The University of Florida Nematode Assay Laboratory (NAL) received a rhizosphere soil sample for a routine diagnosis purpose from Beef Farm Prairie, Osceola County in Florida. The sample was taken from a site dedicated to limpograss (
The owner of this property had recognized a patchy appearance of the field and yield reduction on the limpograss, and sent soil samples for plant-parasitic nematode diagnosis to the NAL in November 2015. We found several individuals belonging to the genus
Only a handful of species in this genus have been reported in the USA, mainly from Florida and California. In Florida, Sher (1968) reported
In California, Golden (1957) reported
There were very limited reports of
In this study, we report the results of our morphological, morphometric, and molecular characterization of the new species
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
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
List of 28S ribosomal RNA gene partial sequences (D2–D3 segment) of
Species | Accession No. | Location |
---|---|---|
|
KY773577 | Florida |
|
EU620464 | South Africa |
|
KY794704 | Iran |
|
JX261958 | Iran |
|
EU620465 | South Africa |
|
JX144359 | Japan |
|
EF029859 | California |
|
DQ077795 | California |
|
EU620466 | South Africa |
|
EU620467 | South Africa |
|
EU620468 | Belgium |
|
EU620469 | Belgium |
|
KF201167 | The Philippines |
|
KP179327 | Cambodia |
|
KP179333 | Cambodia |
|
KY424320 | China |
|
MK241938 | Indonesia |
|
EF029861 | Arizona |
|
EF029860 | California |
|
JX291142 | Myanmar |
|
KF201165 | The Philippines |
|
DQ328686 | Vietnam |
|
KP671713 | Belgium |
|
JX047010 | USA |
List of 18S ribosomal RNA gene partial sequences (ITS rRNA) of
Species | Accession No. | Location |
---|---|---|
|
KY773578 | Florida |
|
KP179328 | Belgium |
|
KF201166 | The Philippines |
|
DQ309589 | Taiwan |
|
KP179329 | Belgium |
|
KJ923642 | China |
|
KP179331 | Belgium |
|
EU620472 | South Africa |
|
EU620473 | South Africa |
|
EU620471 | South Africa |
|
EU620470 | South Africa |
|
KF201164 | The Philippines |
|
KF201163 | The Philippines |
|
DQ309588 | Taiwan |
|
FR692329 | Italy |
Systematics
Morphometrics of
Female | Male | ||
---|---|---|---|
Character | Holotype | Paratypes | Paratype |
|
– | 19 | 20 |
|
1,906 | 1714 ± 152 | 1506 ± 143 |
(1,582–1,929) | (1,235–1,772) | ||
|
61 | 59 ± 4.9 | 60 ± 6.3 |
(50–69) | (47–72) | ||
|
11 | 11 ± 1.1 | 9.6 ± 0.9 |
(10–14) | (8.6–11.3) | ||
|
4.1 | 4.2 ± 0.4 | 4.3 ± 0.4 |
(3.8–4.8) | (3.8–5.1) | ||
|
13 | 14 ± 0.9 | 15 ± 1.5 |
(13–15) | (13–18) | ||
|
6.7 | 6.2 ± 0.5 | 6.2 ± 0.8 |
(5.1–6.7) | (4.8–7.2) | ||
G1 | 22 | 23 ± 2.3 | – |
(20–26) | |||
G2 | 20 | 20 ± 1.8 | – |
(17–24) | |||
T | – | – | 37 ± 5 |
(29–45) | |||
V | 55 | 53 ± 1.7 | – |
(50–56) | – | ||
M | 51 | 52 ± 1.7 | 54 ± 1.9 |
(51–55) | (51–57) | ||
o | 23 | 22 ± 2.2 | 21 ± 2.9 |
(19–27) | (17–27) | ||
DGO | 4.5 | 4.3 ± 0.4 | 4 ± 0.6 |
(3.8–5.1) | (3.3–5.1) | ||
Lip region diameter | 10 | 10 |
9.9 ± 0.4 |
(9.1–11.2) | (9.2–11) | ||
Lip region height | 3.7 | 4.1 ± 0.3 | 4.3 ± 0.3 |
(3.7–4.8) | (4.0–5.1) | ||
Stylet length | 19 | 20 ± 0.6 | 19 ± 0.5 |
(19–21) | (18–20) | ||
Conus length | 9.8 | 10 ± 0.5 | 10 ± 0.4 |
(9.7–11) | (9.6–11) | ||
Shaft length | 9.5 | 9.3 ± 0.4 | 8.7 ± 0.4 |
(8.6–9.9) | (8.2–9.6) | ||
Stylet knob height | 3 | 2.7 ± 0.4 | 2.7 ± 0.2 |
(2–3.4) | (2.4–3.2) | ||
Stylet knob width | 4.6 | 4.5 ± 0.4 | 4.3 ± 0.5 |
(3.8–5.3) | (3.4–5.0) | ||
Procorpus length | 80 | 63 ± 13.8 | 70 ± 7.2 |
(50–87) | (54–80) | ||
Median bulb length | 17 | 17 ± 1.4 | 16 ± 1.3 |
(15–20) | (14–18) | ||
Median bulb diam. | 13 | 12 ± 0.8 | 11 ± 1.4 |
(11–13) | (7.5–13) | ||
Median bulb valve length | 5.8 | 5 ± 0.7 | 4.7 ± 0.7 |
(3.5–6.3) | (3.8–6) | ||
Median bulb valve width | 3.6 | 2.9 ± 0.4 | 2.9 ± 0.5 |
(2.3–3.6) | (2.1–3.6) | ||
Anterior end to oesophageal-intestinal junction | 177 | 154 ± 12 | 158 ± 13 |
(142–177) | (129–179) | ||
Anterior end to excretory (EP) pore | 146 | 133 ± 7 | 119 ± 11 |
(124–146) | (87–134) | ||
EP pore to oesophageal-intestinal junction | 31 | 25 ± 4.3 | 39 ± 8.9 |
(20–32) | (24–50) | ||
Metacorpus valve from anterior end | 89 | 85 ± 4.1 | 80 ± 4.7 |
(80–94) | (72–87) | ||
Nerve ring from anterior end | 116 | 113 ± 5 | 102 ± 10 |
(105–122) | (73–114) | ||
Oesophageal gland overlap | 294 | 257 ± 34 | 193 ± 23 |
(204–311) | (150–223) | ||
Oesophagus length | 470 | 411 ± 38 | 351 ± 30 |
(330–470) | (303–392) | ||
Max body length | 31 | 29 ± 1.6 | 26 ± 3.1 |
(26–31) | (21–30) | ||
Length from phasmids to terminus | 32 | 29 ± 2 | 32 ± 2.9 |
(25–32) | (27–35) | ||
Tail length | 149 | 125 ± 11 | 102 ± 11 |
(103–149) | (84–119) | ||
Anal body width | 22 | 20 ± 1 | 17 ± 1.8 |
(18–22) | (15–20) | ||
From cloaca to anterior most part of testis | – | 559 ± 92 | |
(420–708) | |||
Vulva to anterior end | 1045 | 912 ± 90 | – |
(803–1056) | |||
Anterior genital branch | 420 | 391 ± 57 | – |
(305–472) | |||
Posterior genital branch | 385 | 333 ± 39 | – |
(289–433) | |||
Ant. Spermatheca length | 26 | 28 ± 4.2 | – |
(20–35) | |||
Ant. Spermatheca diam | 15 | 16 ± 2.6 | – |
(9.3–19) | |||
Post. Spermatheca length | 24 | 24 ± 3.9 | – |
(17–30) | |||
Post. Spermatheca diam | 14 | 14 ± 2 | – |
(8.9–17) | |||
Bursa | – | – | 70 ± 15 |
(45–94) | |||
Cloaca to anterior tip of bursa | – | – | 25 ± 5.8 |
(15–33) | |||
Cloaca to posterior tip of bursa | – | – | 45 ± 9.5 |
(27–61) | |||
Gubernaculum | – | – | 8.9 ± 1.1 |
(7.4–11) | |||
Spicules | – | – | 25 ± 2.9 |
(19–30) |
–, data not available. All measurements are in μm and in the form: mean ± SD (range).
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
Female
The new species
The hemispherical lip region in
Out of the 29 nominal species (Khun et al., 2015),
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
Although D2–D3 expansion segments of 28S rRNA from
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
We have characterized
Our data show the presence of interaspecific variation in the female tail shape in
Previous studies have shown that a slight difference in the morphology and morphometrics might lead to the description of new species, resulting in over estimation of species in the genus
The new species showed 99% D2–D3 sequence homology with 94% query coverage to undescribed species of
As in the case of D2–D3 sequence analysis, the BLAST search using ITS sequence from
Apart from the inconsistences in the molecular data, we studied the morphology and morphometric data of the China isolates from Hui et al. (2016). We found differences between
This is a first report on the occurrence of
The identification of