Crambe Cake to Meloidogyne javanica Control in Lettuce

Crambe is an oilseed, which pressing for oil extraction results in the waste called crambe cake. The aforementioned waste may present potential to control nematodes, since it derives from brassica species. The aim of the current study is to assess the best crambe cake application to control Meloidogyne javanica in lettuce plants. Five experiments were carried out in a greenhouse by adopting different crambe cake application procedures; each experiment comprised five treatments (0 (control), 5; 10; 15; 20 g crambe cake per 1 L soil). Lettuce seedlings were cultivated in soil treated with crambe cake, and inoculated with 5,000 nematode eggs and occasional juveniles (J2). Nematological and vegetative parameters were assessed 45 days after inoculation. Nematode reduction was observed in the experiment that applied doses close to 15 g crambe cake to the soil surface; nematode control recorded 83 and 68% for eggs and J2 total and per root gram, respectively. The same parameters showed up to 82 and 93% reduction when the cake was incorporated to the first 8 cm deep into the soil. The number of eggs and J2 per root system reduced by 93% when the cake was incorporated to the total soil volume. Overall, the crambe cake did not increase plant development; in some cases, phytotoxicity was observed at the highest doses.


Introduction
Plant parasitic nematodes stand out among the most destructive pathogens found in agriculture, since they lead to significant annual losses in susceptible plants.Meloidogyne javanica (Treub) Chitwood and M. incognita (Kofoid & White) Chitwood are reported as the nematodes most affecting lettuce crops, mainly in tropical and subtropical countries, where the continuous lettuce cultivation in certain areas leads to significant nematode population increase due to successive pathogen cycles (Pinheiro et al., 2013).
Root-knot nematodes are sedentary endoparasites; females have piriform body and produce, on average, 500 eggs per life cycle, which, under favorable conditions, is completed in four weeks.During the parasitism process, female nematodes induce specific sites for feeding, called giant cells, which present high cellular hypertrophy and hyperplasia (Pinheiro et al., 2013) and lead to the emergence of root nodes, called galls.Managing these parasites is a complex task due to the small number of cultivars presenting high resistance levels (Fiorini et al., 2007;Dias-Arieira et al., 2012).Crop rotation using non-host or antagonist plants is recommended as management practice (Moraes et al., 2006;Santana et al., 2012); however, producers who intensively use cultivation areas show low acceptability to such practice.
Thus, adding organic matter to the soil is one of the most efficient methods for the sustainable control of nematodes affecting vegetables; different organic wastes showed nematode management potential (Lopes et al., 2009;Nazareno et al., 2010;Roldi et al., 2013;Dias-Arieira et al., 2015).The organic matter addition also presents other benefits such as natural enemy population increase and improvements in the physical and chemical properties of the soil, including base saturation, porosity and water conductivity, which allow plants to develop better, as well as to become more resistant to these pathogens (McSorley & Gallaher, 1995;Oka, 2010).Wastes from animal husbandry and agro-industrial processes stand out among the investigated organic matters.Cakes derived from oilseed pressing for vegetable oil or biodiesel production purposes stand out among agro-industrial wastes.It is worth highlighting the filter cake, which is generated in alcohol production plants, as well as the castor bean cake, which derives from the pressing of grains for biodiesel production purposes; both cake types proved to be effective in nematode management studies (Albuquerque et al., 2002;Lopes et al., 2009;Roldi et al., 2013).
Although crambe cake shows nematode control potential, it may also present phytotoxic effects (Tavares-Silva et al., 2015).In addition, there is lack of information about efficient doses able to help managing these pathogens.Therefore, the aim of the current study was to assess different crambe cake application forms and doses to control M. javanica in lettuce plants.

Methods
The experiments were conducted in a greenhouse located at the geographic coordinates 23º47′28.46″S and 53º15′23.46″W, altitude 430 meters; they followed a completely randomized design, with five treatments (0 (control), 5; 10; 15; 20 g crambe cake/L soil) and five replications.Three experiments were conducted separately; they differed from each other according to the way the cake was applied, namely: superficially, incorporated to the first 8 cm deep into the soil, or incorporated to the total soil volume.The experiments with applied crambe cake applied superficially and incorporated to the first 8 cm were conducted in two different environments: greenhouse with plastic cover and 50% shading screen (Experiment 1) and greenhouse with plastic cover and 75% shading screen (Experiment 2); the last experiment, crambe cake incorporated to the total soil volume, was conducted in a greenhouse with plastic cover and 50% shading screen, only.The experiments were carried out between January and April 2017.
Lettuce seedlings cv.Vera were initially produced in polyethylene trays containing BioPlant ® commercial substrate.Plants showing the first fully-expanded pair of leaves were transplanted to pots containing 1 L of soil:sand (2:1) mixture, which was previously autoclaved at 120 ºC for two hours; the soil was characterized as dystrophic Red Latosol.
The crambe cake was applied to the substrate (mixture), on the transplantation day, at the previously mentioned doses and treatment types.A cake sample was subjected to laboratory chemical analysis; results showed 43.40 g/kg, nitrogen, 5.83 g/kg phosphorus, 1.32 g/kg potassium, 0.33 g/kg calcium, 0.08 g/kg magnesium, 8.43 g/kg sulfur, 23.75 g/kg iron, 3.60 g/kg manganese, 0.88 g/kg copper, 4.42 g/kg zinc, 3.67 g/kg boron, 54.37% organic carbon and 93.51% organic matter.
Each plant was inoculated with 5,000 M. javanica eggs and eventual second-stage juveniles (J2) three days after transplantation.The herein used inoculum was obtained from a pure nematode population kept in tomato roots (cv.Santa Clara) and it was extracted according to the methodology by Hussey and Barker (1973), adapted by Boneti and Ferraz (1981).The suspension was calibrated for 2,500 eggs and eventual J2/ml, using a nematode count slide (Peters' slide) under light microscope.The inoculum was deposited in two equidistant holes in the soil around the root crown.
Plants were collected 45 days after inoculation; shoots and roots were separated.The root system was carefully washed and placed on absorbent paper to remove water excess; next, the root fresh weight was measured.Subsequently, nematodes were extracted according to the previously mentioned methodology.Finally, the number of eggs and J2 was assessed in Peters' slide, under light microscope; the recorded value was divided by the root weight in order to find the number of eggs and J2 per root gram.The shoot fresh and dry weights were assessed; shoot dry weight was recorded after the shoot was dried in a forced air circulation oven (65 ºC) until it reached constant weight.jas.ccsenet.Data were through re

Results
Both expe control-t (Experime 1A).The t maximum whereas in    (2015), in which the application of 20 g cake to 2 L soil increased the shoot fresh weight from 27 to 50%, as well as the root weight from 14 to 23%, in lettuce plants.Some factors-such as the chemical composition of the crambe cake, which was not presented in the study by Dias-Arieira et al. (2015), the plant permanence period (which was 60 days in the aforementioned study), and the soil volume used in the experiment-may have contributed to the difference recorded in these results.
Data suggest that it is necessary taking precautions at the time of use high organic compound doses, because high concentrations of some micronutrients, such as manganese and iron, may favor the cellular redox balance, lead to oxidation, and trigger morphological, biochemical and physiological symptoms, which may lead to decreased plant development (Hell & Stephan, 2003).In addition, glycosinolates and their derivatives, which may be responsible for nematode control, can have allelopathic effect on plant development (Eberlein et al., 1998).Thus, the incorporation of Brassica napus and crambe wastes reduced germination, delayed seedling emergence, and decreased root length and shoot dry weight in maize (Spiassi et al., 2011).Phytotoxic effect was also observed in tomato plants when 1% (v: v) crambe cake was applied to 0.33 L pots (Walker, 1996).However, the aforementioned author reported decrease in this effect after three weeks of compound reaction to the soil; it led to the hypothesis that the initial crambe cake fermentation process may release toxic compounds, as it was observed in other organic matter sources (Sediyama et al., 2008).In addition, the root fresh weight may have had its growth reduced due to high nutrient availability near the root system, which did not stimulate root growth (Raij, 2011).
It is emphasizing that the plant shoot was not affected when crambe cake was incorporated to the first 8 cm deep into the soil or to the total soil volume.In addition, plants grown in commercial areas are often subjected to less stress, because there is no physical constraint to their vegetative development and due to microorganisms able to speed up organic matter decomposition.Thus, complementary studies at field level should be conducted in order to confirm the best crambe cake dose to be applied, as well as to assess the previous fermentation of the material to be used.
In this way, it was concluded that crambe cake reduced the nematode population in the root system, regardless of the application form.The highest doses (20 g in 1 L soil) caused phytotoxicity and compromised the vegetative development of lettuce plants.However, the incorporation of crambe cake to the soil reduced the negative effect on the plant.