First report of Mesocriconema sphaerocephalum (Taylor, 1936) Loof, 1989 associated with wild grass in Botswana

Abstract During a survey on the biodiversity of plant-parasitic nematodes of natural areas in Botswana, Mesocriconema sphaerocephalum was discovered around the rhizosphere of the wild grass. The nematodes were extracted using the tray method and then fixed according to the available protocols. The morphological characters fit well with the M. sphaerocephalum. Besides, molecular aspects using 18S and 28S rDNA were studied. The phylogenetic analysis of 18S and 28S rDNA placed the examined population with other populations of M. sphaerocephalum in a group. According to the knowledge, this is the first report of M. spaherocephalum from Botswana.

The genus Mesocriconema belongs to the family Criconematidae (Taylor, 1936;Thorne, 1949) comprises over 90 species (Geraert, 2010;Powers et al., 2016). Mesocriconema xenoplax (Loof and De Grisse, 1989;Raski, 1952) is the type species distributed worldwide (Geraert, 2010). Members of Mesocriconema known as the ring nematode which are ectoparasite and cause yield loss in the high density (Nguyen et al., 2019). However, their ecological behavior has not yet been studied well. During a survey on nematodes of the natural areas of Botswana, M. sphaerocephalum (Loof, 1989;Taylor, 1936) was recovered from the wild grass in Botswana. Specimens were collected at the North-West District of Botswana (S 20° 8′24.882″, E 21° 12′45.475″) from the rhizosphere of wild grass plants. To our knowledge, this is the first report of M. sphaerocephalum from Botswana.

Materials and methods
Nematode extraction, processing, and LM pictures The specimens were extracted using the tray method and were fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin using the De Grisse (1969) method. The classification provided by Geraert (2010) was used for the taxonomical study of Meso criconema. Pictures were taken with a Nikon Eclipse 80i light microscope provided with differential interference contrast optics (DIC) and a Nikon Digital Sight DS-U1 camera (Nikon, Tokyo, Japan). Micrographs were edited using Adobe ® Photoshop ® CS.
DNA extraction, PCR, and phylogenetic analysis DNA extraction was done using the Chelex method (Straube and Juen, 2013). Five specimens of each species were hand-picked with a fine tip needle and transferred to a 1.5 ml Eppendorf tube containing 20 μ l double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. In total, 30 microliters of 5% Chelex ® 50 and 2 µL of proteinase K were added to each of the microcentrifuge tubes that contained the crushed nematodes and mixed. These separate microcentrifuge tubes with the nematode lysate were incubated at 56°C for 2 hr and then incubated at 95°C for 10 min to deactivate the proteinase K and finally spin for 2 min at 16,000 rpm (Shokoohi et al., 2020). The supernatant was then extracted from each of the tubes and stored at −20°C. Following First report of Mesocriconema sphaerocephalum: Shokoohi this step, the forward and reverse primers, SSU F04 (5′-GCTTGTCTCAAAGATTAAGCC-3′) and SSU R26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Blaxter et al., 1998); D2A (5″-ACAAGTACCGTGAGGGAAAGTTG-3″) and D3B (5″-TCGGAAGGAACCAGCTACTA-3″) (De Ley et al., 1999), were used in the PCR reactions for partial amplification of the 18S and 28S rDNA region, respectively. PCR was conducted with 8 μ l of the DNA template, 12.5 μ l of 2X PCR Master Mix Red (Promega, USA) for the Botswanan specimens, 1 μ l of each primer (10 pmol μ l −1 ), and ddH 2 O for a final volume of 30 μ l. The amplification was processed using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with the following program: initial denaturation for 3 min at 94°C, 37 cycles of denaturation for 45 sec at 94°C; 54°C and 56°C annealing temperatures for 18S and 28S rDNA; extension for 45 sec to 1 min at 72°C, and finally an extension step of 6 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 4 μ l of product from each tube was loaded on a 1% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with RedGel and visualized and photographed on a UV transilluminator. The amplicons of each gene were stored at −20°C. Finally, the PCR products were purified for sequencing by Inqaba Biotech (South Africa). The ribosomal DNA sequences were analyzed and edited with BioEdit (Hall, 1999) and aligned using CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were generated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The HKY + Γ (gamma distribution of rate variation with a proportion of invariable sites) model was selected using jModeltest 2.1.10 (Darriba et al., 2012;Guindon and Gascuel, 2003). Analysis using the HKY + Γ model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 10 6 generations for 18S and 28S rDNA. The trees were visualized with the TreeView program. Also, as

Morphological characterization
The morphological and molecular analyses confirmed that the species was M. sphaerocephalum.  Table 1). Females of M. sphaerocephalum are characterized by having body curved ventrally (Fig. 1D, G); lip region with two annuli, slightly flattened labial disc (Fig. 1A); first body annulus much smaller than the second one with smooth edge, sloping posteriorly, (Fig. 1A); cuticle annuli at mid-body 4.2 to 4.6 µm wide. Lateral field with anastomoses, forming zigzag lines (Fig. 1C); stylet robust, knobs 8.6 to 8.9 µm length and 3.2 to 3.4 µm diameter (Fig. 1A, B); vulva located near posterior end; tail rounded in all specimens (Fig. 1E, F). Male not found.

Measurements of
The forward SSU F04 and reverse SSU R26 primers of 18S rDNA (Blaxter et al., 1998); forward Note: All measurements are in µm and in form: mean ± SD (range), except for ratio. Figure 1: Mesocriconema sphaerocephalum (Taylor, 1936;Loof, 1989).   (Fig. 2). The molecular characterization of several sequences of M. sphaerocephalum suggested that they formed a monophyletic group. Findings in the current study were in agreement with the phylogenies of Mesocriconema species studied using 28S rDNA (Nguyen et al., 2019). Two permanent microscope slides containing 10 females of M. sphaerocephalum were deposited in the Nematology collection of the University of Limpopo, South Africa. According to the literature, this is the first record of M. sphaerocephalum from Botswana. In conclusion, the ecological behavior of this species needed to be studied to find out the economic importance of the pest.