Rotylenchus wimbii n. sp. (Nematoda: Hoplolaimidae) associated with finger millet in Kenya

Abstract Rotylenchus wimbii n. sp. was found associated with finger millet in Kenya and is described based on light microscopy, scanning electron microscopy, and molecular information. Sequence analysis was performed on ITS, 18S, and D2-D3 of 28S of ribosomal DNA and COI of mitochondrial DNA. This new species is characterized by a moderate female body size of 0.6 to 0.8 mm, a continuous hemispherical lip region with four annuli, 3 to 4 irregular blocks on the basal lip annule, absence of longitudinal cuticular striations in anterior region, four lateral lines forming three equal bands which are areolated mainly at pharynx level, a robust stylet of 23 to 27 µm of which 45 to 53% is cone part, and with rounded to sometimes indented knobs, a secretory-excretory pore around level of pharyngo-intestinal junction, didelphic-amphidelphic reproductive system, vulva without distinct epiptygma, indistinct to empty spermatheca, tail usually truncated with 5 to 9 annuli, phasmids located at 7 to 17 annuli anterior to anus, and absence of males. Molecular phylogenies, in combination with species delimitation, supported the distinctiveness of Rotylenchus wimbii n. sp. and revealed some mislabeled Rotylenchus brevicaudatus sequences in GenBank.

Within the plant-parasitic nematode (PPN) genus Rotylenchus Filipjev, 1936 (Nematoda: Hoplolaimidae), 104 valid species have been recognized (Nguyen et al., 2019). Rotylenchus spp. have been reported from all the continents, being found associated with numerous important crops ranging from rice and cereals to tubers, vegetables, and even ornamental plants and trees. As obligate root parasites feeding on root hairs or epidermal or cortical cells of a plant, they can cause prominent damage to the host plant, with symptoms ranging from stunted and reduced growth to wilting, chlorosis, reduced root system and root lesions, often leading to significant losses in crop yield (Castillo and Vovlas, 2005;Manzanilla López and Marbán Mendoza, 2012;Sikora et al., 2018). To facilitate the identification of Rotylenchus species, Castillo and Vovlas (2005) have developed a tabular or matrix key using a total of 11 characters of this nematode group. This identification system has so far been widely accepted and its widespread use has also recently been facilitated by a web-based key that draws its basis from cluster analysis (Nguyen et al., 2019). In combination with the morphological features, molecular information such as ITS, 18S, and 28S of ribosomal DNA and COI of mitochondrial DNA have been used for their identification.
In the current paper, we characterize a newly discovered Rotylenchus wimbii n. sp. found associated with finger millet, Eleusine coracana (L.) Gaertn. (Planta: Poaceae) in Kenya. The potential importance of this nematode as a pest of finger millet was evidenced by its dense population in the soil sample and its presence observed in the finger millet root system (revealed by the fuchsin staining method). Nematode characterization was carried out based on morphological information obtained from light microscopy (LM) and scanning electron microscopy (SEM) studies. Illustrations, morphometrics, and molecular information of ITS, 18S, and 28S of rDNA and COI of mtDNA are also provided for the novel species.

Sample collection and nematode extraction
A soil sample mixed with some roots was collected using a shovel from 15 to 25 cm soil depth in a zig-zag pattern from a finger millet field [Eleusine coracana (L.) Gaertn.] in Kipkaren Estate, Eldoret, Kenya in February, 2019. The GPS coordinates of the field location are 00°30.395' N; 035°14.825'E. Nematodes were extracted from 100 ml of soil by using modified Baermann's method (Whitehead and Hemming, 1965). The extracted nematodes were stored at 4°C during the course of analysis.

Root staining
Host roots were stained using acid fuchsin (Byrd et al., 1983). For this, clean roots were bleached in 2.5% NaOCl for 5 min, followed by rinsing the roots in running tap water, and boiling them in 30 ml distilled water with 1 ml of stock staining solution (0.35 g acid fuchsin, 25 ml acetic acid, and 75 ml distilled water) in a microwave for 30 sec. After cooling, excess fuchsin was drained and roots were washed with running tap water and de-stained by immersing them in 70% acidified glycerol, and finally observed under a stereo microscope for presence of stained PPN.

Morphological characterization
For morphological studies, live nematodes were heatrelaxed by quickly passing over a flame in a drop of water on a glass slide until nematode movement stopped and examined, photographed, and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an Olympus C5060Wz camera as described in Singh et al. (2018). After recording morphological information, each specimen was recovered from the slide and its genomic DNA was extracted. For fixing, the nematode suspension obtained after extraction was concentrated in a drop of water in a glass embryo dish, followed by addition of a few drops of Trump's fixative [2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M Sorenson buffer (Sodium phosphate buffer at pH = 7.5)] into it. The nematodes were then immediately heated in a microwave (700 watts) for about 4 sec and left for 1 hr at room temperature and finally at 4°C for 24 hr. This was followed by gradually transferring the nematodes to anhydrous glycerin, ready to be mounted on glass slides as described in Singh et al. (2018). For SEM, specimens fixed in Trump's fixative were washed in 0.1 M phosphate buffer (pH = 7.5) and dehydrated in a graded series of ethanol solutions, critical-point-dried with liquid CO 2 , mounted on stubs with carbon tabs (double conductive tapes), coated with 25 nm gold, and photographed with a JSM-840 EM (JEOL) at 12 kV .

Phylogenetic analysis
The phylogenetic relationships of the new species with other related species were analyzed based on the partial sequences of ITS, 18S, 28S, and COI. Phylogenetic programs implemented in Geneious Prime 2020.0.5 were used. The obtained consensus contigs were subjected to BLAST search to check for closely related species on GenBank and all the collected sequences for each gene fragment were aligned using MUSCLE alignment of Geneious Prime 2020.0.5 using default parameters, followed by manually trimming of the poorly aligned ends. The best nucleotide substitution model of each gene alignment was determined by jModelTest 2.1.10 and using the selected models, phylogenetic trees were created using Bayesian phylogenetic analyses (MrBayes 3.2.6), which was run under 1 × 10 6 generations (4 runs) and Markov chains sampled at every 100 generations, and 20% of the converged runs regarded as burn-in (Huelsenbeck and Ronquist, 2001). To check for additional distinctiveness of our sequences in phylogenetic trees, the species delimitation plugin of Geneious Prime 2020.0.5 (Masters et al., 2011) was used to calculate Rosenberg's PAB, which tests the probability for reciprocal monophyly of sequence clusters (Rosenberg, 2007).

Description Female
Body moderately large (0.6-0.8 mm), habitus spiral to 6-shaped when heat relaxed. Lateral field differentiation starting as single areolated band, gradually forming two areolated bands up to level of metacorpus valve and further continuing as three areolated bands up to isthmus level, after which bands becomes smooth till tail terminus. Longitudinal cuticular striations in anterior region absent. Labial region hemispherical with 4 annuli, not offset from body but appears to have a slight depression under LM. En face showing rounded labial disc, marked from rest of labial region, not elevated, slit like oral aperture, lateral sectors with two amphidial apertures and smaller than subdorsal and subventral sectors. Basal lip annule with 2 to 3 longitudinal striations (from six face views) forming 3 to 4 irregular blocks, but in one specimen with five striations forming six irregular blocks. Stylet robust with large, rounded, and sometimes indented knobs. Dorsal pharyngeal gland opening at 4 to 6 µm from stylet base. Pharynx with well-developed median bulb, valves, slender isthmus, and gland overlapping intestine by maximum 30 µm dorsally. Secretory-excretory pore often at level of pharyngo-intestinal junction. Hemizonid distinct, just above secretory-excretory pore, about two body annuli long. Reproductive tract didelphicamphidelphic, each branch equally developed with outstretched ovaries containing rows of developing eggs, vulva at 55 to 60% of body length from anterior end, without distinct epiptygma. Spermatheca indistinct to rounded, not filled. Tail very short with commonly truncated tip or sometimes slightly hemispherical to rounded tip. Phasmid pore like, 7 to 17 annuli anterior to anus.

Male
Not found.

Diagnosis and relationships
Rotylenchus wimbii n. sp. is characterized by a moderate body size of 0.6 to 0.8 mm, a hemispherical, continuous lip region with four annuli, basal lip annule with 3 to 4 irregular blocks, absence of longitudinal cuticular striations at anterior region, lateral field with four lines forming three equal bands which are areolated around pharynx level, a robust stylet of less than 30 µm of which 45 to 53% is cone and with rounded to sometimes indented knobs, didelphicamphidelphic reproductive system, vulva without distinct epiptygma, indistinct to empty spermatheca, very short truncated tail with 5 to 9 annuli, and pore like phasmids located at 7 to 17 annuli anterior to anus. According to Castillo and Vovlas (2005), the matrix code of this species is A4, B1, C1, D4, E1, F2, G3, H (commonly truncated), I2, J2, K1.
This new species is morphologically closest to Rotylenchus abnormecaudatus Van den Berg andHeyns, 1974 andRotylenchus brevicaudatus Colbran, 1962. The females of these three species have similar body sizes, lip region of more or less  hemispherical to rounded and very slightly offset to continuous with 4 to 5 annuli, stylet length below 30 µm, lateral field areolation only at pharynx level, absence of longitudinal cuticular striations, vulva position of 52 to 62% of body length from anterior end, phasmids positioned at 7 to 17 annuli anterior to anus, and short tail with only 5 to 11 tail annuli. The new species differs from R. abnormecaudatus in having 4 vs 4 to 5 lip annuli, a slightly longer cone part of stylet (45-53% vs 42-45%), and often truncated vs irregularly rounded tail terminus. Rotylenchus abnormecaudatus has also been reported with a peculiar and irregular arrangement of annuli and lateral field ending in the posterior part of tail which is not seen in other species including R. wimbii n. sp. While males of R. wimbii n. sp. and R. abnormecaudatus are not known and subsequently, no sperm is observed in the female spermathecae, males of R. brevicaudatus have been reported and the female spermatheca are often filled with sperm. Rotylenchus wimbii n. sp. further differs from R. brevicaudatus by the presence of 2 to 3 longitudinal striations on the basal lip annule vs 6 to 12 such longitudinal striations as reported by Sher (1965) from the study of six face views of the 20 female topotypes supplied by R.C. Colbran, and commonly truncated vs hemispherical tail terminus. Information on the number of longitudinal striations on basal lip annule for R. abnormecaudatus is not available.
The new species is also comparable to Rotylenchus cypriensis Antoniou, 1981, Rotylenchus mabelei    Number of tail annuli 7 ± 1 (5-9) Note: All measurements, except ratios and V%, are in µm and in the format: mean ± standard deviation (range).
Van den Berg andDe Waele, 1989, andRotylenchus unisexus Sher, 1965, all of which are species without males and reported to occur in South Africa and Kenya. The new species can be separated from R. cypriensis in continuous vs well offset lip region and absence vs presence of a ventral mucron at the tail tip. It can be differentiated from R. mabelei by a slightly smaller body (0.6-0.8 vs 0.9-1.1 mm), a lesser number of tail annuli (5-7 vs 14-18), and the position of phasmids with respect to anus (7-17 anterior vs 3 anterior to 5 posterior of anus). Finally, our new species can be differentiated from R. unisexus by the longitudinal striae on basal lip annule (3-4 vs 16-18 striae), tail annuli number (5-9 vs 9-13), and phasmid position (7-17 annuli anterior vs 6-7 annuli anterior of anus). An additional comparison of the new species with female morphological characters and morphometrics of all the Rotylenchus species that have been reported from Africa till date is provided in Table 2.

Etymology
The species epithet refers to its host. Wimbi originates from Swahili and is used as a common name for finger millet in Eastern Africa.

Type host and locality
The new species was found parasitizing the host plant Eleusine coracana (L.) Gaertn.

18S of rDNA
Six sequences of up to 903 bp without sequence variation were produced (MW074378-MW074383). The closest Rotylenchus sequence available from the databank was a R. unisexus sequence (MK809263) with 98.9% similarity (9 differences in 810 bp). No phylogenetic tree was made using 18S due to limited availability of 18S sequences of Rotylenchus from GenBank.

ITS of rDNA
Five sequences of up to 973 bp without sequence variation were produced (MW074373-MW074377) which were found closest to a R. brevicaudatus (DQ309587) sequence from Taiwan with 96.9% similarity (25 differences in 818 bp). These sequences were aligned with 68 identified (25 species) and five unidentified Rotylenchus sequences. The resulting phylogenetic tree (Fig. 4)    R. incultus and R. unisexus, which were described from crops in Zimbabwe and Kenya respectively, all other species were described from studies in South Africa indicating the relatively unexplored species diversity of Rotylenchus in the African continent. A few other Rotylenchus species such as Figure 5: BI phylogenetic tree generated from the analysis of COI of mtDNA sequences using GTR + G + I nucleotide substitution model. Bayesian posterior probabilities are given next to each node and sequences of Rotylenchus wimbii n. sp. are in bold.