Morphological and molecular characterization of Pratylenchus species from Yam (Dioscorea spp.) in West Africa

Abstract The root-lesion nematodes (RLN), Pratylenchus spp., are among the major plant-parasitic nematodes affecting yam (Dioscorea spp.) production in West Africa. The distribution and diversity of RLN species associated with yam was investigated through a soil and tuber survey of the main producing areas in Nigeria and Ghana. Pratylenchus spp. were detected in the yam rhizosphere in 59% of 81 soil samples from Ghana and 39% of 114 soil samples from Nigeria. Pratylenchus spp. were detected in 24 of 400 tubers examined, in combination with root-knot nematodes (Meloidogyne spp.) and their associated damage of galls and crazy roots (79%), and with yam nematode (Scutellonema bradys) and their associated damage of dry rot (17%), although no specific additional symptoms were observed for Pratylenchus spp. Species of Pratylenchus were identified by their morphological features and by sequences of the D2-D3 region of the 28 S rDNA gene and the mitochondrial cytochrome oxidase I gene (COI). Pratylenchus brachyurus was the most frequent RLN species in both the rhizosphere and tubers of yam. Pratylenchus hexincisus was recovered from one tuber collected in Nigeria. While further investigations are required to establish the host status of yam for this nematode, this appears to be the first record of P. hexincisus on yam. The present taxonomical status of P. scribneri and P. hexincisus is discussed.

Yam (Dioscorea spp. L.) is an economically important crop of tropical and sub-tropical areas of the world. West Africa accounts for over 93% of the total production of this tuber with Nigeria and Ghana being the main cultivating yam countries. In these countries, yam is an important staple food providing a valuable source of carbohydrates, proteins and minerals for over 380 million people from an estimated annual production of 67 MT (Nweke et al., 1991;Orkwor, 1998;Nweke, 2016;FAO, 2018). The most important yam species cultivated for food are D. rotundata Poir., D. cayenensis Lam., D. alata L., D. dumetorum (Kunth) Pax., D. bulbifera L. and D. esculenta (Lour.) Burk. Also, yam plays an important socio-cultural role among communities and its cultivation and sale serve as a major income-generating activity for the people in yam-growing areas (Onwueme and Charles, 1994). Yam production is constrained by numerous biotic factors, however, of which plant-parasitic nematodes are among the most damaging. They affect yield and tuber quality, reducing yam production and tuber storability (Ayensu and Coursey, 1972;Bridge et al., 2005;Coyne and Affokpon, 2018). The major plantparasitic nematodes known to cause serious damage on yam tubers are the yam nematode (Scutellonema bradys (Steiner and LeHew, 1933;Andrássy, 1958), root-knot nematodes (Meloidogyne spp.) and rootlesion nematodes (RLN) (Pratylenchus spp.) Bridge and Starr, 2007;Kolombia et al., 2016b;Coyne and Affokpon, 2018). RLN, however, have been much less studied, even though they are known to cause dry rot symptoms in tubers, indistinguishable from the symptoms caused by S. bradys .
Pratylenchus coffeae (Zimmermann, 1898) Filipjev andSchuurmans Stekhoven, 1941 is the most important RLN of yam, occurring in Central America, the Caribbean Islands and the Pacific Islands (Acosta and Ayala, 1975;Coates-Beckford and Brathwaite, 1977;Bridge, 1988;Moura and Monteiro, 1995;Bridge et al., 2005;Muniz et al., 2012;Coyne and Affokpon, 2018). In Africa, P. brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, P. pseudopratensis (Seinhorst, 1968) and P. sudanensis (Loof and Yassin, 1971) are known to cause damage to yam (Coyne et al., 2003;Mudiope et al., 2007; with indications that they are relatively common in the yam rhizosphere and on tubers (Adegbite et al., 2008;Kolombia et al., 2020). It was also observed that Pratylenchus spp. were associated with the galls and crazy roots caused by root-knot nematodes, or with dry rot caused by S. bradys, although with no specific additional symptoms (Kolombia et al., 2016a). Being a stenomorphic genus, Pratylenchus is easily recognizable at the genus level (low and flattened labial region, esophageal gland lobe overlapping the intestine mostly ventrally, posterior vulva V = 70-80%, with one ovary), while morphological identification at the species level is problematic due to the low number of diagnostic features and high intraspecific variability (Luc, 1987;Duncan et al., 1999;Castillo and Vovlas, 2007). To establish the diversity of Pratylenchus spp., associated with yam, surveys were conducted in the main yam producing areas in Nigeria and Ghana. The Pratylenchus populations obtained from yam tuber tissue and yam rhizosphere were morphologically characterized and molecularly confirmed by sequencing of the D2-D3 of 28 S rDNA and mitochondrial COI genes.

Nematode samples
Nematode populations used in this study were obtained soil and tuber sampling undertaken across agro-ecological zones in Ghana and Nigeria during surveys conducted between 2012 and 2015 (Table 1).
Nematodes from 195 yam rhizosphere and 400 tubers were recovered using the Whitehead tray immersion technique (Hooper et al., 2005). Extraction from rhizosphere was set using 100 ml soil sub-samples including all roots retrieved from soil per sample. Tubers were peeled using a kitchen peeler, chopped and three sub-samples of 5 g tuber peels were used for the extraction (Coyne et al., 2006). Extracted nematodes were collected on 28 μ m sieves, rinsed and divided: one part was heat killed and fixed in 4% formalin, the other part was fixed directly in DESS solution (Yoder et al., 2006). In total, 127 nematodes, including 75 specimens from yam tubers, were used for species identification.

Morphological characterization
Nematodes from 27 samples fixed in formalin were processed to anhydrous glycerin following the glycerin-ethanol method (Seinhorst, 1959) as modified by De Grisse (1969). Permanent slides were prepared and used to record morphometrics and morphological features (Castillo and Vovlas, 2007;Inserra et al., 2007) using an Olympus BX51 DIC microscope equipped with a Nikon digital camera. Additional morphological and morphometrical data were recorded from temporary slides made from DESS fixed specimens, before DNA extraction (see Table 1).
PCR products were electrophoretically separated on a 1% agarose gel and stained with ethidium bromide. PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System Kit

Phylogenetic analysis
Both D2-D3 of 28 S rDNA and COI of mtDNA se quence datasets were aligned using MUSCLE (Edgar, 2004) with default settings. Outgroup taxa of each dataset were chosen based on previously published data (Subbotin et al., 2008;Liu et al., 2016). The best fit models of DNA evolution were estimated using the program jModeltest 0.1.1 (Posada, 2008) under the Akaike information criterion (AIC). Bayesian phylogenetic analysis (BI) was undertaken using MrBayes 3.2.6 for 1 × 10 6 generations with a general time-reversible model with a gamma distribution for the remaining sites (GTR + I + G), four runs, 20% burn-in, and subsampling frequency of 500 generations (Huelsenbeck and Ronquist, 2001) for both D2-D3 and COI.

Results
Occurrence and morphological characterization of Pratylenchus spp. from yam The density of Pratylenchus spp. from the rhizosphere varied from 2 to 704 individuals per 100 ml soil and roots in Ghana, and from 2 to 398 individuals in 100 ml of soil and roots in Nigeria. From 400 tubers examined, Pratylenchus spp. were recovered from just 6% of the 400 tuber peels ( Figure 1C). Twenty-four tubers were infected with Pratylenchus spp., of which, 19 tubers (79%) also had galling and crazy root damage caused by the root-knot nematode (Meloidogyne spp.), 4 tubers (17%) showed dry rot symptoms caused by the yam nematode (Scutellonema bradys) while no symptoms were observed in one tuber, which had a density of 50 specimens of Pratylenchus brachyurus, per 5 g of yam peels ( Figure 1D). Densities of Pratylenchus spp. were as higher as 340 nematodes in tubers with symptoms and up to 525 individuals per 5 g of yam peels in tubers with dry rot and galling, respectively. Twenty-eight populations from 12 yam tubers and 16 rhizosphere samples were studied using morphological and molecular data, which resulted in the identification of Pratylenchus brachyurus and P. hexincisus (Taylor and Jenkins, 1957) and P. zeae (Graham, 1951). Kintampo S1: Two species were recorded from the same sample P. brachyurus (n = 3) and P. zeae (n = 6); T :Sample from yam tuber, otherwise, sample are from rhizosphere. *State (Nigeria)/Region (Ghana); α :LGA = Local Government Area (Nigeria)/District (Ghana); T :Sample from yam tuber, otherwise, sample are from rhizosphere. § :Kintampo S 1: Two species were recorded from the same sample P. brachyurus (n = 3) and P. zeae (n = 6).
Pratylenchus brachyurus was the most prevalent RLN species in Ghana and Nigeria, present in 11 of the 12 tubers used for species identification and 88% of Pratylenchus-positive rhizosphere samples. Twenty-five specimens per 5 g of yam peels of Pratylenchus hexincisus were recovered in just one tuber showing galls from Nigeria, and P. zeae was detected in 12% of the rhizosphere samples from Ghana (26 nem/100 ml soil) and Nigeria (3 nem/100 ml soil).
Female: Body small 390-679 μ m long, stout to moderately slender. Habitus almost straight when heat-relaxed. Lateral fields usually with four longitudinal lines; sometimes 4 to 6 lateral lines at mid body or 2 additional lateral fields faint or broken. Cephalic region slightly offset from body, with two lip annuli. Robust stylet (16.3-20.9 μ m long) with stout and rounded basal knobs, 3.8-6.6 μ m wide, with irregular shape on the surface. The dorsal esophageal gland opening (DEGO) at 2.0-4.3 μ m posterior the stylet base. Median bulb muscular, rounded to oval. Excretory pore just anterior to region of esophago-intestinal junction, but often indistinct. Esophageal glands overla pping intestine ventrally and sometimes laterally. Reproductive system monodelphic-prodelphic, ovary with oocytes in one row, occasionally two rows. Spermatheca usually indistinct, if present, well developed, rounded to spherical, filled with sperm cells in a few specimens. Vulva at 77-88% of body length. Post-vulval uterine sac generally shorter than body diameter length (12.3-34.9 μ m long). Vulvaanus distance about twice the tail length. Tail slightly tapering, terminus mostly bluntly rounded, varying from somewhat narrower, flat to slightly indented; terminus smooth.
Males: Not observed. P. brachyurus populations described were collected from yam tubers and rhizosphere from five districts in Ghana and ten Local Government Areas (LGA) in Nigeria.
From the morphology and the morphometrics, the studied populations are in agreement with the original description of P. brachyurus, and to subsequent descriptions (Roman and Hirschmann, 1969;Corbett, 1976;Castillo and Vovlas, 2007). However, the spermatheca was filled with sperm cells in two specimens (of the same sample), which has not previously been observed. In addition, in one specimen, the vulva was located at 77% of the body, while the vulva is normally located at 81-88% of the body. Pratylenchus hexincisus Taylor and Jenkins, 1957. (Figure 4 and Table 4).
Males: Not observed.

Remarks:
The population used in this study is from one location (Otukpo) in Nigeria collected from a yam tuber. The studied population was in agreement with the original description of P. hexincisus and to subsequent descriptions (Castillo and Vovlas, 2007;Inserra et al., 2007).
Female: Body slender, short 381-561 μ m long, and near-straight when heat-relaxed. Cephalic region continuous with body and bearing three annuli. Lateral fields with four lines at mid body. Stylet 14.6-16.9 µm long, with broad, anteriorly flattened basal knobs. Esophageal glands overlapping intestine ventrally and laterally. Ovary usually long. DEGO at 3 μ m posterior to the stylet base. Excretory pore just anterior to the esophago-intestinal junction. Spermatheca rounded, without sperm. Vulva at 70-73.2%. Post-vulval uterine sac short, about 1 body diam. long. Tail tapering, with 18-21 annuli terminating in an almost pointed tip.
Males: Not observed. Remarks: Based on the morphology and the morphometrics, the studied populations were in agreement with the original description of P. zeae and to the neotype female and other descriptions of P. zeae (Fortuner, 1976;Castillo and Vovlas, 2007).

Molecular characterization of Pratylenchus spp. from yam
The D2-D3 of 28 S rDNA gene The D2-D3 alignment included 80 Pratylenchus sequences, and two outgroup sequences. Thirteen new D2-D3 sequences were obtained in the present study. Following the numbering proposed by Subbotin et al. (2008), the BI tree contained five highly supported clades except for clade III ( Figure 6).
Sequences of P. brachyurus from this study, together with P. brachyurus sequences from GenBank were grouped in a well-supported subclade C of the clade III. The intraspecific variation of P. brachyurus was 2-51 bp (0.3-6.6%) and nucleotide difference between P. brachyurus and the most similar sequence, P. penetrans, was 152-177 bp (19.5-23%).

The mitochondrial COI gene
The COI sequences alignment was 422 bp in length and included 58 sequences of Pratylenchus including eight newly generated sequences, and four outgroup taxa (Meloidogyne, Hirschmanniella, Pratylenchoides and Radopholus). The BI tree contained five highly supported clades following numbering proposed by Subbotin et al. (2008) (Figure 7). Sequences of P. hexincisus from yam formed a well-supported clade with P. hexincisus sequences from China, Italy and the USA and P. scribneri sequences from China and the USA, with P. loosi (PP 0.84) as sister species. The sequences of P. hexincisus generated in this study and P. hexincisus sequences from Italy (KY828322)   Pratylenchus zeae sequences formed a wellsupported clade (VI) together with P. zeae sequences from GenBank. The intraspecific sequence variations of P. zeae were 0-37 bp (0-9.6%) and the interspecific sequence difference was 99-112 bp (25.9-28.6%) with P. parazeae, the closest related species.
Pratylenchus species, and in particular P. coffeae are known to cause "dry rot" on yam tubers, a condition similar to that caused by S. bradys, based on what is known for P. coffeae and P. sudanensis Bridge and Starr, 2007;Coyne and Affokpon, 2018). However, symptoms of P. brachyurus or its effects on yam production are not well known, Given the predominance of P. brachyurus in yam tubers and yam rhizosphere, it appears that this species is a major RLN on yam in West Africa. However, more work is necessary to clearly establish the effect of this species on yam growth, yield and tuber quality. The ability of P. brachyurus to survive a long period without a host and its polyphagous nature, could make its management particularly difficult, without the use of resistant cultivars.
Pratylenchus zeae, retrieved only from the yam rhizosphere of one sample in Ghana and one in Nigeria, is a commonly occurring species on other crops in West Africa (Fortuner, 1976; Plowright and  Hunt, 1994;Coyne et al., 1996;Castillo and Vovlas, 2007). Pratylenchus zeae was reported on yam in Nigeria (Bridge, 1973) but has never been reported on yam rhizosphere in Ghana. Its absence from tuber tissue, however, indicates that yam tubers may not support P. zeae and that its occurrence in this case may be related to other plant species occurring together with the sampled yam.
Pratylenchus coffeae, one of the major plantparasitic nematodes of yam in the Americas and the Pacific Islands was not recorded in any of the samples collected from Ghana and Nigeria. A similar observation was reported by Kwoseh et al. (2005) in Ghana. This remarkable absence from P. coffeae supports the statement of Duncan and Moens (2013) that "P. coffeae is a pest of yam, interestingly, not in Africa", despite being present on other crops in both localities (Duncan et al., 1999;Pourjam et al., 1999;Speijer et al., 2001;Bridge et al., 2005;Kwoseh et al., 2005;Coyne and Affokpon, 2018). Although Osei Traditional taxonomy can have serious limitations for differentiating species of Pratylenchus (Luc, 1987;Subbotin et al., 2008). In the current study,  Inserra et al. (2007).
In summary, P. hexincisus and P. scribneri have similar, indeed overlapping, morphometric characteristics and shared morphological characters, leading to a confuse and difficult identification. Hence, a topotype population of P. scribneri is needed to solve the identity and validity of P. scribneri and P. hexincisus, as suggested by Inserra et al. (2007) and Subbotin et al. (2008). Likewise, the D2-D3 sequence of P. agilis Thorne and Malek, 1968 is also similar to P. hexincisus sensu Inserra et al. (2007), as provided and mentioned by Subbotin et al. (2008). Loof (1978) had already doubted the validity of P. agilis and the species was considered as species inquerendae (Frederick and Tarjan, 1989). This was confirmed by ITS sequences and isozyme analysis. Pratylenchus agilis was proposed as a junior synonym of P. scribneri (Hernández et al., 2000), although Waeyenberge et al. (2000) indicated differences between P. scribneri and P. agilis with respect to ITS-rDNA length and the RFLPs.
Evidently, additional morphological and molecular characterizations are required to further analyses the species group of P. scribneri, P. hexincisus and P. agilis.