Solanum linnaeanum and Solanum sisymbriifolium as a sustainable strategy for the management of Meloidogyne chitwoodi

Root-knot nematodes (RKN), Meloidogyne spp., are important crop pests that cause severe losses in crop production worldwide, reducing both productivity and crop quality. Meloidogyne chitwoodi Golden, O'Bannon, Santo & Finley, 1980 is considered a quarantine organism by the European and Mediterranean Plant Protection Organization (EPPO) causing damage in tomato and potato crops. The development of nonchemical and sustainable management strategies to reduce nematode damage is crucial. The resistance of Solanum linnaeanum Hepper & P.-M.L. Jaeger and S. sisymbriifolium Lamarck cv. Sis 6001 to M. chitwoodi was evaluated based on gall index (GI), the Bridge & Page (1980) rating chart and reproduction factor (RF). Both plant species were resistant to M. chitwoodi. Solanum linnaeanum had an average of 519 small root swellings/plant, with 45% adult nematodes inside the roots, all males. Solanum sisymbriifolium had GI ≤ 2 and RF ≤ 1 with a high percentage (69%) of nematodes inside the roots that did not develop beyond the sexually undifferentiated second-stage. The use of S. linnaeanum as a new source of resistance is a good alternative for the control of RKN in the quest to develop nonchemical and sustainable management strategies to protect crops.

www.nature.com/scientificreports/ and European legislation (Directive 69/465/CEE; Directive 2009/128/EC) is very strict regarding the use of nematicides in the field, focusing mainly on environmental safety issues and health risks. The increase of environmental concerns and regulatory restrictions on the application of chemical products in conventional systems have meant that the use of plant resistance to control PPN has increased. The most effective, environmentally friendly, and economical means of controlling Meloidogyne spp. is the use of resistant cultivars 10 . However, up to now, there are no potato cultivars resistant to RKN 9 .
Therefore, other control measures are being developed, including the use of plants as trap crops as an alternative to chemical pesticides [17][18][19][20] . A trap crop is a plant species attractive to pests from another crop but in which the pest fails to survive or reproduce. Many wild Solanum species display resistance to Meloidogyne spp., although the resistance is often not complete, and the nematodes may form a few galls or eggs. A potentially useful source of resistance occurs in S. sisymbriifolium Lamarck 21,22 .
Solanum sisymbriifolium, originates from warm, temperate South America, is an annual or perennial erect, rhizomatous, shrubby weed with an extensive root system and spiny leaves, currently distributed throughout the world and invasive in some countries. Interest in the study of this plant has increased since it was proved to be a good trap crop against potato cyst nematodes (PCN), Globodera spp. It was introduced in The Netherlands following research that identified the species as the most suitable candidate among a diversity of species tested as potential trap crops 17,20,[22][23][24][25] . Several studies have been done with this plant and it has proved to have effects on hatching, mortality, infectivity and/or reproduction in several nematode species, including PCN, RKN and root-lesion nematodes (RLN) 18,20,23,24,[26][27][28] . The effects of S. sisymbriifolium on nematodes depend on the cultivars used, the genus and species of nematode present and on biotic and abiotic conditions 10 .
In Portugal, S. sisymbriifolium is not part of the native flora 29 , but other plants of the same genus, for instance S. linnaeanum Hepper & P.-M.L. Jaeger, which is similar to S. sisymbriifolium ( Supplementary Fig. S1), are present in the South of Portugal 30,31 . It is an invasive species of plant, spiny, with an extensive root system, and is probably native from Southern Africa although it is a common weed in North Africa and Southern Europe 30,32 .
Solanum sisymbriifolium is also known to be a source of resistance, or partial resistance, to some diseases and plant pests, including fungi, bacteria, nematodes and insects. Solanum linnaeanum has been much less studied but has potential as a source of resistance to some fungi and viruses [33][34][35] .  www.nature.com/scientificreports/ The main goals of the present study were: to evaluate the potential value of two wild Solanum plants, S. linnaeanum and S. sisymbriifolium, in controlling agricultural pests such as RKN; to assist in the development of sustainable and ecofriendly management methods of key enemies in agriculture, in order to reduce dependence on chemical pesticides; and to improve crop productivity. The resistance shown by S. linnaeanum and S. sisymbriifolium cv. Sis 6001 was evaluated against M. chitwoodi. Although some studies have been made with S. sisymbriifolium and PPN and it is already used as a trap crop in some places, not much is known about S. linnaeanum and its effects on nematodes. New sources of resistance may result in the development of sustainable and nonchemical management strategies to protect crops against PPN. This is the first report of S. linnaeanum being used in Portugal for the management of PPN.

Results
The environmental conditions used, and the period of the experiments, proved to be appropriate and sufficient for the development and reproduction of the nematodes. The results showed that there was no reproduction of M. chitwoodi in S. linnaeanum and that there was only a little reproduction in S. sisymbriifolium cv. Sis 6001, when compared with the reproduction in the susceptible plants used as controls, tomato cv. Coração de boi and potato cv. Désirée ( Fig. 1 and Supplementary Fig. S1).
Soil sterility was confirmed by the absence of galls and egg masses in uninoculated plants. The values of GI = 5 and RF > 1 (Table 1) obtained in the controls, tomato and potato, confirmed the viability of the inocula and that environmental conditions were favorable for penetration, development and reproduction of M. chitwoodi.
The same was verified by the index in the Bridge & Page (1980) rating chart 36 , which varied between 4 in tomato plants, 40% roots infested with larger knots but main roots clean, and 5 in potato plants, 50% roots infested with knotting on parts of main roots. All inoculated plants presented symptoms of yellowing, wilting and leaf drop, related to the presence of nematodes.
Solanum linnaeanum was considered resistant to M. chitwoodi (Table 1 and Supplementary Table S1). The index in the Bridge & Page (1980) rating chart 36 was 0, because no egg masses developed on the roots. Observation of the stained root systems showed the presence of many small root swellings (average of the 10 replicates was 519 small root swellings/plant) and nematodes inside the roots (total numbers values between 329 and 883 per plant) (Supplementary Table S2). Almost all the nematodes inside the roots were second-stage juveniles (J 2 ) (44%) or adults (45%) and all the adults were males (100%) (Fig. 2).
The values of GI = 0 and RF = 0 (Table 1 and Supplementary Table S1) 17,36 . However, when plants were analyzed separately some variability could be observed between them. Only two replicates (in a total of 10 replicates, 5 per assay) showed the presence of galls, one with one gall and without egg masses and another with 11 galls with 9 egg masses containing 187 eggs in total (Supplementary Table S1). Despite this, there are no statistically significant differences between numbers of galls, egg masses, eggs and RF between S. linnaeanum and S. sisymbriifolium cv. Sis 6001. Observation of stained roots of S. sisymbriifolium cv. Sis 6001 showed some nematodes (total numbers values between 4 and 117 per plant) (Supplementary Table S2). The majority of nematodes inside the roots were J 2 (69%); of the remainder, 17% were adults, of which 63% were males (Fig. 3).

Discussion
The choice of the classification system of Sasser et al. (1984) and the rating chart of Bridge & Page (1980) for the resistance studies of S. linnaeanum and S. sisymbriifolium cv. Sis 6001 to M. chitwoodi was because they can be applied to any crop and they also allow results from different studies to be compared without the need to include the susceptible controls 36,37 .
The time that any Meloidogyne species needs to complete a generation depends on external factors, such as temperature, humidity, light and the quality and condition of the host plant in relation to its age and nutritional status. All of these factors are crucial as they determine how and when the effects of nematodes on the plant will be most visible 38 . In our laboratory, it was observed that M. chitwoodi had a slower rate of development than other Meloidogyne isolates. Thus, the plants were uprooted 70 days after inoculation (DAI) instead of the usual Table 1. Resistance degree (RD) of Solanum linnaeanum, S. sisymbriifolium cv. Sis 6001 and respective controls (tomato and potato), 70 days after inoculation with 5000 eggs of Meloidogyne chitwoodi per plant (averages of ten replicates). GI (Gall Index) on a scale of 0-5; RF (Reproduction Factor) = Pf/Pi where Pf = final population and Pi = initial population; Resistance degree (RD): R = resistant (GI ≤ 2 and RF ≤ 1); S = Susceptible (GI > 2 and RF > 1); a Controls. Data from five plants per treatment of two independent experiments (n = 10) were submitted to ANOVA, and comparison of means by LSD test (P < 0.05) was carried out for GI and RF. Values with the same symbol are not significantly different. www.nature.com/scientificreports/  www.nature.com/scientificreports/ 60 DAI. Any differences found between species or replicates can only be attributed to characteristics inherent to the plants as the assay conditions were always the same. The control of the population density of PPN in the soil is best achieved by resistance in the plant, characterized as the ability of a plant species to inhibit nematode development or reproduction 39 . So far, the capacity of J 2 penetration into the roots has not been considered as a mechanism for characterization of the resistance. Generally, resistance to nematodes in plants becomes obvious after J 2 penetration into the roots (i.e. post-infection) 40 . The J 2 that penetrate the roots of resistant plants may die or leave the roots, but they can also develop to the adult stage, as females without egg production (or the eggs produced are not viable) or as males 41 . Various procedures for determining the resistance of plants to nematodes are used. The most common procedures are the percentage or number of galls or egg masses formed as well as the RF (Pf/Pi) values 37 . The low numbers of nematodes that penetrated the roots of S. linnaeanum and S. sisymbriifolium cv. Sis 6001 are probably due to substances produced by the roots of these plants, which prevented their penetration. Conceição  In S. linnaeanum, in the present work, there was greater nematode penetration of the roots than in S. sisymbriifolium cv. Sis 6001, but there was no reproduction in any of the plants, and all nematodes that developed into adults were males. This reaction is a form of active resistance (i.e. post-infection) in which, although the J 2 can penetrate the roots, they do not find favorable conditions for their normal development and reproduction. Adverse nutrition conditions in younger stages can, by themselves, change the physiology of certain nematodes, eliminating formation of females, egg production or population increase 14 . The larger number of males in the roots may indicate that the plant has a type of resistance that prevents the nematode from feeding normally.
In S. sisymbriifolium cv. Sis 6001, although some nematodes penetrated and established in the roots, there was actual reproduction in only one replicate of the first bioassay. The reduced number of nematodes found in the roots may be related to the existence of exudates or compounds produced by the roots of this cultivar that prevent J 2 penetration 19 . This type of resistance occurs before the nematode penetrates the root (i.e. pre-infection) and is called passive 43 . From analysis of the results of other authors, S. sisymbriifolium presents great variability, both between and within cultivars. In some cases, resistant and susceptible plants may co-exist within the same cultivar 17 . In Ali et al. (1992), for M. incognita, although S. sisymbriifolium was invaded by nematodes which formed some (4-5) swellings (not real galls), the nematodes did not develop beyond J 2 and there was no egg mass production 21 . Similar results were also reported by Fassuliotis & Bhatt (1982), possibly due to a tracheid discontinuity, due in turn to the formation of a small, thin-walled giant cell. However, for M. javanica, using regenerated plants rather than seeds, S. sisymbriifolium demonstrated low resistance 44 . Some reports do not mention the S. sisymbriifolium cultivar and Meloidogyne species used and S. sisymbriifolium may react differently to different species of Meloidogyne. As reported by Daunay & Dalmasso (1985), S. sisymbriifolium was a poor host for M. arenaria and M. incognita but it was a better host for M. javanica 45 . Therefore, caution should be taken when choosing the S. sisymbriifolium cultivar to control RKN because the results can change with the cultivar and/or the nematode species considered. The effects of some S. sisymbriifolium cultivars, or their plant extracts, on hatching, infectivity and/or reproduction of PCN, RLN and RKN has also been demonstrated by other authors with positive results 17,18,26,27 .
Although S. sisymbriifolium is tolerant of cold and heat 21,46 and is already used as a trap crop in some countries, this plant has already become invasive in some places. However, it is not native to Portugal and is also attacked by pests common to some of our crops 47 . Taking also in consideration, as already mentioned, that S. sisymbriifolium cultivars have great intra-and inter-cultivar variability 42,44 and given the results obtained in this work, S. linnaeanum may be a better plant to use against nematodes. It already exists in Portugal and is also a source of resistance to other pests and plant diseases such as Verticillium wilt 33,34 . Despite this, care must also be taken with the Meloidogyne species considered and with the plants of S. linnaeanum used. Tzortzakakis and collaborators (2006) tested S. linnaeanum against M. incognita and M. javanica and, in both cases, the nematodes reproduced, although in M. incognita the numbers of galls and egg masses were significantly lower than in the controls 48 .
The plant species we have considered are resistant, or partially resistant, to some diseases and plant pests. Solanum linnaeanum exhibits resistance to Verticillium wilt and Liu and collaborators (2015) succeeded in the transference of this resistance to eggplants through the introgression of the disease resistance gene 34 . In the same way, if the S. linnaeanum and S. sisymbriifolium resistance genes are isolated, they can be used to confer resistance to plants susceptible to M. chitwoodi, such as tomato and potato plants. They have a potential to be used as sources for resistance to M. chitwoodi in breeding programs.
In this study, it was demonstrated that S. linnaeanum can reduce RKN densities in the soil. Its roots attract M. chitwoodi juveniles, removing them from the soil but at the same time reducing the reproduction of the nematodes. The fact that the nematodes developed into males, without females, avoids the risk of reproduction or leaving a dangerous, viable population of nematodes in the soil. Since the number of J 2 that hatch and penetrate the roots is high, the population density of nematodes in the soil decreases and even those that hatch but do not enter the roots will die. In that way, the use of S. linnaeanum as a trap crop, in a crop rotation system, or even as rootstocks, may be an appropriate component of Integrated Pest Management, keeping RKN populations at levels low enough not to cause economic losses, thereby increasing production and crop quality and avoiding or limiting the use of synthetic nematicides. Using a trap crop does not disturb the ecological balance in the soil and  50 . This culture of M. chitwoodi is a pure isolate started initially from a single egg mass; its identification was confirmed by esterase phenotype analysis at the beginning and at the end of each assay 51 .
Plant materials. Solanum lycopersicum cv. Coração de boi (tomato), S. linnaeanum and S. sisymbriifolium cv. Sis 6001 were grown from seeds. Our stock of S. linnaeanum is a wild isolate whose seeds were harvested from a plant growing on the roadside in the Algarve. The seeds were germinated at 25-27 °C on moist filter paper in Petri dishes and transplanted singly into 5 cm diameter plastic pots containing 60 cm 3 of a steam-sterilized mixture of loam soil and sand (1:2 v/v). Solanum sisymbriifolium seeds were germinated in a glasshouse in polystyrene plates containing sterile peat. Fifteen days after germination the seedlings of this species were transplanted singly into pots filled with 500 g of steam-sterilized soil mix (sand:soil:peat 1:1:1 v/v). The potato plants, S. tuberosum ssp. tuberosum L. (cv. Désirée), were obtained from pieces of potato tubers with sprouts in pots with the same mixture of soil (sand:soil:peat 1:1:1 v/v). All plants were kept in a glasshouse under the same conditions as described above.
Pathogenicity tests. Five four-weeks-old plants from each species were inoculated with 5000 M. chitwoodi eggs (initial population density, Pi). To confirm the soil sterility one plant of each species was potted in the sterilized soil mix and not inoculated. Five susceptible tomato plants cv. Coração de boi and five susceptible potato plants cv. Désirée were also inoculated to confirm the viability of the inocula. Pots were kept in the conditions already mentioned and the plants were watered daily. Seventy DAI the plants were uprooted and the root systems washed. The root systems were stained with phloxine B (0.0015% solution) for 15 min 52 , and galls and eggs masses were counted. Eggs were extracted as described above and counted to determine the final population (Pf). Resistance rating of the species were based on Gall Index (GI) and Reproduction Factor (RF = Pf / Pi), according to the modified quantitative scheme of Canto-Sáenz (1985) 41,53 . The evaluation of the degree of resistance of the plants was based on GI and RF 37 . Roots that had less than 100 egg masses were stained with acid fuchsin 54 , and the numbers of the different developmental stages of M. chitwoodi were recorded. Resistance ratings of these plants were based on the RKN rating chart of Bridge & Page (1980) 36 . This scale is based on the percentage and types of roots galled from 0 (0%, no galls) to 10 (100% galled). The roots were observed using a routine stereo microscope Leica M80, at a magnification of 60 x, and the nematodes found were transferred onto a glass slide and observed with an optical microscope Leica DM2500, at a magnification of 400 ×. The identification of the different developmental stages was done by comparing their morphological characteristics with those described for M. incognita (Supplementary Fig. S2) 41,55 .
The assay was done twice, using the same conditions each time.
Statistical analysis. The data (values obtained for galls, egg masses and eggs counts and RF), were confirmed to meet the statistical assumptions of normality and homogeneity of variances (one way ANOVA), and were submitted to analysis of variance and the means compared by LSD (P < 0.05) using Statistic 10 software (Statsoft Inc.).