Hatching and Mortality of Meloidogyne enterolobii Under the Interference of Entomopathogenic Nematodes In vitro

The experiments were carried out in incubator chambers (biochemical oxygen demand—BOD) at 25°C, 70% of relative humidity, of the Laboratory of Agricultural Nematology of the Department of Plant Protection of the College of Agronomic Sciences at the Paulista State University “Júlio de Mesquita Filho”, Botucatu-SP.

The pure population of M. enterolobii used in the experiments was obtained from tomato plants kept in greenhouse as an inoculum source and multiplied in ‘Rutgers’ tomato in 1,000 cm³ pots, containing substrate composed of soil, sand and organic matter in the proportion of 1: 2: 1 (v: v: v), pre-sterilized by autoclaving (120°C for 2hr). The nematode eggs were extracted from tomato roots by the method of Hussey and Barker (1973) and concentration of the suspension determined with the aid of the Peters chamber under a light microscope.

The populations of EPNs were obtained from the EPNs Collection of the Bank of the Biological Institute of São Paulo, São Paulo, Brazil. The infective juveniles (IJs) of S. brazilense, S. feltiae, S. rarum, H. amazonensis, and H. bacteriophora were multiplied in third to fifth larval instar of Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae). Five larvae per petri dish (9 cm in diameter) were coated with filter paper moistened with a nematode suspension at the concentration of 500 IJ/larvae. The dead larvae of D. saccharalis were transferred to traps of White (White, 1927) and stored in incubator (BOD) at 25°C. The IJs were collected in a water film (1 cm depth) in Erlemeyers, which were kept in a BOD chamber at a temperature of 18°C, 70% of relative humidity and used up to 48 hr after collection.

For the tomato in pots test, the ‘Rutgers’ tomato seeds were placed for germination in polypropylene trays containing 128 cells containing substrate. After germination, tomato seedlings were transplanted into pots for 1000 cm³ containing substrate composed of soil, sand, and organic matter in the proportion of 1: 1: 1 (v: v: v), sterilized in an autoclave (120°C for 2 hr).

The EPNs S. brazilense, S. rarum, S. feltiae, H. amazonensis and H. bacteriophora reduced hatch, number of galls, egg mass, nematodes total number and reproduction factor of M. enterolobii. However, only the species S. brazilense, S. rarum and H.bacteriophora affected juvenile survival, providing higher mortality on the 2nd day (S. brazilense and S. rarum) and on the 4th day (H. bacteriophora) evaluation.

The ovicidal activity of the EPNs tested can be attributed to the presence of the symbiotic bacterium. The eggs of M. enterolobii in the suspension can be served as a stimulus for the EPNs to release their bacteria. These bacteria may have produced substances toxic to the nematode eggs, reducing juvenile hatching (Grewal et al., 1999; Hu et al., 1999; de Freitas Ferreira et al., 2011), since the eggs of plants parasitic nematode are permeable (Perry et al., 1992).

The suspension, containing EPNs inoculated on tomato seedlings, provided a reduction in the parasitism of M. enterolobii, reflecting the reduction of gall number, egg mass, nematodes total number, and reproduction factor. Grewal et al. (1999) and Hu et al. (1999) observed that the toxic metabolites produced by bacteria act on the ability of Meloidogyne’s J2 to locate the roots. Such metabolites may have influenced the parasitism of M. enterolobii in tomato roots, causing a smaller number of nematodes in the root system, consequently, reducing the number of galls and egg masses.

The toxic effects of the metabolites produced by the symbiotic bacteria do not act constant in the suspension. In the course of time, occurs bacteria decline in the environment, as they do not have survival forms and, consequently, decline of the released molecules (Burnell and Stock, 2000), reducing the toxicity to juveniles of M. enterolobii. However, even with increasing mortality after 96 hr, it did not differ from the control, since, probably, occurred the natural mortality of juveniles that were exposed at 25°C in a suspension with accelerated metabolism.

The protein molecules produced by the bacterium Photorhabdus spp. (Ruiz Machado et al., 2018) present in the H. amazonensis and H. bacteriphora nematodes have been reported as presenting toxicity to Aphelenchoides rhytium (Massey), Bursaphelenchus spp., Caenorhabditis elegans, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934, Kohno et al. 2007), due to the production of the secondary metabolite, 3,5-dihydroxy-4-isopropylstilbene that has nematicidal properties (Grewal et al., 1999). The other nematodes S. feltiae, S. rarum and S. brazilense have a symbiotic relationship with the bacteria of the Xenorhabdus genus. Bode (2009) found that Xenorhabdus nematophila and Xenorhabdus bovienii produce ketones, amides and also more complex compounds like xenocoumarins (antibiotics) that can provide toxic effect to various organisms.

Monteiro et al. (2014) reported that castor bean pie has a nematicidal effect on Aphelenchoides besseyi (Christie, 1942), since among the pie compounds, the ketone group is predominant. However, only S. feltiae has an association with the bacterium X. bovienii (Ehlers et al., 1997), the other EPNs studied have symbiosis with other species of bacteria of the same genus. However, it is generally believed that all bacteria of the Steinernema genus produce near or similar metabolites, since bacteria species of the Xenorhabdus genus are able of producing a wide range of bioactive compounds, including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds (Fukruksa et al., 2017). Studies are needed to relate the compounds produced by the bacteria present in S. brazilense and S. rarum.

Therefore, it was verified that the reduction in hatching of M. enterolobii and the reduction of gall index, egg mass index, nematodes total number and reproductive factor of M. enterolobii in the presence S. brazilense, S. rarum, S. feltiae, H. amazonensis, and H. bacteriophora. The results demonstrate the potential of the use of EPNs in integrated management programs of M. enterolobii.