Juvenile stages 2 (J2) of phytonematode Meloidogyne incognita were obtained from egg masses of females present in intentionally infested tomato roots, from AGROCALIDAD (Tumbaco - Ecuador), the eggs were incubated until hatching of the juveniles in the second stage. The J2 were captured by means of a 100 μL micropipette (micro-fishing) until a sufficient population was completed for laboratory mortality tests.

For the extraction of eggs the methodology of McClure et al. (1973) and Taylor and Sasser (1978) was followed, for which 20 g of infested tomato roots were weighed, cut into pieces of approximately one centimeter, then disinfected with 4% sodium hypochlorite, rinsed with sterile distilled water and liquefied for 20 seconds at 3000 rpm. Finally, the liquid was filtered and sieved in two 60 and 500 mesh sieves; the samples of this last sieve were diluted to 20 mL. The eggs were disinfected with sodium hypochlorite, rinsed and incubated at average laboratory temperature (20°C).

For this essay, the species Pleurotus ostreatus NE1 and Trichoderma harzianum NE2 grown in the substrates (wheat straw and rabbit manure) were considered. The criteria for the selection of these two species of fungi and substrates were as follows: a) Pleurotus ostreatus NE1 was the best fungus from the laboratory mortality test since it was able to immobilize and kill nematodes in the shortest time (24 hours), b) Trichoderma harzianum NE2 was taken into account even though it was not the best in the laboratory test, however, this genus is referred to in many investigations as a plant growth promoter, c) the rabbit manure substrate being organic matter, is assumed to exert double action when carrying the fungus and fertilizing the plants, d) the wheat substrate, because it was one of the best substrates that allowed the development of these two species of fungi in the laboratory.

Due to the temperature requirements of tomato plants, the test was established in a greenhouse, where the average recorded temperature was ± 24°C, the tomato variety used was Daniela, sown in one-litre polystyrene pots (Flex foam), which were filled with soil from the place that had the following characteristics: pH: 8,4 (slightly alkaline), MO: 0.6% (low); NH4: 15.6 (low); P: 31.8 (high); K: 0.95 (high). Approximately 2000 Meloidogyne incognita J2 phytonematodes were inoculated into each of the pots, the same ones that were obtained from root galls of 4-month-old tomato plants purposely infested. They were washed with plenty of water to remove the soil, then they were disinfected with 4% sodium hypochlorite for 5 minutes and with the help of a scalpel and dissecting needle, the nodules and egg masses were removed and deposited in Petri dishes with sterile distilled water plus chloramphenicol (0.01 g 100 mL^-1) for 2 minutes and finally they were placed in a container with sterile distilled water at room temperature until the eggs hatched (6-10 days).

Once the pots were irrigated to field capacity, 5 g plant^-1 of each of the selected bioformulates (Nematofagous fungi plus solid substrates) selected from the previous stage (Pleurotus ostreatus NE1 developed in wheat straw and rabbit manure, Trichoderma harzianum NE2 developed in wheat and rabbit manure) were applied. Finally, in the pots with sterile Canadian peat, tomato seedlings with 4 leaves were transplanted. In 180 tomato plants were evaluated the following parameters: plant height, stem diameter, number of leaves, number of galls in the roots, fresh and dry weight of the aerial and roots at 60 days after transplant.

A complete random design (CRD) with three replications was used, the coefficient of variation and the Tukey’s Studentized Range (HSD) Test were determined at 5% using the INFOSTAT program, trough ANOVAS. The correlation analysis was determined with the Pearson test and the Z-test was applied for the hypothesis test.

When analyzing the effect of evaluated treatments on the galling and development of tomato in greenhouse, we can observe mainly a very close tendency between the Chemical control (Fenamiphos) with the selected bionematicides, reflected in a summary of all results obtained ± SD (Standard deviation) and the level of significance of each one (Table 1).

Data are average value of three replications, mean values of different superscript letters are significantly different and same letters are not statistically different by Tukey’s Studentized Range (HSD) Test (p < 0.05).

Table 1
Average results obtained in tomato seedlings grown in the greenhouse 60 days after transplant (media ± SD)

The analysis of variance for plant height at 60 days after transplant showed a significant difference for treatments, with a coefficient of variation of 4.32%. According to the Tukey test at 5%, it was observed that the chemical control (Fenamiphos) occupies the level “a” with 34.90 cm, while the level “c” was occupied by the absolute control with 27.60 cm. In addition, according to the analysis of variance for stem diameter and number of leaves, there was no significant difference.

According to the analysis of variance for number of galls at 60 days after transplant, there was a highly significant difference between treatments, with a coefficient of variation of 20.76%. Using the Tukey test at 5% in the treatments: Chemical control (Fenamiphos), Trichoderma-rabbit manure, Pleurotus-rabbit manure, Trichoderma-wheat straw and Pleurotus-wheat straw, a lower amount of galls per plant was observed, occupying level “a” (since 0.27 to 2,47); while the absolute control occupies level “b” with the highest number of galls per plant (18.27). These results showed that these antagonistic fungi are an effective option for the protection of this crop against the phytonematode Meloidogyne incognita.

In the same way, the analysis of variance for fresh weight of the aerial part of the plant at 60 days after transplant showed a highly significant difference for treatments, with a coefficient of variation of 8.08%. According to the Tukey test at 5%, the chemical control (Fenamiphos) occupied level “a”, with 59.61 g, while the treatment Pleurotus-rabbit manure occupied level “ab” with 52.56g and finally level “c” was the absolute control with 38.58 g.

The analysis of variance for fresh weight of the root at 60 days after transplant showed a highly significant difference for treatments, with a coefficient of variation of 8.10%. According to the Tukey test at 5% Trichoderma-rabbit manure treatment occupied level “a” with 11.15 g, followed by Pleurotus-rabbit manure (level “ab”) and Pleurotus-wheat straw (level “bc”) with 10.12 and 9.29g respectively and the last level “c” was occupied by absolute control with 6.66 g.

The analysis of variance for dry weight of the aerial part of the plants at 60 days after transplant showed a highly significant difference for treatments, with a coefficient of variation of 9.34%. The Tukey test at 5% showed that the chemical control (Fenamiphos) treatment occupied level “a” with 10.09 g, followed by Pleurotus-rabbit manure and Pleurotus-wheat straw (level “ab”) with 8.68 and 8.38 g respectively. Trichoderma-rabbit manure occupied level “abc” with 8.04 g and at the level “c” the absolute control was found with 6.25 g.

The analysis of variance for root dry weight at 60 days after transplant was highly significant for treatments, with a coefficient of variation of 13.40%. The Tukey test at 5% indicated that with the chemical control (Fenamiphos), greater root weight was achieved, represented by level “a” with 3.39 g, while the treatment Trichoderma-rabbit manure occupied level “ab” with 3.25 g and the absolute control occupied level “b” with 1.75 g.

In addition, it is appreciated that the treatments formed by the bioformulates constituted by Pleurotus ostreatus-rabbit manure and Trichodermaharzianum-rabbit manure contributed with fresh and dry weights of the aerial part and roots similar to chemical control shows that biological measures can be implemented with the purpose of diminishing the use of pesticides of chemical synthesis, which produce vegetables with traces of dangerous substances, risking the health of producers, consumers and the environment.

According to Figures 1 and 2, a correlation between number of galls and dry weight of the aerial and root parts of the plant was determined, reflected in the values of r with 0.640 and 0.792 respectively.

According to the hypotheses raised in this research, consisting of:

Ho (null): Bionematicides formulated with native fungal strains are not able to reduce the number of galls in tomato plants grown in the greenhouse by at least 60% compared to a synthetic nematicide.

Hi (alternating): Bionematicides formulated with strains of native fungi are able to reduce the number of galls in tomato plants grown in the greenhouse by at least 60% compared to a synthetic nematicide.

According to the results obtained, the value -1.65 indicates that the alternating hypothesis is accepted, that is to say that the bionematicides formulated with strains of local nematophagous fungi, controls the galling of the roots of tomato plants grown in greenhouses in at least 60% of a synthetic nematicide (Table 2) (Figure 3).

Figure 1
Correlation between number of galls and dry weight of the aerial part of the plant. ** Highly significant correlation

Figure 2.
Correlation between number of galls and dry weight of the plant root. ** Highly significant correlation

Table 2
Z test to determine the effect of bionematicides and the number of galls on the roots of plants

Figure 3.
Tomato seedlings in greenhouse phase (A), Root infested with Meloidogyne incognita (absolute control) (B), Roots inoculated with bionematicides (C and D).

According to the results obtained, the potential damage caused by Meloidogyne incognita was significantly reduced, whose effect was probably increased by the addition of organic matter (rabbit manure), and so, the development of the seedlings was evidenced in a higher radicular and aerial dry weight and lesser number of galls. Trichoderma harzianum-rabbit manure, Pleurotus ostreatus-rabbit manure and P. ostreatus-wheat straw allowed obtaining higher fresh and dry weight values of the plant at 60 days after the greenhouse transplant. The benefit of the addition of organic matter to the soil has been widely recognized, which is able to reduce some diseases and pests such as phytonematodes (Singht & Sitaramaiah, 1970).

The results obtained in this research can be related to those achieved in the study on the potential role of bioinoculants and organic matter for the management of the root knot nematode in infested chickpea, where it was demonstrated that the combined application of biological agents and organic matter is very useful for the producers of this legume to reduce root knot disease, in addition to minimizing environmental disturbances, caused by pesticides of synthetic origin and preserve the flora, fauna and microbial diversity in soil (Taylor & Sasser, 1978).

Other studies show that organic matter is beneficial not only when combined with biological controllers, but also with plants with nematicidal action such as neem. As evidenced by the work on sustainable management of root knot disease in tomato with neem cake and Glomus fasciculatum in Aligarh, India, the nematicidal potential of the organic matter and the neem cake and the biological agent, G. fasciculatum and its positive influence on tomato growth, was determined when inoculated individually, as well as in combination, while providing sources of nutrients for the proper growth of plants, as the intensity of the disease caused by the knot nematode decreased (Akram, Rizvi, Sumbul, Ansari, & Mahmood, 2016).

Likewise, the positive effect of Trichoderma harzianum and Nerium indicum has been proven either alone or in combination, that it reduced the severity of the disease complex caused by the nematode Meloidogyne javanica and Rizoctonia solani. It is stated that, the incorporation of biofertilizers significantly reduced the multiplication of the nematode, root-galls, egg mass/root system, and also reduced the root rot of R. solani to the hundred. It was concluded that the combined application of N. indicum and T. harzianum showed maximum improvement in growth variables compared to their individual application, as well as untreated control. In addition, the maximum inhibition in the incidence and multiplication of the nematode of the root knot was observed (Vásquez & Álvarez, 2011).

Probably, another explanation is the resistance induced in plants, caused by some microorganisms, as shown by the study carried out with the genus Arthrobotrys oligospora, the same one that grew inter and intracellularly in roots of barley and tomato, but did not penetrate the vascular tissues of plant roots. In addition, it also induced plant defense reactions without impairing their development, making them more resistant to phytoparasitic nematodes and/or other pathogens (Askary & Martinelli, 2015).