum sisymbriifolium\u003c/em\u003e. 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Starr.Wallingford: CABI Publishing), pages 301\u0026ndash;325. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/9781845934927.0301\u003c/span\u003e\u003cspan address=\"10.1079/9781845934927.0301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuo, K., Chen, J., Lin, B., Wang, J., Sun, F., Hu, L., \u0026amp; Liao, J. (2017). A novel \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e effector MeTCTP promotes parasitism by suppressing programmed cell death in host plants. Molecular Plant Pathology 18, 45\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mpp.12374\u003c/span\u003e\u003cspan address=\"10.1111/mpp.12374\" 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":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"root-knot nematode, defeated resistance gene, resistance, Solanum lycopersicum","lastPublishedDoi":"10.21203/rs.3.rs-5364816/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5364816/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMeloidogyne enterolobii\u003c/em\u003e represents a major threat to the global tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) production due to its ability to \u0026ldquo;break-down\u0026rdquo; the resistance conferred by the dominant \u003cem\u003eMi\u003c/em\u003e-1.2 gene. However, a subgroup of \u0026ldquo;defeated\u0026rdquo; resistance genes in various pathosystems exhibits residual effects characterized by an enduring interference in quantitative levels of disease expression induced by novel virulent pathogens. Thus far, residual effects of the \u0026ldquo;defeated\u0026rdquo; \u003cem\u003eMi\u003c/em\u003e-1.2 gene to \u003cem\u003eM. enterolobii\u003c/em\u003e have not been properly investigated. Herein, two comparative assays using contrasting near-isogenic lines (NILs) for presence/absence of the \u003cem\u003eMi\u003c/em\u003e-1.2 locus were carried out using a guava race population of \u003cem\u003eM. enterolobii\u003c/em\u003e. Seedlings of two pairs of contrasting NILs \u0026lsquo;Nemadoro\u0026rsquo; (homozygous dominant; \u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) / \u0026lsquo;Rio Grande\u0026rsquo; (homozygous recessive, \u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) and \u0026lsquo;Del Rey\u0026rsquo; (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) / \u0026lsquo;Calipso\u0026rsquo; (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) were inoculated with \u0026asymp; 2,000 \u003cem\u003eM. enterolobii\u003c/em\u003e eggs. The homozygous dominant (\u003cem\u003eMi\u003c/em\u003e-1.2/\u003cem\u003eMi\u003c/em\u003e-1.2) NILs displayed values for the quantitative parameter NEGR (number of eggs\u0026thinsp;+\u0026thinsp;occasional J2 per gram of root tissue) similar or even superior to their corresponding recessive (\u003cem\u003emi\u003c/em\u003e-1.2/\u003cem\u003emi\u003c/em\u003e-1.2) NILs. A slight positive impact of the resistance gene in the reproduction factor (RF) value was observed only for one pair of contrasting NILs (\u0026lsquo;Del Rey\u0026rsquo; / \u0026lsquo;Calipso\u0026rsquo;), which was restricted to one bioassay. The employment of NILs in our bioassays allowed us to hypothesize that the \u003cem\u003eMi\u003c/em\u003e-1.2 gene, although extremely effective against populations of at least 13 \u003cem\u003eMeloidogyne\u003c/em\u003e species, does not confer significant residual effects against \u003cem\u003eM. enterolobii\u003c/em\u003e race from guava.\u003c/p\u003e","manuscriptTitle":"Assessing the residual effects of the “defeated” tomato Mi-1.2 gene against a Meloidogyne enterolobii (guava race) population via comparative assays with contrasting near-isogenic lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 06:33:35","doi":"10.21203/rs.3.rs-5364816/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b93346f3-ef05-4d8d-8459-bd7083c62ade","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-08T18:24:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-05 06:33:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5364816","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5364816","identity":"rs-5364816","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}