Universitat Politècnica de Catalunya
Departament d’Enginyeria Agroalimentària i Biotecnologia
Programa de Doctorat Tecnologia Agroalimentària i Biotecnologia
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Durability of resistance to Meloidogyne spp. mediated by R-genes in solanaceous and cucurbitaceous crops
PhD dissertation
Helio Adán García Mendívil
PhD Supervisor: Dr. Francisco Javier Sorribas Royo
Castelldefels, España
The work presented in this thesis has been developed in the Integrated Plant-Parasitic Nematode Research Group of the Department of Agri-Food Engineering and Biotechnology of the UPC (Universitat Politècnica de Catalunya-BarcelonaTech, in the framework of the research projects:
"Efecto de la resistencia de genes R y la inducida por hongos endofitos en la epidemiología de Meloidogyne y la producción y calidad de la cosecha en solanaceas-cucurbitaceas". Funded by Ministerio de Economća y Competitividad (AGL2013-49040-C2-1R).
"Estrategias de gestión de germoplasma vegetal resistente a Meloidogyne para evitar la selección de virulencia". Funded by Ministerio de Economća y Competitividad (AGL2017-89785-R).
And with help of the pre-doctoral grant provided by the Consejo Nacional de Ciencía y Tecnología (No. 411638).
Durability of resistance to Meloidogyne spp. mediated by R-genes in solanaceous and cucurbitaceous crops
Helio Adán García Mendívil
Abstract
Watermelon, Citrullus lanatus var. lanatus, and eggplant, Solanum melongena, are two major crops commonly grafted in order to overcome soilborne diseases. However, there are currently no commercially available rootstocks resistant to root-knot nematodes (RKN), Meloidogyne spp., infection in the Cucurbitacea family, and also a narrow diversity in the Solanaceae, mostly in tomato and pepper. In order find alternatives to address this problem, the main objective of this thesis was to determine the durability of resistance to Meloidogyne of Citrullus amarus and Solanum torvum as potential rootstocks for watermelon and eggplant, respectively. In the first part of this document, the work conducted with the cucurbits is presented in chapters one and two, while chapters three and four correspond to work done with solanaceous.
Durabilty of Citrullus amarus resistance to Meloidogyne: the response of two Citrullus amarus accessions, BGV0005164 and BGV0005167, was assessed against different Meloidogyne arenaria, M. incognita, and M. javanica isolates in pot experiments and against M. incognita in plastic greenhouse. (i) In the pot experiments, plants were inoculated with a second-stage juvenile per cm3 of sterile sand and maintained in a growth chamber at 25 C for 50 days. The watermelon cv. Sugar Baby was included as a susceptible control for comparison. At the end of the experiments, the number of egg masses and eggs per plant was determined, and the reproduction index was calculated as the percentage of the number of eggs produced in the C. amarus accessions with regard to that produced in the susceptible cv. Sugar Baby. (ii) In the plastic greenhouse experiment, the ungrafted watermelon cv. Sugar Baby and watermelon grafted onto each of the C. amarus accessions and onto the watermelon rootstock cv. Robusta were cultivated from May to August 2016 in plots with nematode densities from 46 to 1392 J2 per 250 cm3 of soil at transplantation. At the end of the experiment, the galling index and the number of eggs per plant were determined, and the reproduction index was calculated. (iii) Additionally, the compatibility of the two accessions with the watermelon cv. Sugar Baby and the effect on fruit quality (weight, size, shape, firmness, pH, total soluble solids, and flesh color) were assessed under a hydroponic system in a greenhouse. The commercial rootstocks cv. Cobalt and cv. Robusta were also included. Moreover (iv) The response of ungrafted and grafted watermelon cv. Sugar baby onto the C. amarus accessions BGV0005164 and BGV0005167 submitted to increasing densities of M. incognita and M. javanica was studied in pot experiments to determine the maximum multiplication rate, the maximum population density and the equilibrium density of the root-knot nematode species and the effect on shoot dry biomass of watermelon. (v) In plastic greenhouse conditions, the ungrafted and grafted watermelon onto both C. amarus accessions, and onto the C. lanatus rootstock cv. Robusta were cultivated for two consecutive years in the same plots to assess the level of resistance to M. incognita and crop yield. (vi) Additionally, after the second crop, the putative selection for virulence in the nematode subpopulation originated in the ungrafted and grafted watermelon was assessed in pot experiments. The results showed that (i) all the Meloidogyne isolates produced fewer egg masses and eggs per plant in the accessions than in Sugar Baby. Both accessions performed as resistant against M. arenaria, and from highly to moderately resistant to M. incognita and M. javanica in pot experiments. (ii) In the plastic greenhouse experiment, both C. amarus accessions performed as resistant to M. incognita. (iii) Both C. amarus accessions were compatible with the watermelon cv. Sugar Baby, but only the BGV0005167 accession did not influence the fruit quality. (iv) The maximum multiplication rate, the maximum population density and the equilibrium density values of both Meloidogyne species were lower in grafted than ungrafted watermelon. (v) In the plastic greenhouse experiment, the nematode densities in soil at transplantation ranged from 1 to 53 J2 per 100 cm3 of soil in 2017 and did not differ between grafted and ungrafted watermelons. At the end of the crop, the galling index and the number of eggs per plant was higher in ungrafted than in grafted watermelon both years. The C. amarus accessions performed from highly resistant to resistant to M. incognita, and the rootstock cv. Robusta from moderately resistant in 2017 to slightly resistant in 2018. All grafted watermelons yielded more kg per plant than the ungrafted in both years. (vi) The repeated cultivation of grafted watermelon onto C. amarus accessions did not select for virulence. In conclusion, the BGV0005167 accession is a promising rootstock for managing the three tropical root-knot nematode species without influencing watermelon fruit quality. The results of this study highlight the poorer host status of CI64 and CI67 accessions to M. incognita and M. javanica compared to watermelon; the stability of the C. amarus resistance; and the beneficial effect of C. amarus on watermelon yield when cultivated in Meloidogyne infested soils.
Durabilty of Solanum torvum resistance to Meloidogyne: several experiments were carried out to assess the performance of commercial Solanum torvum cultivars against Meloidogyne incognita and M. javanica isolates from Spain. (i) The response of S. torvum rootstock cultivars Brutus, Espina, Salutamu and Torpedo against M. incognita and Mi1.2 (a)virulent M. javanica isolates was determined in pot experiments, and of cv. Brutus to an N-virulent isolate of M. incognita, compared with that of the eggplant cv. Cristal. (ii) The relationship between the initial and final population densities of M. javanica on ungrafted and grafted ‘Cristal’ onto the S. torvum ‘Brutus’ was assessed, together with the effect on dry shoot biomass. (iii) Finally, the population growth rate and the resistance level of the four S. torvum cultivars against M. incognita was assessed under plastic greenhouse conditions in two cropping seasons. (iv) The eggplant Solanum melongena cv. Cristal, either ungrafted or grafted onto the S. torvum rootstock cv. Brutus was cultivated for two consecutive years in the same plots in a plastic greenhouse to assess the level of resistance to M. incognita and crop yield. (v) At the end of the second crop, the putative selection for virulence of the nematode subpopulations coming from infected ungrafted and grafted eggplant was assessed in the eggplant and in S. torvum in a pot experiment. The results showed that: (i) all S. torvum rootstocks responded as resistant to the M. incognita isolates and from highly resistant to susceptible against M. javanica isolates. (ii) The maximum multiplication rate of M. javanica on the ungrafted or grafted eggplant were 270 and 49, respectively, and the equilibrium density were 1318 and 2056 eggs and J2 per 100 cm3 soil, respectively. The tolerance of the ungrafted eggplant was 10.9 J2 per 100 cm3 soil, and the minimum relative dry shoot biomass was 0.76. (iii) The population growth rate of M. incognita on eggplant cv. Cristal differed from that of the S. torvum cultivars in both cropping seasons. (iv) Nematode population densities at transplantation in 2017 ranged from 2 to 378 J2 per 100 cm3 of soil and did not differ between ungrafted and grafted eggplant. At the end of each crop, higher galling index and number of nematodes in soil and in roots were registered in ungrafted than grafted eggplant. The grafted eggplant performed as resistant in 2017 and as highly resistant in 2018. Eggplant yield did not differ irrespective of grafting in 2017 after being cultivated for 135 days, but it differed after 251 days of cultivation in 2018. (v) In the pot experiment, S. torvum performed as resistant to both M. incognita subpopulations. However, the M. incognita subpopulation obtained from roots of S. torvum produced 49.4% less egg masses and 56% less eggs per plant in the eggplant than the nematode subpopulation obtained from roots of the eggplant cv. Cristal. These results suggest that S. torvum is a valuable rootstock for managing the two Meloidogyne species irrespective of the (a)virulence status, and revealed that the infective and reproductive fitness of the nematode decrease without having been selected for virulence.
General introduction
Durability of resistance to Meloidogyne spp. mediated by R-genes in
solanaceous and cucurbitaceous crops
Helio A. García-Mendívil
PhD Dissertation
Meloidogyne spp.
Biology
Root-knot nematodes (RKN), Meloidogyne spp., are the most damaging plant parasitic nematodes worldwide. This genus comprises more than 100 species, however, most of the crop yield losses are caused by four of them: the tropical species M. arenaria, M. incognita and M. javanica, and the temperate species M. hapla (Jones et al. 2013). The widened global warming can favor the expansion and proliferation of the RKN tropical species in areas where the temperate RKN species predominates. This fact evidence the importance of focusing research efforts for designing management strategies to the tropical RKN species.
RKN are obligated parasites that require a suitable host plant for life cycle completion (Fig. 1). The infective pre-parasitic vermiform second-stage juveniles (J2) of Meloidogyne moves through the soil and penetrate the root behind the tip, by using their protractible stylet and releasing secretions containing cell-wall-degrading enzymes (Abad et al. 2003). J2 then migrate intercellularly between the cortical cells towards the root tip where they make a U-turn to later enter into the vascular cylinder and moves until stablishing a feeding site. Each J2 is able to induce the redifferentiation of five to seven parenchymatic root cells into multinucleate and hypertrophied feeding cells, the so-called giant cells, from which get the nutrients needed for its life cycle completion (Nyczepir and Thomas 2009). The accumulation of these giant cells is responsible for the characteristic galled tissue present in infected root systems, and for the disturbance of plant development, defenses, and metabolism (Shukla et al. 2018). Once infection occurs, juveniles become sedentary and assume a “sausage” shape as increases its size. J2 then moults three times achieving the adult stage. The tropical species, M. arenaria, M. incognita and M. javanica, reproduce parthenogenetically. The sedentary pearl-shaped adult female keeps feeding from the giant cells and is able to lay c. 300-500 eggs inside a gelatinous mass. Under unfavourable conditions (high nematode density, scarcity of food or stressed plants) the juveniles develop into males. Interestingly, some studies have found to increase stimulation towards maleness by cropping several cucurbit species (Fassuliotis 1970; Walters et al. 2006; Expósito et al. 2019).
Figure 1: Diagram of the life cycle of the root-knot nematode, Meloidogyne. J2: second-stage juvenile; J3: third-stage juvenile; J4: fourth-stage juvenile (adapted from Moens et al. (2009)).
Meloidogyne spp. is a poikilothermic organism, meaning that temperature influence its life cycle and determine its length (Tyler 1933). The nematode development occurs between 10 and 32 C, and needs to accumulate an amount of degree-days (K) at a certain basal temperature (Tb) to complete its life cycle (Trudgill 1995). For instance, it has been reported that Meloidogyne spp. requires between 600 and 700 accumulated degree-days with a base temperature of 10 C to complete its life cycle in tomato (Ferris 1985). However, the thermal time requirements for life cycle completion can differ between RKN populations of a given species depending of its geographic origin, showing its ability to adapt to environmental factors, i.e., optimal temperatures of 25-30C and 32-34C were found in populations of M. javanica from Australia and California, respectively (Ferris and Van Gundy 1979). Such adaptive ability may be one of the main reasons of its success in spreading globally, and also evidence its importance for modern agriculture.
Economic importance
The occurrence of soilborne diseases and pests have increased in recent years as consequence of the intensive cultivation needed to supply a growing population in limited land resources (Judy A. Thies, Buckner, et al. 2015). A comprehensive summary of the estimation of yield losses caused by RKN in several crops have been published by Greco and Di Vito (2009). The cucurbit and solanaceous crops are two of the crop families most frequently cultivated worldwide that are severely affected by RKN. Regarding cucurbit crops, maximum yield losses of 88, 53 and 35% in cucumber, zucchini and watermelon, respectively, have been estimated under greenhouse conditions (Giné et al. 2014; Vela et al. 2014; López-Gómez et al. 2014, 2015). RKN are also one of the most damaging soilborne pathogens for solanaceous crops, specially under protected cultivation, with maximum yield losses of 94, 95 and 100% reported for pepper, eggplant and tomato, respectively (Giné et al. 2017; Hallmann and Meressa 2018).
Population dynamics and yield losses
Population dynamics studies the factors that determine the temporal oscillation of densities of individuals from the same specie living in a certain area. The nematode population density at sowing or transplanting of a crop is related with its productivity. The proper modeling of population dynamics allows to estimate the densities variability over time in relation with influencing factors, and therefore, to relate them with the yield losses that nematodes could cause. Modeling the damage levels enable to calculate parameters such the tolerance limit (T), the maximum population density above which yield losses start to occur, and the maximum yield losses (m). These parameters, along with the maximum reproduction rate (a)(Pf/Pi at low densities (Ferris 1985), and the equilibrium density, that initial population at which nematode receive just enough supply of nutrients to maintain the population density at the same level from begin at the end of the growing season; permits to evaluate the importance of a determined nematode from a growing area, or the effect of management strategies. These quantitative studies constitute the basis for populations’ evolution understanding of specific patosystems with typical agro-environmental conditions from determine growing area, and to design and implement effective and durable management strategies (Sorriba and Verdejo-Lucas 2011; Fig 2).
Figure 2: Relation between initial and final densities in experiments with a nematode on a good host, intermediate hosts, poor hosts, and a nonhost. Pi and Pf: initial and final densities on logarithmic scales. Equilibrium density: Pf = Pi (adapted from Seinhorst (1970)).
Population density fluctuations are affected by denso-independent and denso-dependent factors. Denso-independent factors, such climate, environmental conditions, and intraspecific competition, influence from outside of population, while denso-dependent include interspecific competition and the action of antagonist or predators. Two phases can be distinguish: one during the host plant cultivation in which nematode have enough nutrients to increase its population density, and the second during either no cropping periods in which there is no food supply and population density do not increase, or decrease depending on the nematode and the duration of periods with no host cultivation.
The multiplication growth rate (Pf/Pi), that is, the relationship between the nematode population density at transplanting (Pi) and at the end of the crop (Pf) is consider a good indicator of population growth. In absence of competition between individuals for limiting resources, the Pf/Pi maximize (a), and the relationship with Pi its a straight line (Pf = aPi; thus Pf/Pi = a). However, increases in Pi induce competition between individuals for healthy plant tissue, can induce alteration in sex differentiation or to reduce in female fecundity, thus Pf/Pi decreases. In the case of nematodes that reproduce parthenogenetically, such Meloidogyne spp., the relationship between Pf/Pi and Pi follows an inverse potential function (Pf/Pi = aPi-b, where a is the maximum multiplication rate, and -b is the decrease rate of the population as Pi increase). The maximum multiplication rate (a) is a good indicator of the plant host status to Meloidogyne. Higher values of a indicate that the plant is a good host and low values, a pooor host (Ferris 1985).
The population growth rate can also be useful to estimate the Pi at which Pf/Pi = 1, the equilibrium density (E). This can be calculated from the regression equation obtained from linearizing the relationship between the Pi and the multiplication rate (Pf/Pi). The population growth rate is also a useful indicator that allows to compare between plant species and/or germplasms, as well as the effectiveness of control methods (Talavera et al. 2009; A. Giné and Sorribas 2017; Expósito et al. 2018); Fig 3).
Figure 3: Relation between initial population density (Pi) and multiplication rate (Pf/Pi) of RKN on two hypothetical germplasms (adapted from Ferris (1985)).
Regarding to crop yield losses, there are two parameters that can be calculated by estimating the relationship between increasing levels of Pi and the relative crop biomass or yield according to the Seinhort’s damage model (Seinhorst 1998):
The nematode population density above which yield loss start to occur is defined as the nematode damage threshold level or the tolerance limit of the crop (T). This value, along with the the minimum relative yield (m) are important parameters to characterize the response of a crop plant to a nematode population. The parameter T manifests itself at small nematode densities and m at larger ones (Greco and Di Vito 2009); Fig 4).
Figure 4: Relationship between relative yield and the initial nematode population density of three hypothetical germplasms with different values of T and m (adapted from Schomaker and Been (2013)).
Plant resistance
The most commonly used strategy for RKN management has been, until recent years, the use of fumigant and non fumigant nematicides (Nyczepir and Thomas 2009). However, regulations such the European directive 2009/128/EC and the U.S. Clean Air Act ( 2012)), have brought special attention on the research and development of environmentally friendlier management strategies. A promising alternative strategy involves the use of resistant plants, those whose have the ability to suppress infection development, and/or reproduction of plant-parasitic nematodes (Roberts 2009). Plant resistance has proven to be effective and economically profitable (Sorribas et al. 2005), and also reduce yield losses of the follwing crop (Ornat, Verdejo-Lucas, and Sorribas 1997; Talavera et al. 2009; Westphal 2011; Judy A. Thies, Ariss, et al. 2015; A. Giné and Sorribas 2017; Expósito et al. 2019).
The resistance levels of a determined plant germplasm can be categorized in relation to a susceptible standard (Hadisoeganda 1982). Expressed as the percentage of RKN reproduction compared to that in a susceptible standard, this parameter is called the reproduction index (RI; Fig 5).
Figure 5: A diagrammatic representation of the continuum of susceptibility and resistance to nematode reproduction within a crop germplasm pool (adapted from Starr and Mercer (2009)).
Despite its advantages, the expression of plant resistance can be affected by several factors, such the genetic background of the plant cultivar and/or the nematode species or population (Cortada et al. 2008). The selection of virulent nematode populations due to repeated cultivation of the same resistance gene (Verdejo-Lucas et al. 2009; A. Giné and Sorribas 2017; Expósito et al. 2019). Moreover, there are only a few resistance genes introgressed into commercial solanaceous cultivars at present, and none in cucurbits. That are, the Me1, Me3 and N gene in pepper, and the Mi1.2 gene in tomato. Soil temeprature can also affect the expression of plant resistance. It is known that the Mi1.2 gene is affected at soil temperatures above 30 °C, while the resistance of the N gene is reported to be partially lost at 32 °C (Araujo et al. 1982; Thies and Fery 1998). Thus,plant resistance is a valuable management tool but it should be used in a proper manner to maximize its effectiveness. Moreover, more research are needed to identify new resistance sources for increasing the R-genes that could be used in rotation schemes .
Grafting vegetables onto resistant rootstocks is an effective method to control soilborne pathogens when no commercial cultivars are available (Miguel et al. 2004; Cohen et al. 2007; Thies et al. 2016; Kumar, Bharti, and Saravaiya 2018). This control method was essentially rediscovered in the past two decades and quickly expanded to become a common practice at the present (Judy A. Thies, Buckner, et al. 2015; Kyriacou et al. 2017). Grafting has expanded mainly in the Cucurbitaceae and the Solanaceae families (Moncada et al. 2013). The use of anatomical and physiological compatible graft combinations improve the plant performance under biotic or abiotic stress conditions when compared with that of the ungrafted scion, allowing rapid response to new pathogen races without the prolonged screening and selection required for breeding resistance into cultivars (Davis et al. 2008). Although the tolerance and resistance to abiotic and biotic stresses, respectively, along with an increasing productivity have been the main drivers of rootstock selection and breeding, the effect of grafting on fruit quality is also another important factor to take into account (Kyriacou et al. 2017).
Some strategies proposed to avoid the selection for RKN virulence or to reduce the level of virulence and crop yield losses consider to include resistant and susceptible crops in rotation sequences (Talavera et al. 2009), the use of crops of two different resistant plant species (Expósito et al. 2019), or to pyramid different R-genes (Djian-Caporalino et al. 2014). However, as literature reviewed points out, most of the research done have been focusing on tomato and pepper. Thus, the aim of this thesis was to increase the diversity of resistance sources against RKN by potentially effective rootstocks for two species of the most economically important botanical families cultivated, the cucurbitaseous watermelon (Citrullus lanatus var. lanatus), and the solanaseous eggplant (Solanum melongena). A brief description of these two crops and their potential rootstocks is presented in the next two sections.
Watermelon
Importance
Watermelon is the most cultivated cucurbit crop worldwide. The global production in 2017 was ca. 118 million t in ca. 3.4 million ha, 38% of the arable land destined to cultivate for this botanical family, with an estimated value of 48.9B (FAOSTAT 2017); Fig 6). In spite of its origin theorized in the African continent, most of the watermelon are produced in Asia, with 67% of total world production in China. Within the European Union (EU), Spain was the main watermelon producer during 2017, with 20,026 ha harvested to produce 1.1 million t of fruit marketed with an estimated value of 331M, 68% of which were exported (MAPA 2019). The watermelon fruit composition is 93% water, with small amounts of protein, fat, minerals, and vitamins. The major nutritional fruit components are carbohydrates (6.4 g/100 g), vitamin A (590 IU), and lycopene (4,100 μg/100g, range 2300–7200), an anticarcinogenic compound found in red flesh watermelon, even in higher amounts than in tomato, pink grapefruit, or guava (Wehner 2008).
![Origin and production quantities of watermelons in 2017 by country [FAOSTAT, 2019; @Renner2019].](https://lh3.googleusercontent.com/pw/ACtC-3dtr0vFepGytI3IyvDSVPYt0w3r3nl2UJBKqLpMEAlGoRXoaVOkm-QIAAar49tVVCx5GM9lnNBMfMHjHEAyscxQ_QHbJFwDb2YXBJE1xtQA86slfYulB_QSHmR_rd5Dnmv0znTMlkiX2FlgztOd2ipQ=w1112-h557-no?authuser=3)
Figure 6: Origin and production quantities of watermelons in 2017 by country (FAOSTAT, 2019; Renner et al. 2019).
Origin
Watermelon is a diploid creeping monoecious crop that belongs to the Cucurbitaceae family. The taxonomy of the genus Citrullus have had major misapplication of names until recent years, including that of watermelon, C. lanatus, itself (Chomicki and Renner 2015); (Renner, Chomicki, and Greuter 2014). The Southern African region is reported to be the center of diversity and probably the center of origin of most of the Citrullus species (Dane and Lang 2004; Rubatsky 2001; Robinson and Decker-Walters 1997). However, four hypotheses have been proposed for the origin of watermelon: i) that it descends from the northern African colocynth (C. colocynthis, (Singh 1978; Sain, Joshi, and Divakara Sastry 2002; McCreight et al. 2013). ii) That it derives from the South African citron melon, C. amarus, previously referred as C. lanatus var. citroides (Robinson and Decker-Walters 1997; Maynard and Maynard 2000; Rubatsky 2001; Chomicki and Renner 2015). iii) That it stems from the West African C. mucosospermus (Guo et al. 2013; Chomicki and Renner 2015), and iv) Recent research with polygenomic analyses of nuclear gene sequences suggest that the white-fleshed Sudanese Kordophan melon (Citrullus vulgaris) is the closest relative of domesticated watermelon that was originated and domesticated in north-eastern Africa (Renner et al. 2019); Fig 6).
Cultivation challenges
The modern cultivated watermelon has a narrow genetic base (Szamosi et al. 2009; Solmaz et al. 2010; Nimmakayala et al. 2014) attributed to many years of domestication and selection of its desirable horticultural traits such as red-scarlet flesh color and sweetness (Levi, Thomas, Wehner, et al. 2001; Hwang et al. 2011; Lambel et al. 2014; Nimmakayala et al. 2014). The continued use of cultivars with narrow genetic base for breeding resulted in reduced gene diversity among American watermelon accessions (Wang et al. 2015; Zhang et al. 2016). This has derived in susceptibility of crops to random and emerging biotic and abiotic stresses (Levi, Thomas, Keinath, et al. 2001; Mo et al. 2016). In spite of being consider a less suitable host for RKN than other cucurbit crops, watermelon maximum yield losses of 37% have been recently reported in Spain (López-Gómez et al. 2014). Moreover, as the next section will better describe, modern watermelons are commonly grafted onto commercial rootstocks owing its resistance to fusarium, however, these rootstocks are not resistant to Meloidogyne (Kokalis-Burelle and Rosskopf 2011; López-Gómez, Talavera, and Verdejo-Lucas 2016; Giné et al. 2017).
Grafting
A detailed history of cucurbit grafting have been done by Davis (2008). The primary motive for grafting cucurbits is to avoid damage caused by soilborne pests and pathogens when genetic or chemical approaches for disease management are not available (Oda 2002). Research on cucurbit grafting began in the 1920s with the use of Cucurbita moschata as a rootstock for watermelon, but then bottle gourd (Lagenaria siceraria) soon became the preferred rootstock. By the year 1998, approximately 95% of watermelon and oriental melons were grafted onto squash or bottlegourd rootstocks in Japan, Korea, and Taiwan (Oda and Lee 2003). Possibly because of the widespread use of bottle gourd rootstocks, there are reports of plants affected by Fusarium wilt, probably the most common and damaging soilborne disease of cucurbit crops worldwide, caused by Fusarium oxysporum Schltdl. Moreover both rootstocks are susceptible to Meloidogyne infection (Kokalis-Burelle and Rosskopf 2011; López-Gómez, Talavera, and Verdejo-Lucas 2016; Giné et al. 2017). Hence, an alternative genetic source of resistance to both pathogens must be found and properly characterized in order to be able to design an effective and widely applicable RKN management strategy.
A potential alternative rootstock for watermelon, is its close relative citron or preserving melon, C. amarus. The flesh of the citron is white or green, and may vary from bland to bitter tasting. Its rind is used to make pickles, and the fruit are feeding to livestock (Wehner 2008). Conversely to watermelon, citron melon exhibits wider genetic variation (Levi, Thomas, Wehner, et al. 2001) suggesting its genetic worthiness as a source of valuable genes for breeding (Ngwepe, Mashilo, and Shimelis 2019). This rootstock have show to be resistant to several soil pathogens such as fusarium wilt, gummy stem blight, powdery mildew, potyviruses, and some species and populations of Meloidogyne (Gusmini, Song, and Wehner 2005; Huitrón et al. 2007; Guner and Wehner 2008; Tetteh, Wehner, and Davis 2010; Thies et al. 2016). In spite of it, there is little information about it use to control RKN in the EU, thus, research on its response to local population must be done before include it in a management strategy.
Eggplant
Importance
Eggplant, Solanum melongena, is the third most widely cultivated Solanaceous fruiting crop after potato and tomato. The global production in 2017 was ca. 52 million t in ca. 1.8 million ha, 7% of the arable land destined to cultivate for this family, with an estimated value of 36.2B. Most of eggplants production is made in China and India, with ca. 87% of global production done there in 2017. Italy, Spain and Romania were the top three eggplant producer countries in the EU, with 286,473, 225,912 and 124,429 t produced, respectively (FAOSTAT (2017); Fig 7). Eggplant is not high in the majority of health-related micronutrients, but it is very low in fat an calorie and a rich source of nutritionally and pharmaceutically useful compounds, such a number phytonutrients, especially hydroxycinnamic acid (HCA) conjugates, potentially involved in consumer health, fruit taste and texture (Meyer et al. 2015; Chapman 2019).
Figure 7: Production quantities of eggplants in 2017 by country (FAOSTAT, 2019).
Origin
The Asian eggplant is a widely grown species from the Solanaceae family. Eggplant is especially popular in the Southeast Asia and the Mediterranean region . Several non-exclusive theories have been proposed regarding eggplant’s origin. The general consensus (Weese and Bohs 2010; Knapp, Vorontsova, and Prohens 2013) is that the African/Middle Eastern species S. incanum was transported into Indo-China where the true wild progenitor, S. insanum, evolved, from which S. melongena is derived (Chapman 2019).
Cultivation challenges
The commercial eggplant cultivars also have a narrow genetic diversity that, is even poorer than for other solanaceous crops such as tomato and pepper. A list of eggplant diseases and pests, and resistances described has been well summarized by Daunay (2008). Among the most common soil borne diseases affecting eggplants are bacterial, fusarium and verticillium wilt, caused by Ralstonia solanacearum, Fusarium oxysporum f. sp. melongenae, Verticillium dahliae and V. albo-atrum. Moreover, it is also susceptible to RKN.
Grafting
Eggplant grafting is mostly used in intensive production conditions. As Daunay (2008) well summarize, there are currently three types of eggplant rootstock: i) S. melongena lines and hybrids that resist to Fusarium and bacterial wilt, and Phomopsis blight, ii) rootstocks based on the use of S. integrifolium, which also resists to Fusarium and bacterial wilts, and is used directly as a rootstock or as parent crossed with S. melongena varieties for producing interspecific hybrid rootstocks (S. integrifolium × S. melongena), and iii) the third type of rootstocks is composed by Solanum species such S. torvum and S. sanitswongsei. However, within Europe eggplant is grafted mostly onto tomato or tomato interspecific hybrids (L. esculentum × L. hirsutum).
Solanum torvum is a wild relative of eggplant that is resistant to V. dahliae, R. solanacearum, F. oxysporum f. sp. Melongenae, and some RKN populations (Singh and Gopalakrishnan 1997; Bletsos, Thanassoulopoulos, and Roupakias 2003; Daunay 2008; Gisbert et al. 2011). Although resistance of S. torvum rootstocks to M. incognita have been consistently described against several populations from France (Daunay and Dalmasso 1985), India (Shetty and Reddy 1985; Dhivya, Sadasakthi, and Sivakumar 2014), Japan (Hara et al. 1983; Ali et al. 1992), Pakistan (Rahman et al. 2002) and Turkey (Çürük et al. 2009); several studies found discrepancies on the levels of resistance to M. arenaria (Daunay and Dalmasso 1985; Gonzalez et al 2010; Ryu et al. 2011; Uehara et al. 2017; Öçal, Özalp, and Devran 2018) and M. javanica (Daunay and Dalmasso 1985; Boiteux and Charchar 1996; Tzortzakakis, Bletsos, and Avgelis 2006; Öçal, Özalp, and Devran 2018). As far as literature reviewed pointed out, there are no enough studies that widely characterize the response of S. torvum against Meloidogyne populations occurring in Spain.
Most of grafting application have expanded mainly in the Cucurbitaceae and Solanaceae family, both major vegetable crops that are commonly rotated to maximize land use and boost productivity (López-Gómez et al. 2015; Kyriacou et al. 2017). The proper screening of C. amarus and S. torvum rootstocks could give crucial information about potential tools already available to design effective and environmentally friendlier strategies to managing RKN. In particular, this work will focus on Meloidogyne populations ocurrying in Spain, some of them previously described as virulents to the Mi1.2 gene in tomato (Ornat, Verdejo-Lucas, and Sorribas 2007; Verdejo-Lucas, Talavera, and Andrés 2012).
Objectives
The main objective of this thesis was to determine the durability of resistance to Meloidogyne of Citrullus amarus and Solanum torvum as potential rootstocks for watermelon and eggplant, respectively. This objective was divided into specific objectives to determine:
The response of two Citrullus amarus accessions, BGV0005164 and BGV0005167, against different Meloidogyne arenaria, M. incognita, and M. javanica (a)virulent isolates in pot experiments and against M. incognita in plastic greenhouse (Chapter 1).
The performance of ungrafted and grafted watermelon onto C. amarus accessions submitted to increasing densities of M. incognita and M. javanica in pot experiments to determine the maximum multiplication rate, the maximum population density and the equilibrium density of the root-knot nematode species and the effect on shoot dry biomass of watermelon (Chapter 2).
The durability of resistance of C. amarus accessions after two consecutive years in the same plots, the selection for virulence and the fitness cost for the nematode (Chapter 2).
The response of commercial Solanum torvum cultivars against (a)virulent isolates of M. incognita and M. javanica in pot experiments and against M. incognita in plastic greenhouse in two cropping seasons (Chapter 3).
The performance of ungrafted and grafted eggplant onto S. torvum ‘Brutus’ submitted to increasing densities of M. javanica in pot experiments to determine the maximum multiplication rate, the maximum population density and the equilibrium density of the root-knot nematode species and the effect on shoot dry biomass of watermelon (Chapter 3).
The durability of resistance of S. torvum ‘Brutus’ after two consecutive years in the same plots, the selection for virulence and the fitness cost for the nematode (Chapter 4).
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