Description of Hirschmanniella dicksoni n. sp. (Nematoda: Pratylenchidae) from rhizosphere soil of limpograss from Florida, USA

Soil samples were collected from Beef Farm Prairie, Osceola County in Florida, with latitude 28.087400 and longitude 80.897847, and submitted to the NAL for nematode diagnosis in December 2015. Nematodes were extracted from 100 cm³ of soil using centrifugation and sugar floatation techniques (Harrison and Green, 1976).

In total, 20 females and 20 males were used for morphological and morphometric characterization. Nematodes were processed for permanent mounting according to Ryss (2003). Nematodes were picked and transferred to a PCR tube and killed with hot (95°C) 4% formalin. The PCR tube with heat-killed nematodes was immediately placed in a thermo cycler: 95°C for 2 min, 65°C for 10 min, 75°C for 10 min, 85°C for 10 min, and 95°C for 10 min. After the tube reached room temperature, the tube was rinsed with distilled water and the content was transferred to a staining dish. Nematodes were picked out and transferred to a glass slide with cavity filled with a mixture of glycerol and distilled water in proportion of 1:20. Nematodes were infiltrated in glycerin for permanent slide preparation by placing the cavity slide with the nematodes on a hot plate at 70°C for 15 to 20 min and mounted on glass slides following the method described by De Grisse (1969). The specimens were examined for morphological features and morphometrics using a camera installed on ZEISS AXIO microscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). Measurements and pictures were taken using the ZEN lite software on ZEISS Axiocam ERc5s digital camera. Morphological and morphometric characteristics were compared with identification keys for genus Hirschmanniella revised by Khun et al. (2015).

Adults and juveniles of the new species were collected individually by hand and washed with sodium phosphate buffered in 3% glutaraldehyde solution for 24 hr at 4°C. The specimens were then post fixed overnight in 3% Osmium tetraoxide solution and dehydrated in a concentration gradient of ethanol. Dehydrated nematodes were dried with a critical point dryer, attached to a self-adhesive stub, and coated with gold/palladium auto sputter coater. Specimens were studied with a Hitachi SU5000 electron microscope at the Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida, Gainesville, FL.

Total genomic DNA was extracted following the sodium hydroxide method (Wang et al., 1993). A single female was transferred to 20 µl of 0.25 NaOH in 1.5 ml eppendorf tubes, crushed in a sterile glass rod, and incubated overnight at 25°C. The tubes were then heated at 99°C for 3 min and cooled down to room temperature. In total, 5 µl of 1 M HCl, 10 µl of 0.5 M Tris-HCl (pH 8.0), and 5 µl of 2% Triton X-100 were added to the lysate and mixed briefly, and heated at 99°C for 3 min. The digest was cooled to room temperature and 1.5 µl of the digest was immediately used for polymerease chain reaction (PCR). DNA amplification was performed in 25 µl of reaction mix containing 12.5 µl 2× Hot Start Master mix (New England BioLabs, Ipswich, MA), 1.25 µl forward and reverse primers, 8.5 µl sterile nuclease free water (Thermo Fisher Scientific, Gainesville, FL), and 1.5 µl template DNA. For amplification of the ribosomal region spanning the internal transcribed spacer (ITS)1, 5.8 S gene, and ITS2, the primers TW81R (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28F (5′-ATATGCTTAAGTTCAGCGGGT-3′) were used. D2–D3 expansion segment of the large subunit (LSU) rDNA was amplified using the primers sets D2A (5′-CAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′). The thermocycling was carried out using a Master cycler nexus (Eppendorf AG, Hamburg, Germany). The thermocycling reactions for both DNA regions were as follows: 95°C for 15 min, followed by 40 cycles of 94°C for 30 sec, 50°C for 45 sec, and 72°C for 2 min, and a final extension step of 72°C for 7 min.

DNA fragments were separated by electrophoresis in 1XTAE buffer and 1.2% agarose gel, stained with ethidium bromide, and visualized under UV light using the ChemiDOC XRS One 4.5.2 program (Bio-Rad Laboratories, Life Science Group, Hercules, CA). The DNA fragments were purified using QIAquick PCR Purification kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. The purified DNA was quantified using a NanoDrop 1000 (Thermo Science, Grand Island, NY) spectrophotometer and sequenced at the ICBR, University of Florida, Gainesville, FL. To identify the species, raw sequences were checked and edited manually using BioEDIT, v. 7.2.5 (Hall, 1999). Consensus sequences obtained were compared with those deposited in the GenBank database using BLAST engine search for sequence homology. The newly obtained consensus sequences were submitted to the GenBank database under accession numbers KY773577 for D2–D3 of 28S gene and KY773578 for ITS rRNA.

To perform the phylogenetic analysis, D2–D3 LSU, ITS1-5.8S-ITS2 rDNA sequences obtained from H. dicksoni n. sp. identified in this study and those retrieved from the GenBank and NCBI databases (Tables 1, 2) were aligned over the same length in MUSCLE using MEGA v. X (Kumar et al., 2018). The alignment was analyzed to get the base substitution model for these sequences using MEGA X (Kumar et al., 2018). For D2–D3 LSU sequences, the evolutionary history was inferred by using the Kimura 2-parameter model (Kimura, 1980). For ITS1-5.8S-ITS2 rDNA sequences, the evolutionary history was inferred by using the Tamura 3-parameter model (Tamura, 1992). Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log-likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites. The phylogram was generated using base substitution model selected for each alignment and running maximum likelihood model with 1,000 bootstrap replicates using MEGA X (Kumar et al., 2018) to assess the degree of support for each branch on the tree (Landa et al., 2008). Unique data sequences obtained from this study were submitted to GenBank under accession numbers KY773577 and KY773578 for D2–D3 LSU and ITS1-5.8S-ITS2 rDNA sequences, respectively. The D2–D3 sequence from Pratylenchus vulnus was used as outgroup for constructing the phylogenetic tree based on the sequences form D2–D3 expansion segment of 28S rRNA, whereas P. bolivianus was used as outgroup in the case of phylogenetic tree based on ITS1-5.8S-ITS r DNA sequence.

They are characterized by slender body, slightly curved to irregularly shaped, 1,582 to 1,929 μm long. The maximum body diameter is 26 to 31 μm. Lip region is hemispherical (Figs. 1C, 3A), with diameter 9.1 to 11.2 μm and height 3.7 to 4.8 μm, and six to seven transverse annules without longitudinal lines, continuous with body contour. Some lip region annules are incomplete. Cephalic framework is moderately sclerotized. Stylet is 1.9 to 2.1 times as long as lip region diameter; stylet knobs are round, clearly offset, and stylet cone is 1.07 to 1.1 times as long as shaft. The position of excretory pore (EP) is 124 to 146 μm from the anterior end. Oesophageal glands are elongated, tapering posteriorly and overlapping intestine ventrally. Hemizoid is anterior to EP. Lateral incisures on lateral fields begin about 26 annules from anterior end as two fully areolated lines and becoming four lines towards posterior end. Areolation is irregular and incomplete in the middle of lateral field. Female genital branches are opposed and outstretched. Vulva is located at 50 to 56% of the body length. Spermatheca is oval and filled with rounded sperm. Intestine is not overlapping rectum. The shape of tail terminus is variable, with a ventral projection usually with subterminal notch (Figs. 1B, 3F).

They are similar to females in general morphology except for secondary sexual character and shorter body length, ranging 1,235 to 1,772 µm. Male genital system is monorchic and outstretched. Spicules are medium-sized, 19 to 30 μm long, well developed and arcuate ventrally. Gubernaculum is simple, straight or curved, not protruding, ca 7.4 to 11.3 μm or 27 to 38.9% of spicules length. Bursa is leptoderan. The shape of tail terminus is variable, with pointed terminus without a projection and round terminus without a projection (Fig. 2). Tail terminus with subterminal notch is rare in males.

Soil samples were collected from Beef Farm Prairie, Osceola County, Florida with latitude 28.087400 and longitude 80.897847. The new species was collected from a waterlogged wetland rhizosphere soil associated with limpograss.

Holotype female and Paratypes (NemaLAB 0001) and paratypes (NemaLAB 0002) are deposited at the NAL, University of Florida nematode collection.

The species is named in honor of Dr Donald W. Dickson for his over 50 years of outstanding service as a mentor, trainer, and researcher at the University of Florida.

ZOOBANK Code: zoobank.org:pub:6DD36DA0-6544-406D-8F4C-371EE443E115.

The main diagnosis characteristics of Hirschmanniella dicksoni n. sp. are as follows: body length less than 2 mm, hemispherical lip region with 6 to 7 lip annuli, stylet length 19 to 21 µm with round knobs, excretory pore at or anterior of oesophageal–intestinal junction (OIJ) up to 32 µm anterior to the latter, irregularly areolated lateral fields, spermatheca oval and filled with rounded sperm, intestine not overlapping the rectum. Tail length is 103 to149 µm, and tail terminus has a ventral projection usually with subterminal notch. Males are present and are similar to the females except for shorter body length and secondary sexual characters. Male spicules are 19 to 30 µm in length.

Female Hirschmanniella dicksonsi n. sp. closely resembles H. mucronata (Das, 1960; Luc and Goodey, 1964) in morphology and morphometry, but it can be distinguished from H. mucronata by the following characteristics: it has a relatively longer tail (103–149 vs 60–160 μm). Usually the tail terminus has a ventral projection and subterminal notch vs usually pointed or round with or without a projection, but no subterminal notch; relatively smaller c (13–15 vs 13–30 μm), but relatively greater c′ (5.1–6.7 vs 2.5–5.5 μm), relatively longer procorpus (50–87 vs 31–62 μm), relatively longer distance of OIJ from anterior end (142–177 vs 91–150 μm), excretory pore (124–146 vs 83–141 μm), and nerve ring position (105–122 vs 74–107 μm) (Table 3) (Khun et al., 2015).

The new species H. dicksoni n. sp. differs from H. oryzae and H. belli by having a hemispherical lip region with six to seven lips annuli vs no hemispherical lip region with three to four lips annuli and a tail terminus with ventral projection and subterminal notch vs a tail terminus with projection or mucron. Hirschmanniella dicksoni n. sp. also differs in spicules length (19–30 vs 31–36 μm) from H. belli, but has similar spicules length (19–30 vs 18–34 μm) as H. oryzae. However, H. dicksoni n. sp. is similar in stylet length to H. belli (19–21 vs 18–22 μm) and H. oryzae (19–21 vs 13–21 μm), as well as body length (1,582–1,929 μm), (1,420–2,220 μm), (900–1,850 μm), respectively (Khun et al., 2015).

Hirschmanniella dicksoni n. sp. has a longer body (1,582–1,929 μm) than H. brassicae (Duan et al., 1996) (920–1,420 μm), H. microtyla (930–1,450 μm), and H. shamimi (Ahmad, 1972) (1,190–1,540 μm). The new species has a shorter body (1,582–1,929 μm) than H. furcate (Razjivin et al., 1981) (2,830–2,990), H. imamuri (Sher, 1968) (2,150–272 μm), H. obesa (Razjivin et al., 1981) (3,240–3,410 μm) and H. truncata (Razjivin et al., 1981) (2,180–2,430 μm). Hirschmanniella dicksoni n. sp. possesses greater c′ ratio (5.1–6.7) than H. areolata (Ebsary and Anderson, 1982) (3.1–4.6), H. mexicana (6.2 vs 4.8), H. microtyla (3.0–4.9), H. miticausa (Bridge et al., 1983) (3.4–4.9), and H. thornei (Sher, 1968) (3.2–4.8), but smaller c′ ratio (6) than H. zostericola (Allgén, 1934; Luc and Goodey, 1964) (9) (Khun et al., 2015).

The hemispherical lip region in H. dicksoni n. sp. is similar to H. areolate, H. behningi (Micoletzky, 1923; Luc and Goodey, 1964), H. diversa, H. furcate, H. imamuri, H. loofi (Sher, 1968), H. mucronata, H. obesa, H. pisquidensis (Ebsary and Pharoah, 1982), H. spinicaudata, H. thornei, and H. truncate. Based on stylet length, H. dicksoni n. sp. (19–21 μm) is close to H. belli (18–22 μm), H. caribbeana (Van den Berg and Quénéhervé, 2000) (17–20 μm), H. caudacrena (17–23 μm), H. gracilis (20–24 μm), H. kwazuna (Van den Berg et al., 2009) (18–22.5 μm), H. marina (20–25 μm), H. mexicana (20 μm), H. microtyla (17.6–21.5 μm), H. miticausa (Bridge et al., 1983) (18.5–21 μm), H. oryzae (13–21 μm), H. pomponiensis (17–23 μm), and H. santarosae (17–21 μm) (Khun et al., 2015).

Out of the 29 nominal species (Khun et al., 2015), H. dicksoni n. sp. has a longer stylet (19–21 μm) than H. brassicae (14–16 μm) and H. zostericola (15 μm) and shorter stylet than H. areolata (24–27 μm), H. behningi (24–28 μm), H. diversa (22–25 μm), H. furcate (38.0–38.5 μm), H. imamuri (29–32 μm), H. loofi (31–41.6 μm), H. obesa (40.9–41.5 μm), H. pisquidensis (23–25 μm), H. spinicaudata (38–50 μm), H. thornei (26–30 μm), and H. truncata (35.7–38.5 μm) (Khun et al., 2015).

Hirschmanniella dicksoni n. sp. has a tail terminus shape with single ventral projection usually with a subterminal notch, which is similar to H. caudacrena, H. furcate, H. halophile, H. marina, H. mexicana, and H. obesa, but it can be differentiated from these species as follows: H. dicksoni n. sp. differs from H. caudacrena with its hemispherical lip region and ventral tail projection vs no hemispherical lip region and axial tail projection. Hirschmanniella dicksoni n. sp. differs from H. furcate with its shorter body length (1,582–1,929 vs 2,830–2,990 μm), smaller c (12.6–15.3 vs18.3–20.4), shorter stylet length (19–21 vs 38.0–38.5 μm), greater a (50.3–68.6 vs 45.3–47) and b (10.0–14.0 vs 5.9–6.4). Hirschmanniella dicksoni n. sp. differs from H. halophila with its hemispherical lip region and EP anterior to OIJ vs no hemispherical lip region and EP posterior to OIJ. Hirschmanniella dicksoni n. sp. differs from H. marina with its hemispherical lip region, shorter spicules (19–30) vs no hemispherical lip region and longer spicules (34–36 μm). Hirschmanniella dicksoni n. sp. is differentiated from H. mexicana with its greater a (59 vs 37), smaller b’ (4.2 vs 5.5) and c (14 vs 17) ratios, V value (53 vs 49) and its hemispherical lip region and EP anterior to OIJ vs no hemispherical lip region and EP posterior to OIJ. Hirschmanniella dicksoni n. sp. differs from H. obesa with its shorter body length (1,582–1,929 vs 3,240–3,410 μm), smaller c (13.0–15.0 vs 18.2–20.4), shorter stylet length (19–21 vs 40.9–41.5 μm), and greater a (50.0–69.0 vs 39.4–41), b (9.8–13.6 vs 6.3–6.6), and c′ (5.1–6.7 vs 3.1–4.9).

Hirschmanniella dicksoni n. sp. differs from H. spinicaudata with the presence of subterminal notch and ventral projection, EP anterior to OIJ vs absence of subterminal notch and axial mucron and EP posterior to OIJ. Hirschmanniella dicksoni n. sp. also differs from H. spinicaudata with its smaller c (12.6–15.3 vs 17–28), shorter stylet (19–21 vs 38–50), and shorter spicules (22–30 vs 41–54).

The amplification of D2–D3 expansion segments of 28S rRNA yielded a single fragment of approximately 782 bp based on direct fragment sequencing. Sequences from other species of Hirschmanniella obtained from NCBI were used for further phylogenetic studies (Table 1). Based on the BLAST analysis, D2–D3 expansion segments of 28S rRNA of H. dicksoni n. sp. showed 99% similarity (91% query cover) with an undescribed species of Hirschmanniella isolated from the Colorado River in Yuma, Arizona (EF 029861), whereas they showed 96% similarity (90% query cover) with H. belli (EF029860) and 95% similarity (89% query cover) with H. oryzae (JX291141).

Although D2–D3 expansion segments of 28S rRNA from H. dicksoni n. sp. species showed 99% similarity over 100% query cover with two isolates identified as H. mucronata from Jiangsu, China (KR780066 and KR780067), these sequences showed closer similarity with H. oryzae than other isolates of H. mucronata from the Philippines (KF201167) and Cambodia (KP179327 and KP179333). Therefore, we did not include these sequences in the phylogenetic analysis because we did not consider these sequences as H. mucronata. The phylogenetic analysis excluding these sequences from China isolates indicates that the sequences from H. dicksoni n. sp. and other species of Hirschmanniella obtained from GenBank were grouped into three different clades (Fig. 4). The D2–D3 expansion segments of 28S rRNA from H. dicksoni n. sp. clustered together with the undescribed species of Hirschmanniella from Yuma (Arizona) and Vietnam, H. belli from California, and H. oryzae in Clade I. The second clade included H. loofi, H. kwazuna, and H. mucronanta from the Philippines and Cambodia. The isolates of H. halophila, H. diversa, H. santarosae, and H. pomponiensis were grouped in Clade III.

The amplification of ITS1-5.8S-ITS2 r DNA yielded a single fragment of approximately 787 bp based on sequencing. Sequences from other species of Hirschmanniella obtained from NCBI were used for further phylogenetic studies (Table 2). The similarity search using BLAST tool showed that the ITS sequence from H. dicksoni n. sp. was 88% similar to H. oryzae (Vietnam isolate) over 99% query cover. As in the case of D2–D3 sequence analysis, the BLAST search using ITS sequence from H. dicksoni n. sp. also showed 96% similarity with sequences from an isolate of H. mucronata (KR780069) from Jiangsu, China, over 100% query cover. However, KR780069 showed higher similarity with H. oryzae sequence (88%) than with other isolates of H. mucronata sequences (84%) over 100% query cover. In contrast to this, another sequence (KJ923642) identified as H. mucronata from the same study (Jiangsu, China) showed only 85% similarity with H. dicksoni n. sp. over 74% query cover. The sequence KJ923642 showed 99% similarity with sequences from other isolates of H. mucronata, whereas the sequence KR78069 showed only 84% with H. mucronata over 100% query cover. Thus, since the identity of the China isolate KR78069 as H. mucronata was not certain based on the sequence analysis, we did not include this sequence in the phylogenetic analysis. The phylogenetic analysis excluding KR78069 showed that the sequences from H. dicksoni n. sp. and other isolates from GenBank were grouped into two clades. Hirschmanniella dicksoni n. sp. was grouped, as a sister species, with isolates of H. oryzae in Clade I, which was similar to the phylogenetic analysis based on D2–D3 sequence. The second clade included H. loofi, H. kwazuna and H. mucronata from the Philippines, Belgium, Taiwan, and China.