Meloidogyne javanica control in lettuce with fertilizers applied isolated or associated with biological product

Root-knot nematodes limit lettuce crop productivity. Efficient control is achieved through integrated management, and research about the efficiency of new products for nutrition and biological control is required. Thus, the objective of this study was to evaluate the effect of fertilizers, applied alone or in combination with microorganisms based product on the control of Meloidogyne javanica in lettuce. In the first research (research 1), conducted at two different periods, the products Agro-Mos®, Soil-SetTM and CopperCrop® applied on shoots were evaluated; in research 2, the same products were applied isolated on shoots or associated to NemOutTM (Bacillus licheniformis, B. subtilis and Trichoderma longibrachiatum) applied to the soil. In research 1, the fertilizers Agro-Mos®, Soil-SetTM and Copper-Crop® applied alone promoted reduction in reproduction of M. javanica ranging from 31 to 75%, 36 to 79%, and 71 to 75%, respectively. The effect on plant development was variable, obtaining better results of fresh shoot mass applying Copper-Crop®. The combination of products did not have an additional effect on the control of the nematode, on the contrary, in general the products had antagonistic action. However, fertilizers and biological control applied isolated were efficient for nematode control.

Palavras-chave: Lactuca sativa, controle alternativo, nematoide das galhas, manejo integrado. products available on the market are highly toxic and there are no specific chemicals registered for the lettuce crop in Brazil.
Crop rotation with non-host plants or antagonist is one of the most suitable methods for nematode control in vegetables (Pinheiro, 2017). However, it has limitations due to the wide host range of the root-knot nematodes and the resistance of the producer, who should keep the planting area without commercial production for a certain period.
Regarding control of nematodes, research for alternative practices that may help in reducing the population or improving the productivity of the plant even in the presence of nematodes is constant. In this context, products intended for plant nutrition may be a viable alternative since it is known that fertilizer use can directly affect nematodes, interfering with their life cycle (Couto et al., 2016;Rumiani et al., 2016), or indirectly, altering the resistance of the plant to them (Costa et al., 2010;Junqueira et al., 2011;Gomes et al., 2016;Miamoto et al., 2017).
Biological control is another alternative that has been emphasized. It consists on the application of microorganisms that have some direct or indirect effect on the nematodes. The activity of the rhizobacteria of the genus Bacillus spp., as well as the fungi of the genus Trichoderma, has been investigated, mainly for the control of the root-knot nematode (Sharon et al., 2011;Yu et al., 2015;Zheng et al., 2016;Hu et al., 2017). However, several products marketed as soil conditioners based on these organisms have not yet been investigated about the effect on nematodes.
Considering the information previously discussed, this study aimed to evaluate the efficiency of nutrients and microorganisms based products to control M. javanica in lettuce.

Search 1 -Fertilizer applied to shoot in control of Meloidogyne javanica
The research was carried out in a greenhouse at Universidade Estadual d e M a r i n g á , i n U m u a r a m a -P R (23º47'28"S, 53º15'23"W, 430 m altitude), in completely randomized design with four treatments and five replicates. The research was conducted in two different periods, the first between April and August, 2015 (called Research 1, Trial 1), under mean minimum and maximum temperatures 16.8 and 26.3°C, respectively. And the second one between November 2015 and March 2016 (called Research 1, Trial 2), whose temperatures were 21.1 and 30.3°C, respectively. Tomato seedlings (Solanum lycopersicum), cultivar San Marzano, were produced in 128-cell polystyrene trays, containing Bioplant ® commercial substrate and, 25 days after germination, were transplanted to pots containing 2 liters of a soil:sand mixture (2:1), previously autoclaved (120°C/2 hours). Ten days before transplantation, 1.14 g lime and 2.80 g NPK (20-20-20) were applied to the soil. Two days after transplantation, the seedlings were inoculated with a suspension of 4 mL containing 2,000 eggs and eventual second stage juveniles (J2) of M. javanica, in four open perfurations in the soil around the seedling. The inoculum was obtained from a pure population of the nematode, maintained in roots of tomato cv. Santa Clara and previously extracted according to the methodology proposed by Hussey & Barker, adapted by Boneti & Ferraz (1981). This part of the research had the objective of simulating a naturally infested soil in which the nematode is found in egg masses or in residual roots.
Lettuce plants were irrigated daily and kept in a greenhouse until 45 days after transplantation. Subsequently, plants were collected, separating shoots and roots. Roots were carefully washed, placed on absorbent paper to remove excess water and fresh mass was determined. Afterwards, nematodes were extracted according to the methodology already mentioned, and the total number of nematodes was evaluated in a Peters chamber under light microscope. This total number of nematodes was divided by the root mass, obtaining the number of nematodes/g of roots. In Research 1, Trial 2, besides the parameters already mentioned, fresh mass and dry mass of shoots were evaluated. Dry mass of shoots was obtained after drying the material in an oven with forced air circulation at 65ºC during three days.
Data were submitted to analysis of variance at 5% probability. For nematological parameters, the original data were transformed by √(x + 0.5) to meet the normality assumptions, according to Shapiro Wilk. Then, means were compared by Tukey test at 5% probability, using the statistical program Sisvar (Ferreira, 2011).

Search 2 -Combination of fertilizers and Nem-Out TM for control of Meloidogyne javanica
This research was similar to research 1, however, it was carried out between January and May 2016, whose temperatures were 20.0 and 29.5°C respectively, using pots containing 1 L of autoclaved soil:sand substrate (2:1). In addition, this research was in a 4 x 2 factorial, with four treatments in shoots (water, Agro-Mos ® , Soil-Set ™ and Copper-Crop ® ), with or without Nem-Out™ [Bacillus licheniformis, B. subtilis, Trichoderma longibrachiatum, with total microbiological count of 3.75 x 10 8 CFU g -1 ; protease, silanase, cellulase of which amounts were not stated by the manufacturer] at a dose of 5 kg/hectare, as recommended by the manufacturer (Improcrop ® do Brasil Ltda.), applied to the planting furrow at the time of transplantation. Fifty-six plants were evaluated, one per experimental unit.
In this research, the same parameters described in Research 1, Trial 1 were evaluated. Statistical analysis was performed as already reported in research 1, however, products applied to shoots were compared by Tukey test and the application of Nem-Out TM by the Bonferroni T test, both at 5% probability.

Research 1 -Fertilizers applied to shoots for the control of Meloidogyne javanica
There was no statistical difference in Trial 1 for the variable root mass (Table 1). In Trial 2, Agro-Mos ® and Soil-Set™ promoted an increase in root mass compared to the control. Agro-Mos ® presented the highest mean for this parameter (11.47 g), followed by Soil-Set™ treatment (8.52 g) (Table 1). Several factors possibly influenced the difference between these results, being the most probable the temperature, whose minimum and maximum averages in trial 1 were 16.8°C and 26.3°C, while in trial 2 were 21.1 and 30.3°C, respectively.
All treatments reduced the total number of M. javanica in both trials and the number of nematodes per gram of root in Trial 2. However, in trial 1, only the treatment Copper-Crop ® differed from the control for the number of nematodes/g of roots (Table 1), which may be due to differences in plant root system development. The treatment with Agro-Mos ® promoted 31 and 75% reductions in the total nematode population in trials 1 and 2, respectively.
Despite the shortage of studies aiming the control of nematodes with Agro-Mos ® , this product controlled M. javanica in soybean when applied alone on shoots (Miamoto et al., 2017), contributing not only to the control of the pathogen, but also to the development of the crop. In addition, recent studies revealed that Agro-Mos ® decreases the number of third and fourth stage juveniles (J3 and J4) of M. javanica within tomato roots, although it did not interfere with the number of J2 that penetrated the roots (Chidichima et al., 2018), which may be an indication that the product delays the development of the nematode in treated plants. Agro-Mos ® has phosphorylated mannanoligosaccharide from the cell wall of Saccharomyces cerevisiae as active ingredient, considered an inducer of resistance in plants (Junqueira et al., 2011).
In addition to nematode control, the efficiency of Agro-Mos ® in the control of other pathogens has been studied and the results have been positive in the management of fungal diseases of shoot, including anthracnose in guava and leaf diseases in strawberry (Gouvea et al., 2009;Gomes et al., 2016). The application of Agro-Mos ® resulted in increased activity of chitinase and glucanase proteins, which have the capacity to hydrolyze fungal cells (Gouvea et al., 2009). The product also reduced the incidence of Moniliophthora perniciosa in cocoa seedlings, again with increased activity of some enzymes related to plant defense responses (Costa et al., 2010).
The application of Soi-Set TM reduced the total nematode population of M. javanica by 36 and 79% in trials 1 and 2, respectively (Table 1). Previous works showed reduction of Pratylenchus brachyurus in two experimental seasons in soybean and M. javanica in the same crop in one experimental season (Miamoto et al., 2017). This product is composed of sulfur, copper, iron, manganese and zinc, and also organic carbon. It is known that micronutrients are fundamental in plant metabolism, acting on the phenol and lignin content, as well as membrane stability (Marschner, 2012). Furthermore, some micronutrients may be related to nematode control or to the induction of plant resistance.
Manganese, for example, activates enzymes of the shikimic acid route, affects the phenylalanine ammonia lyase (PAL) enzyme and stimulates peroxidases necessary for lignin biosynthesis (Marschner, 2012). Copper, in turn, acts in the control of RNA and DNA synthesis. Copper is part of proteins and enzymes vital to plant metabolism and participates in several physiological processes, such as photosynthesis, respiration and carbohydrate distribution (Kabata-Pendias, 2011). This element is also cofactor of several enzymes in plant cells, by providing ions and, consequently, influencing cell wall composition and promoting increased synthesis of peroxides, quinones and phenolic compounds, which may induce plant resistance (Marschner, 2012). These attributions can elucidate the obtained results, since the nematodes use mechanical and enzymatic strength to degrade plant cell wall in the penetration process, movement in the cortical parenchyma and feeding. In the case of a more lignified cell wall, such activities will require greater energy expenditure, thus reducing nematode activity and, possibly, the reproduction. In addition, the presence of Zinc in the product formulation may have contributed to the results, since in the work carried out by Couto et al. (2016), the application of Zinc promoted the reduction of M. incognita population in tomato. Besides, Soil-Set TM was efficient in the preventive control of tomato bacterial spot (Xanthomonas spp.), reducing disease severity when applied to shoots (Rodrigues et al., 2016).
Copper-Crop ® was the product that promoted the greatest reduction of M. javanica reproduction in lettuce, which was equivalent to 71 and 85% in trials 1 and 2, respectively ( Table  1). The effect of Copper-Crop ® for disease management is limited to the protective effect against fungi and shoot bacteria. However, it is known that copper is an element with direct effect on such pathogens, inhibiting their activity at pre-penetration. However, it is not possible to extrapolate this mode of action to nematode control, since there is no direct contact between the product and the parasite. The copper activity in the defense mechanisms activation process discussed earlier can be applied here as well. Furthermore, it has recently been observed that the product reduced the formation of J3 and J4 of M. javanica in tomato seedlings (Chidichima et al., 2018).
Due to lack of studies, it is difficult to know the mechanisms of action by which these products control nematodes.
Additional studies, involving mainly the expression of resistance enzymes and alteration of root exudates, are necessary to elucidate recurrent questions about the mode of action of fertilizers on nematodes, especially for those applied to shoots.
In trial 2 it was possible to observe that Copper-Crop ® and Soil-Set™ increased fresh mass of shoot (74.55 and 60.29 g, respectively), relative to the control (29.90 g). All treatments were efficient in increasing shoot dry mass (Table 2). These results were already expected since they are products for plant nutrition. The effect of the different nutrients in the development of plants was elucidated in detail by Marschner (2012).

Research 2 -Combination of fertilizers applied to shoot with Nem-Out TM for the control of Meloidogyne javanica
There was no interaction between the factors for fresh root mass. Agro-Mos ® reduced the root system mass when compared to the control and Copper-Crop ® (Table 3). Interaction was observed between the factors for both nematological parameters. Nem-Out TM applied alone reduced nematodes total and nematodes/g of root, which corroborates with results observed in the soybean crop, in which the product was efficient in controlling M. javanica and P. brachyurus, when applied alone (Miamoto et al., 2017). In addition, Nem-Out TM was efficient in the management of Pratylenchus spp. in sugarcane, in researches under controlled conditions (Oliveira et al., 2011). One of the factors that may explain the efficiency of Nem-Out TM for nematode control is the presence of bacteria of the genus Bacillus, which have been shown to have multiple mechanisms of action, including the production of metabolites toxic to nematodes, competition for nutrients in the rhizosphere, changes in root exudates released by the plant and the ability to induce plant resistance to pathogens (Oliveira et al., 2014;Yu et al., 2015;Zheng et al., 2016;Govindasamy et al., 2017). Moreover, the presence of the fungus Trichoderma longibrachiatum of which the nematicidal effect on M. incognita is due to the high capability  to colonize the rhizosphere, competing with the nematode, and by its direct parasitism (Zhang et al., 2015). In fact, several species and isolates of Trichoderma have shown positive results for the management of plants parasitic nematodes, and their mode of action is attributed to alteration of root exudates, parasitism of eggs and juveniles, stimulus to vegetative development and induction of resistance in plants (Hoyos-Carvajal et al., 2009;Sharon et al., 2011;Zhang et al., 2015). All products used as foliar fertilizers promoted reduction in the total nematode population, in the absence of Nem-Out TM (Table 3), corroborating the results of Research 1. On the other hand, in the presence of Nem-Out TM , there was no difference between the treatments with fertilizers and the control. For nematodes per gram of root, the products applied alone did not differ and, in the presence of Nem-Out™, only Soil-Set TM showed reduction of M. javanica when compared to the control (Table 3). However, the application of Nem-Out™ reduced nematodes per gram of root in the treatmeants control, Agro-Mos ® and Soil-Set TM (Table 3).
Researches on the evaluation of shoot fertilizers on the control of M. javanica are still very scarce, considering that they are registered products with the purpose of supplying the need of nutrients by the plants, and not as pathogen control agents. However, it is known that fertilizers for aerial application can have deleterious effect when in direct contact with microorganisms, including Trichoderma spp. (Dłużniewska, 2008), even though indirect action is poorly studied. On the other hand, there are reports that such products may alter the exudation of substances by the roots, making them more or less attractive to microorganisms (Canarini et al., 2019), since colonization of the rhizosphere depends on the relationship between exudates and microorganisms (Zhang et al., 2014). So, as the fertilizers inhibited the action of the nematode, as presented and discussed in this study, it can be hypothesized that, in some way, they also alter the activity of the bacteria and the fungus that makes up the Nem-Out TM , altering the attractiveness of exudates or causing induction of resistance, in the case of these organisms establish some endophytic relationship. This hypothesis would explain the fact that both products control the nematode when applied alone, but without evident additional effect when associated with each other.
Since studies are conducted under controlled conditions, field research is needed to confirm whether nutrientbased products can be a strategy for composing integrated nematode management.