Resistance sources to root-knot nematodes Meloidogyne javanica, M. incognita and M. enterolobii in sweet potato

One of the main obstacles for food production in many developing countries, as in Brazil, is the damage caused by root-knot nematodes, mainly those belonging to the genus Meloidogyne. This study aimed to assess the resistance levels of 44 sweet potato genotypes to M. javanica, M. incognita race 1 and M. enterolobii. These researches were carried out in 2014, under greenhouse conditions in BrasíliaDF, Brazil. A completely randomized design with six replicates of one plant/plot/treatment was used. We determined the gall index (GI) and egg mass index (EMI) in the root system of each plant, the number of eggs and juveniles per gram of root with galls and the nematode reproduction factor. M. javanica was less aggressive and reproduced in only 9.09% of the evaluated genotypes; M. incognita race 1 was intermediate (47.73%); whereas M. enterolobii was more aggressive, with a population increase in 79.55% of the genotypes. The genotypes CNPH 1200, CNPH 1219, CNPH 1292, CNPH 1392, CNPH 60 and ‘Coquinho’ were the most resistant to the three species and can be used in breeding programs for multiple resistance to root-knot nematodes.


Research
Horticultura Brasileira 38 (2) April -June, 2020 T he sweet potato (Ipomoea batatas), although being a rustic plant, hosts nematodes of the genus Meloidogyne. These nematodes cause great concern regarding production losses in the tropics, subtropics and warm regions all around the world (Atkinson et al., 2012;Bernard et al., 2017;Karuri et al., 2017).
In Brazil, the most important species in sweet potato cultivations are M. incognita and M. javanica (Charchar & Ritschel, 2004;Chaves et al., 2013) and, according to Cervantes-Flores et al. (2002a), resistant genotypes to multiple species of Meloidogyne have been rarely recorded. However, M. enterolobii, synonym of M. mayaguensis, is gaining prominence due to its ability to infect plants that are resistant to other species of Meloidogyne (Carneiro et al., 2006a(Carneiro et al., , 2006b, among them the sweet potato (EPPO, 2014;Rutter et al., 2019).
The root-knot nematodes can reduce absorbent roots, with reduction on foliage and growth of the sweet potato plant, besides predisposing to the formation of longitudinal cracks in the roots, affecting not only yield, but also quality, conservation and the visual appearance of the commercial product (Perry & Moens, 2006;Bernard et al., 2017).
Several strategies have been used to control root-knot nematodes in sweet potato, among them the use of chemical nematicides, in countries where there
The aim of this study was to assess the levels of resistance of 44 sweet potato genotypes to M. javanica, M. incognita race 1 and M. enterolobii, to be used in breeding programs as resistance genes sources to root-knot nematodes.

MATERIAL AND METHODS
The experiments were conducted between December 17, 2013 and April 14, 2014, in a greenhouse at Embrapa Hortaliças, Brasília-DF, Brazil (15º55'44"S, 48º08'29"W, 999 m altitude), whose climatic classification according Köppen, is tropical savanna with rain concentration in summer and dry in winter.
Forty four sweet potato genotypes, from the Active Germplasm Bank of Embrapa Hortaliças, were evaluated for resistance to M. javanica, M. incognita race 1, which is the nematode race that most occurs in vegetables cultivations in Brazil, including sweet potato, and M. enterolobii. The genotypes were chosen due to its superior characteristics and potential to become new cultivars, and not yet characterized to nematode resistance. For each species of nematode, an independent experiment was installed, using a completely randomized design with six replications. Each experimental plot consisted of one plant grown in a 2-L plastic pot, containing substrate on the proportion 1:1:1:1 of subsurface soil (a clay Oxisol, typically encountered in the Savanna Biome region in Brazil), washed sand, cow manure and carbonized rice husk mix, autoclaved at 121°C for 60 min. We added to 300 g of this mixture 300 g N-P-K, 4-30-16 formulation, and 3,000 g of calcined dolomitic lime. The seedlings were obtained from healthy vines with four intermodal buds from each genotype, and planted individually.
The identification of the rootknot nematodes, M. javanica, M. incognita race 1 and M. enterolobii, was accomplished by morphological examination of the perineal region of adult females and comparison with taxonomic descriptions and keys of Yang & Eisenback (1983);Rammah &Hirschmann (1988) andEisenback &Hirschmann-Triantaphyllou (1991). To analyze the phenotype of esterase isozyme we used the technique proposed by Carneiro & Almeida (2001). The Meloidogyne incognita race was identified according to the differential host test of Taylor & Sasser (1978). standards of resistance and susceptibility, respectively for M. javanica and M. incognita race 1, (Charchar & Ritschel, 2004;Marchese et al., 2010;Massaroto et al., 2010). In addition, the tomato 'Santa Cruz' (Solanum lycopersicum) was used as susceptibility standard.
Thirty days after planting the vines, eggs and juveniles of second stages (J2) were extracted from the tomato 'Santa Cruz' roots, inoculated previously with each species to be evaluated, according to methodology of Boneti & Ferraz (1981). After the calibration, the inoculum was distributed over the soil, around the plants, with a concentration equivalent to 5,000 eggs + J2/plant. Plants were irrigated daily, and about one month after inoculation, side dressing was carried out using 3 g of Osmocote® (19-06-10 N-P-K) per liter of substrate.
Plants were harvested 80 days after inoculation, and determined the gall index (GI) and egg mass index (EMI) in the root system of each plant, according to the grades scale proposed by Taylor & Sasser (1978) (0= roots without gall or egg masses; 1= presence of 1 to 2 galls or egg masses; 2= presence of 3 to 10 galls or egg masses; 3= presence of 11 to 30 galls or egg masses; 4= presence of 31 to 100 galls or egg masses and 5= presence of more than 100 galls or egg masses on the root system). The final population of the nematodes in the root system and in the portions of the tuberous roots with galls, were also quantified, extracting the eggs and nematodes using the method of Boneti & Ferraz (1981). The final population (Fp) was quantified, counting the eggs and J2 under an optic microscope. The results were divided by the fresh weight of the root and the part of the tuberous roots with galls and expressed as eggs + J2 per gram of root (FWRG). The nematode reproduction factor (Rf = Fp/Ip) was calculated by dividing the final and initial populations (inoculated). Genotypes presenting Rf less than 1 were considered resistant, and susceptible those presenting Rf greater or equal to the unit (Oostenbrink, 1966).
The data were transformed to √x+1, to meet the assumptions of normal distribution and homoscedasticity, being presented the original values.
Data were subjected to a one-way analysis of variance (ANOVA), for each characteristic, and means were grouped using the Scott-Knott clustering test at a significance level of 0.05, using Genes software (Cruz, 2013).

RESULTS AND DISCUSSION
We observed a predominance of variation of genetic order in relation to the environmental variation for most of the evaluated characters, for the different species of nematodes, according to values higher than the unit for the rate between the genotypic and experimental coefficients of variation (CVg/CV), indicating favorable situation for the characterization of the resistance levels of the genotypes evaluated in the experiments (Tables 1, 2 and 3). eggs + J2 per gram of root and in the portions of tuberous roots with galls, in addition to the highest reproduction factor (17.26).
Cervantes-Flores et al. (2002b) evaluated 26 sweet potato genotypes and also verified that most (88.46%) were resistant to M. javanica, while the cultivar Beauregard was susceptible. Gomes (2014) also observed resistance of the sweet potato 'Brazlândia Branca' to this nematode. Silveira & Maluf (1993) evaluated 36 sweet potato clones regarding the resistance to M. javanica and identified 23 resistant materials. These authors also verified that the cultivars Brazlândia Roxa, Brazlândia Rosada and Coquinho were resistant. However, the cultivars Brazlândia Branca and Princesa, found being resistant in this research, in that study were considered susceptible. Charchar & Ritschel (2004) verified that 85.99% of 357 sweet Means followed by same letters in the columns do not differ by Scott-Knott hierarchical clustering algorithm, at a significance level of 0.05 for the means/grouping test. CV= environmental coefficient. CVg/CV= genotypic and environmental coefficients relation. GI= Gall Index; EMI= Egg Mass Index (0= without galls or egg mass; 1= 1-2 galls or egg masses; 2= 3-10 galls or egg masses; 3= 11-30 galls or egg masses; 4= 31-100 galls or egg masses and 5= more than 100 galls or egg masses in the root system) (Taylor & Sasser, 1978); Rf= reproduction factor, calculated by dividing the final and initial populations (inoculated); Reaction: degree of resistance (R= resistant and S= susceptible) considering resistant the genotypes with Rf lower than 1 and, susceptible, those that presented Rf higher or equal to 1 (Oostenbrink, 1966); FWRG= eggs + J 2 per root gram part of tuberous root with galls. Clones CNPH 46, CNPH 66 and CNPH 1220, did not differ statistically from the previous ones and were only slightly infected. However, due to the reproduction factors greater than 1, were classified as susceptible. Although being these genotypes not extremely resistant, if they have other superior characteristics, they could be tested to,  Maluf et al. (1996) evaluated the resistance of 226 sweet potato clones to M. javanica and M. incognita races 1, 2, 3 and 4, based on the number of egg masses per root, and observed that the frequencies of resistant genotypes were higher to M. javanica and lower to M. incognita race 2.
Wanderley & Santos (2004) studied the resistance of 35 sweet potato cultivars to M. incognita, based on reproduction factor, and observed that 15 were resistant. Similarly, Chaves et al. (2013) evaluated the reaction of 25 sweet potato genotypes to M. incognita race 2, based on the reproduction index, and verified that 28% of the genotypes were slightly resistant; 52% moderately resistant; 16% highly resistant and that only one genotype was susceptible. Massaroto et al. (2010) evaluated the reaction of 50 sweet potatoes accessions to the infection by M. incognita race 1, through the index of egg mass by radicular system, and verified that 15 were highly resistant. Moderate resistance was observed in the cultivar Brazlândia Rosada, corroborating the result found in this work.
Gomes (2014) Silveira & Maluf (1993) also evaluated the reaction of 36 sweet potato genotypes to the production of egg masses of races 1, 2, 3 and 4 of M. incognita and verified that 7, 1, 9 and 2 genotypes were resistant to these races, respectively. None genotype was simultaneously resistant to the Means followed by same letters in the columns do not differ by Scott-Knott hierarchical clustering algorithm, at a significance level of 0.05 for the means/grouping test. CV= environmental coefficient. CVg/CV= genotypic and environmental coefficients relation. GI= Gall Index and EMI= Egg Mass Index (0= without galls or egg masses; 1= 1-2 galls or egg masses; 2= 3-10 galls or egg masses; 3= 11-30 galls or egg masses; 4= 31-100 galls or egg masses and 5= more than 100 galls or egg masses in the root system) (Taylor & Sasser, 1978); Rf= reproduction factor, calculated by dividing the final and initial populations (inoculated); Reaction: degree of resistance (R= resistant and S= susceptible) considering resistant the genotypes with Rf lower than 1 and, susceptible, those that presented Rf higher or equal to 1 (Oostenbrink, 1966); FWRG= eggs + J 2 per root gram part of tuberous root with galls.  Means followed by same letters in the columns do not differ by Scott-Knott hierarchical clustering algorithm, at a significance level of 0.05 for the means/grouping test. CV= environmental coefficient. CVg/CV= genotypic and environmental coefficients relation. GI= Gall Index and EMI= Egg Mass Index (0= without galls or egg masses; 1= 1-2 galls or egg masses; 2= 3-10 galls or egg masses; 3= 11-30 galls or egg masses; 4= 31-100 galls or egg masses and 5= more than 100 galls or egg masses in the root system) (Taylor & Sasser, 1978); Rf= reproduction factor, calculated by dividing the final and initial populations (inoculated); Reaction: degree of resistance (R= resistant and S= susceptible) considering resistant the genotypes with Rf lower than 1 and, susceptible, those that presented Rf higher or equal to 1 (Oostenbrink, 1966); FWRG= eggs + J 2 per root gram part of tuberous root with galls.
but showed resistance to M. javanica. Cultivar Coquinho, the only one resistant to both species of nematodes, is an early cycle cultivar having good root yield, is known as resistant to rootknot nematodes, but presents irregular root shape, ranging from elongated to rounded, pale yellow and unattractive peel; therefore, not widely cultivated, but it could be an alternative to compose an integrated management system in areas infested with root-knot nematodes, or to be used in crossings. Melo et al. (2011) evaluated t h e r e s i s t a n c e o f e i g h t s w e e t potato genotypes to M. enterolobii. The authors verified that cultivars Brazlândia Rosada, Brazlândia Roxa and Brazlândia Branca were susceptible and that only two genotypes were resistant, UFLA0749 and UFLA0753. According to these authors, due to the existence of susceptible genotypes to M. enterolobii but resistant to other rootknot nematode species, indicate that the resistance to M. enterolobii, apparently, is mediated by genes other than those that confer resistance to other species of Meloidogyne. Gonçalves (2011) studied 142 sweet potato clones to the resistance to M. enterolobii and found 30 resistant genotypes. In that study, cultivars Brazlândia Rosada, Brazlândia Roxa and Brazlândia Branca were also susceptible.
Although several studies found in the literature about sweet potato genotype's reaction to root-knot nematodes, there exist few information related to M. enterolobii. In the present study many new accessions were evaluated, which provide more options to breeders; but even for those coincident accessions, different results in some researches were verified. These differences may be due to the concept of resistance that, in some cases, is determined by the egg mass index or by the gall visualization and, in some cases, by the reproduction factor. Moreover, experimental factors as temperature, the time the roots remain in the soil before inoculation, the planting date, the life cycle of the nematode, the population density and the stage of development of the root system can interfere in the nematode multiplication and, as a consequence, in the resistance expression (Lawrence et al., 1986). So, even for genotypes evaluated in earlier works, it is important to study the genotypes to confirm or not the resistance pattern, and to define whether these genotypes are resistant to other species of nematodes. About the parameters and concepts, the best parameter to assess resistance is based on the Reproduction Factor, according Oostenbrink. Regarding the Gall Index and Egg Mass Index, these are variables