Entomopathogenic nematodes (EPNs) exhibit a cosmopolitan distribution and have been isolated on five continents in different habitats around the world (Griffin et al., 1990; Hominik et al., 1996). In Mexico, EPNs, particularly species of the genera
Soil samples were collected from 14 plots of sugar cane located in the state of Tepalcingo, Morelos in Mexico using the technique proposed by Stock et al. (1999), and a modified method described by Orozco et al. (2014). For soil sample collection, within a plot, samples were taken at five points, with three subsamples per site; for sampling, a grid of 3 m2 was established around the sugar cane plant, and 1 kg of soil was collected at a depth of approximately 15 cm; the soil samples were placed in plastic bags. The sites were sampled in September 2014.
Soil analysis was performed to determine soil physicochemical characteristics such as texture, pH, electrical conductivity, and % organic matter (MO) in the bioremediation laboratory of CEIB/UAEM. EPNs were extracted with the insect baiting technique as described by Orozco et al. (2014). Each soil sample was baited by placing 12 fifth-instar
Morphological and morphometric identification was performed using an optical microscope equipped with a micrometric eyepiece. The nematodes were extracted through the dissection of wax worm larvae, and the IJs were collected from the white trap according to the method proposed by Nguyen and Smart (1996).
For identification with molecular techniques, three hundred IJs were frozen in liquid nitrogen and macerated with a plastic pestle. Their DNA was purified with a Puregene® DNA Purification Kit following the manufacturer’s instructions. An 850 bp fragment corresponding to the D2D3 region of the 23S rDNA gene was PCR amplified with the primers pair D2F (5′CCTTAGTAACGGCGAGTGAAA-3′) and 536 (5′-CAGCTATCCTGAGGGAAAC-3′) (Nguyen et al., 2006). The reaction was conducted in a final volume of 50 µl containing each primer at 0.5 µM, 0.16 mM dNTPs, 1.5 mM MgCl2, 2 units of Taq DNA polymerase (Thermo Fisher Scientific Inc.) and 1X reaction buffer. The PCR product was verified by electrophoresis in a 1% agarose gel, and the bands were excised and purified using a QIAquick® Gel Extraction kit (QIAGEN, Germany). The PCR conditions were 94 °C for 2 min initial denaturation; 37 cycles of 30 s of denaturation at 94 °C, 45 s of hybridization at 48 °C and extension at 72 °C for 90 s; with a final extension of 72 °C for 5 min. The PCR product was sent to the Biotechnology Institute of the National Autonomous University of Mexico for sequencing. The D2D3 region was analyzed using the BLAST program of the GenBank database of the National Institutes of Health.
For the extraction of symbiotic bacteria from MC5-2014, two methods were used. In the first method, 5000 freshly emerged IJs were obtained from the white trap, and were placed in Eppendorf tubes. The tubes were centrifuged at 1,000 revolutions per minute (rpm) for 7 min, and the supernatant was discarded. Thereafter, washes were conducted with 5% sodium hypochlorite, and three washes were performed with sterile distilled water (Lee and Stock, 2010). The IJ nematodes were macerated in a mortar and pestle for a period of 30 min and plated in solid Luria-Bertani (LB) and HCT (Bacto Tryptone (Difco) 5; Casamino acids (Difco) 2; pH adjusted to 7.5) media. After sterilization, culture medium consisting of KH2 PO4, 3.4 g/L; MgSO4.7H2 O, 0.012 g/L; MnSO4.4H2 O, 0.003 g/L; ZnSO4.7H2 O, 0.0028 g/L; Fe(SO4)3.7H2 O, 0.02 g/L; CaCl2.2H2 O, 0.147 g/L; and glucose, 3 g/L was used. For the second method of extraction from hemolymph, 10 larvae of
Subsequently, a 0,3 ml insulin syringe whith a 31 × 6 mm needle were used to extract the hemolymph by inserting the needle between the 6th and 7th inter-segments of the larvae. An aliquot of the hemolymph was plated on solid LB and HCT culture media, and the plates were incubated for 24 hr at 27 °C. All colonies were sampled, and the cross-streak technique was used to obtain single colonies. The bacteria were identified by biochemical methods using Gram staining, motility tests, and assays of protease activity and lecithinase and antibiotic production as suggested by Boemare et al. (1997) and Akhurst (1980). For molecular identification of the isolated bacteria, the cells were incubated in LB liquid medium for 12 h at 30 °C, and DNA extraction was then carried out with the Easy-DNA™ kit following the instructions recommended by the manufacturer. Then, amplification of a partial 16S rRNA region (600 bp) was performed by PCR using the primer 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) (Nübel et al., 1996). The PCR conditions were 95 °C for 3 min initial denaturation; 37 cycles of 35 s of denaturation at 95 °C, 42 s of hybridization at 59 °C and extension at 72 °C for 1 min 30 s; with a final extension of 72 °C for 5 min. The PCR products were identified at the Biotechnology Institute of the National Autonomous University of Mexico with the primer 63 f. The sequences were analyzed using the BLAST program of the GenBank database, National Institutes of Health.
Pathogenicity bioassays of bacterial strain MC5-2014 were carried out in 10 larvae of the 5th stage of
Strain Bar 86 of
The virulence bioassays of the bacteria isolated from the MC5-2014 strain were performed on 30 larvae of
Virulence was determined using 6, 10, 14, 18, or 20 infected juveniles (IJs) plus the control, which contained only sterile distilled water. Virulence was determined by obtaining the average lethal concentration (LC50) via PROBIT analysis using the POLO PLUS version 1.0 program. (LeOra Software LLC).
Among the 14 soil samples taken from different locations in the state of Morelos, Mexico, positive results were found in the municipality of Teplacingo, which has an altitude of 1100 mm, a warm subhumid climate, an average annual temperature of 24 °C and a minimum temperature of 14 °C, from a site with sandy loam soil with a pH of 6.4 and an organic material (OM) percentage of 2.64%. We obtained the MC5-2014 isolate from sugar cane crops, and there have been reports of positive isolation in sugar cane cultures of
The male, female and IJ specimens were deposited in the Nacional Collection of Helminthes of the Biology Institute of the National Autonomous University of Mexico (CNHE/UNAM) with registration number 11085.
Comparison of morphometrics with the MC5-2014 isolate.
Characteristic |
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Locality | California | Turkey | Mexico | Pakistan | China | Iran |
Type host/habitat |
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Soil, | Soil, | Soil | ||
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– | – | 964.5 | 1607 | 1042 | 1179 | |
L | – | (972-1530) | (760-1160) | (1362-2015) | (920-1179) | |
(830-1500) | ||||||
– | – | 37 | 88 | 49.5 | 69 | |
W | – | (52-100) | (30-60) | (72-125) | (39.8-58.2) | (54-90) |
554 | ||||||
V | – | – | (410-760) | – | – | – |
52 | ||||||
Eggs | – | – | (50-56) | – | – | – |
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– | – | 843 | 862 | 1396 | 790 | |
L | (830-1470) | (841-1175) | (720-910) | (760-1390) | (1195-1692) | (671-950) |
– | – | 28 | 61.6 | 62.4 | 46 | |
W | (380-800) | (52-72) | (20-30) | (53-75) | (46.3-66.2) | (36-5)0 |
– | 41 | 50 | 49.2 | 40 | ||
Spicules | – | (27-39) | (30-40) | (47-55) | (35.2-60.9) | (34-44) |
G | – | – | 22 | 20.3 | 19.7 | 15 |
– | – | – | (20-30) | (18-22) | (19.9-26.5) | (12-23) |
Note: L: total body length, W: maximum body width, V: vulva, and G: gubernaculum.
Based on the morphometric characteristics of males and females that are commonly used for the identification of nematodes, 10 females and 10 males were observed under a microscope (Nikon eclipse e200), and morphometric measurements were performed (Table 1). The maximum and minimum ranges of the body length were obtained: L, body width: W, spicules, distance from the part before the vulva, and length of eggs. The average values were calculated.
For molecular identification, an 832 bp fragment of the D2D3 region of the 28s ribosomal gene was amplified by PCR. The sequence of the MC5 2014 isolate was deposited in GenBank under accession number MK418537, showing 99% identity with DF5020 of
In Turkey (Zeynep et al., 2017), a nematode associated with
Notably, Torrini et al. (2015) reported a new species of EPN belonging to the genus
For the macerated extraction and the hemolymph extraction methods, the bacterial colonies were isolated at 48 and 72 h, grown on solid LB and HCT agar plates, and initially assigned as MC5-R based on red colonies. The biochemical tests showed that the bacterial strain MC5-R was Gram negative and was positive for motility tests, protease activity and lecithin activity. From the genomic DNA extraction product, a fragment of 600 bp was obtained. Once obtained, the PCR products were sent to the Institute of Biotechnology of UNAM (National Autonomous University of Mexico). The sequence of the isolated MC5-R was deposited in GenBank with the accession number MK463930. A BLAST search indicated 100% similarity with an MK463930 sequence and
The percentage of mortality in
The percentage of mortality for
The mean lethal concentration was 4.732 IJ in
Currently, species belonging to the genus