Reaction of weeds, found in vegetable production areas, to root-knot nematodes Meloidogyne incognita and M. enterolobii

Root-knot nematodes cause great damage to vegetable crops in Brazil, besides having a large range of host plants, such as weeds. Weeds can maintain the inoculums or even favor the multiplication of these nematodes. In this study we evaluated the reaction of selected weed species, present in a vegetable production area, to root-knot nematodes Meloidogyne incognita and M. enterolobii. The trials were conducted in a greenhouse at Embrapa Hortaliças, Brasília-DF, in a completely randomized design with six replicates. Fifteen weed species were evaluated for M. incognita race 1, and 16 weed species were evaluated for M. enterolobii. Two tomato cultivars were evaluated as resistance and susceptibility standards. Gall index (IG), egg mass index (IMO), number of eggs per gram of roots (eggs/g roots) and reproduction factor (FR) were evaluated. M. enterolobii survives and multiplies more easily in weeds collected in vegetable production areas than M. incognita race 1 and, the great majority of weed species evaluated in this study are hosts of both nematode species. Only the species Urena lobata, Sonchus oleraceus, Euphorbia heterophylla, Melampodium perfoliatum and Tagetes sp. were immune to M. incognita race 1. All evaluated species are either hosts or favor the multiplication of M. enterolobii. The species which are the most susceptible to M. incognita race 1, and therefore require greater control of crops infected by this nematode are Ipomoea nil, I. triloba and Eleusine indica, and for M. enterolobii are I. nil, Solanum americanum, Hyptis suaveolens, Portulaca oleracea, I. triloba and Euphorbia heterophylla.

Palavras chave: Hospedabilidade de plantas daninhas, fator de reprodução, disseminação. JB Pinheiro et al. T he diversity of hosts and their interaction with other pathogens make the nematodes one of the main phytopathogens responsible for limiting agricultural productivity worldwide (Moens et al., 2009). These pathogens are widely distributed in the most diverse agricultural areas all over the world, in several annual and perennial crops, causing damages of approximately U$ 157 billions annually (Bellé et al., 2017).
For vegetables, intensive cultivation has promoted the development of various diseases, such as occurrence of nematodes, with significant losses of heavily nematode-infested crops. Despite this pathogen's low natural mobility, constant soil tickler and machinery use, typical vegetable crop production operations, favor this spread. The root-knot nematode (Meloidogyne spp.) is quite destructive on most vegetables. In Brazil, M. javanica and M. arenaria are the most important pathogens on these crops (Pinheiro et al., 2018).
In Brazil, M. enterolobii sin. M. mayaguensis was reported parasitizing guava plants in the state of Bahia, causing several damages . This nematode has been identified in several plant species, including vegetables, in other regions of Brazil since then (Damaceno et al., 2016), even in cultivars resistant to other root-knot nematode species (Melo et al., 2011).
Weeds which grow next to crops can multiply the nematode inoculums and ensure the maintenance of high population densities of these pathogenic organisms in the soil, in the harvest or in the off season, making it difficult to control nematodes in production areas (Mônaco et al., 2009). Thus, characterizing weeds which are nematode hosts for suitable controlling in production areas is extremely important.
Studies on weed hosts to various species of root-knot nematodes can be found in literature, showing contrasting results, though. Moreover, no reports on evaluation of several weed species to M. enterolobii can be found. Given the above, the aim of this study was to evaluate the reaction of weeds, in vegetable production areas, to M. incognita and M. enterolobii.

MATERIAL AND METHODS
The trials were carried out in a greenhouse and the evaluations were done in the laboratory, at Embrapa Hortaliças, Brasília-DF, from October 23, 2014 to January 15, 2015.
The experimental design was completely randomized, with six replicates. Fifteen weed species were evaluated in relation to reaction to M. incognita race 1, and 16 species were evaluated in relation to reaction to M. enterolobii. Weed seeds were obtained from Agrocosmos Company, located in Engenheiro Coelho-SP, and selected based on botanical survey on an area cultivated during several years with vegetables in Brasília.
For both trials, female root-knot nematodes (M. incognita), collected from tomato roots in the experimental area of Embrapa Hortaliças, and females of M. enterolobii obtained from guava crop in Petrolina-PE, were previously identified using isoenzyme standard . Then, these pathogens were submitted to perineal cut and patterns described by Eisenback & Hirschmann-Triantaphyllou (1991) for species identification. Nematodes were, then, multiplied on tomato plant 'Rutgers', kept in a greenhouse. M. incognita was identified using host test, according to Taylor & Sasser (1978). After identification, nematodes were inoculated on tomatoes 'Rutgers', in order to produce and maintain the inoculums. About 45 days after inoculation, second stage eggs and juveniles (J2) were extracted from the plants to be used in the trials.
In both trials, weed specie's seeds were sown in 1.5-L plastic pots containing substrate composed of soil, washed sand, cattle manure and rice husk, in 1:1:1:1 ratio. After 26 days, thinning was carried out, leaving one plant per pot (experimental plot). Then, the seeds were inoculated with suspension of 5,000 eggs and eventual second stage juveniles (J2) of the nematodes in 5 mL water distributed around the base of the plants.
At 56 days after inoculation, egg mass index (IMO) was evaluated. Root systems were washed in running water and stained during 15 minutes in an aqueous solution of phloxine B, 0.5 g/L water (Taylor & Sasser, 1978). Then, the number of eggs on the roots was counted, using a stereoscope microscope.
To evaluate number of eggs per gram of roots (eggs/g roots), the roots were dried at room temperature during five hours, then weighed and processed according to Hussey & Barker (1973) modified by Boneti & Ferraz (1981).
Reproduction factor (FR) was obtained by the relationship between final and initial population densities (FR= Pf/Pi) (Oostenbrink, 1966). As initial population (Pi), inoculums of 5,000 eggs and eventual juveniles (J2) were considered.
A f t e r v e r i f y i n g A N O VA assumptions, data were submitted to analysis of variance and Scott-Knott clustering test at 5% probability, using GENES statistical software (Cruz, 2016).

RESULTS AND DISCUSSION
According to analysis of variance, significant differences (p= 0.05) were observed for all variables in relation to the reaction of weeds to nematodes. The genotypic and phenotypic coefficients of variation ratio, being superior to unit, show the predominance of genetic variation compared to environmental variation (Cruz et al., 2012). This fact shows reliability of estimates obtained for most evaluated traits. The authors observed exception for number of eggs per gram of roots (eggs/g roots) for M. enterolobii, showing that despite the detection of significant differences in weed reaction to this species, the results were unreliable.
Gall index (IG) and egg mass index (IMO) were similar for the evaluated weeds in relation to reaction to M. incognita race 1, showing greater presence of galls and eggs in the susceptible control (tomato 'Rutgers'), followed by I. triloba and I. nil. Lower values for these indexes were obtained for Tagetes sp. and S. oleraceus, followed by the cluster formed by S. rhombifolia, S. americanum, M. perfoliatum In relation to reproduction factor (FR) of M. incognita race 1, in evaluated weeds and controls, the greatest value was obtained for the susceptible control 'Rutgers', showing the initial nematode population multiplied throughout vegetative cycle of the crop by a factor of 34.6 times. The second species in which greater nematode multiplication was noticed was I. triloba. Although not being statistically different from the most resistant species, E. indica and I. nil presented susceptibility reaction according to Oostenbrink (1966 Gall index (IG) and egg mass index (IMO) according to Taylor & Sasser (1978); 0= roots without egg mass and/or galls; 1= roots with 1 to 2 egg masses and/or galls; 2= roots with 3 to 10 egg masses and/or galls; 3= roots with 11 to 30 egg masses and/or galls; 4= roots with 31 to 100 egg masses and/or galls; 5= roots with over 100 egg masses and/or galls; 2 Eggs/g= number of eggs per gram of roots; 3 FR= reproduction factor (FR), final population/initial population; 4 Resistance reaction according to Oostenbrink (1966): I= immune (FR= 0); R= resistant (FR<1) and S = susceptible (FR>1). Averages followed by same letters do not differ from each other, Scott-Knott test (p<0.05). CVg/CV= genotypic and phenotypic coefficients of variation ratio. The greatest number of M. enterolobii eggs per gram of roots was found in the susceptible control (tomato 'Rutgers') and in I. triloba, followed by the cluster formed by I. nil, S. americanum and E. heterophylla. The other species were clustered with lower number of eggs per gram of roots (Table 2). According to the clustering for FR, the highest value was obtained for tomato cultivar 'Rutgers', followed by I. nil, S. americanum, I. triloba, E. heterophylla, and tomato 'Nemadoro' (resistance standard); all of them were considered susceptible, according to Oostenbrink (1966). Using the same methodology, H. suaveolens and P. oleracea were also considered susceptible, although being clustered among species with lower FR. The other weed species were classified as resistant; any of them was considered immune to this nematode.
Considering the 11 common weed species in both trials, besides the two controls, we verified that M. enterolobii was more aggressive, managing to maintain or multiply on more weed species than M. incognita race 1. Considering these weed species, eight were considered resistant or immune to M. incognita race 1, and only five were considered resistant or immune to M. enterolobii. Only E. indica showed to The reaction of the weed species to M. enterolobii was directly related to botanical family. All tested Asteraceae species (Acanthospermum australe, Ageratum conyzoides, Bidens pilosa and Tagetes sp.) showed the same behavior: all they were resistant; the three amaranth species (Alternanthera tenella, Amaranthus hybridus and A. viridis) were also resistant; the two Convolvulaceae species were susceptible; and the two Poaceae species (Digitaria horizontalis and Eleusine indica) were resistant. These results show similar response of the weed species, belonging to the same family, to this nematode. This result is positive for further studies and nematode management strategies in the field. Weed differentiation at family or genus level is already sufficient to add information on nematode management, regardless of whether weed control is chemical, using herbicides; handpicking or mechanical, using a hoe, a harrow, a grid or growers.
To m a t o ' N e m a d o r o ' , w h i c h presented immunity reaction to M. incognita race 1, in relation to reproduction factor, with E. heterophylla and Tagetes sp, did not show the same immunity reaction to M. enterolobii. This result was expected for tomato, since this gene comprises resistance to M. incognita, M. javanica and M. arenaria, but does not show resistance to M. hapla and M. enterolobii. Meloidogyne enterolobii is known as a nematode which causes damages even to crops which carry genes showing resistance to other species of Meloidogyne spp. (Tigano et al., 2010).
Some studies about reaction of weeds to M. incognita can be found in literature. Mônaco et al. (2009) have studied the reaction of 57 species to M. incognita race 1. Considering these species, E. heterophylla, A. hybridus, A. viridis, I. nil, S. rhombifolia, S. americanum and S. oleraceus were also evaluated in this study. Ipomea nil, S. oleraceus and S. americanum showed the same reaction in both studies. Euphorbia heterophylla was classified as resistant in the first study, A. hybridus and A. viridis, as susceptible; and S. rhombifolia, as immune; however, in this study, the first was considered immune and the others resistant. Cordeiro et al. (2014) evaluated the reaction of 10 weed plants to M. incognita, without identifying the race, and among those which were common to this study, E. heterophylla, S. americanum, B. pilosa, all were considered resistant, which is in accordance with this study, considering the last two ones; whereas, E. heterophylla showed to be immune.
Thus, Bellé et al. (2017), studying the reaction of 34 weed plants to M. incognita, without identifying the race, verified that any species was immune to the nematode, 10 species were resistant and the others, susceptible. I. nil, E. indica, E. heterophylla, A. hybridus, A. viridis, B. pilosa, S. rhombifolia and S. americanum behaved the same way in this study. The first was considered susceptible, which is in accordance with this study; however, the others showed a different reaction. E. indica and E. heterophylla, which were classified as susceptible and resistant, respectively, in Bellé et al. (2017), were classified as resistant and immune, respectively, in this study; whereas all the other ones were considered susceptible in the cited study, and resistant in this study.
Similarly, Silva et al. (2013), evaluating the reaction of 23 weed species to M. incognita and M. javanica, without identifying races, concluded the same reaction pattern to two nematode species, considering 11 weed plants susceptible and 12 resistant. A. hybridus, H. suaveolens, S. americanum, B. pilosa and E. heterophylla were also evaluated in this study, and the reaction of B. pilosa was the same verified in this study; however, the others behaved differently: the three first ones were considered susceptible and in this study they were considered resistant. E. heterophylla was considered susceptible, in these evaluations it was considered immune. The same authors also evaluated S. cordifolia, another guanxuma species, which showed resistance standard, in accordance with this study for S. rhombifolia.
Contrasting reaction patterns of some weeds found in literature for M. incognita can be explained using different conditions of evaluations of the experiments, such as, inoculation time after sowing, inoculum density, origin of isolate, identification or not of the races, time of evaluation after inoculation, among other factors.
In relation to reaction of weed plants to M. enterolobii, Almeida et al. (2011) collected weed plants in areas infested by this nematode in guava plants (Psidium guajava), are common in this study B. pilosa and S. americanum. The results showed that both plants presented eggs and juveniles in their roots, as in this study. In a similar study, also collecting weed plants in an area under guava cultivation, infested by M. enterolobii, Carneiro et al. (2006) studying 10 species of weed plants and cultivated, among these weeds B. pilosa and Tagetes minuta, verified the presence of females of these nematodes in B. pilosa and absence of T. minuta, similar situation observed in this study. Souza et al. (2006), also using 14 weed plant species, among them A. hybridus and S. americanum, observed the presence of females of this nematode in the roots, confirming that this species of nematodes is polyphagous and that knowing the plants which host or multiply this nematode is important for the control.
In general, most weed species evaluated in this study were hosts for both nematodes. Only U. lobata, S. oleraceus, E. heterophylla, M. perfoliatum and Tagetes sp. were immune, it means, they neither hosted nor allowed the multiplication of M. incognita race 1. However, all evaluated species were host or provided multiplication of M. enterolobii. The species which were the most susceptible to M. incognita race 1, and that, therefore, need to be controlled more carefully in crops infected by this nematode were I. nil, I. triloba and E. indica. Regarding M. enterolobii were I. nil, S. americanum, H. suaveolens, P. oleracea, I. triloba and E. heterophylla. In addition, M. enterolobii species are more easily maintained and multiply in common weeds in vegetable crops than M. incognita race 1.