Journal of Nematology 37(1):94–104. 2005. ©The Society of Nematologists 2005. Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), aNeedle Nematode Parasitizing Pine in Georgia Z. A. H andoo, 1 L. K. C arta, 1 A. M. S kantar, 1 W. Y e , 2 R. T. R obbins, 2 S. A. S ubbotin, 3 S. W. F raedrich, 4 and M. M. C ram4 Abstract: We describe and illustrate anew needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, ( Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with abody length of 5.4–9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end atruncate appearance; long odontostyle (124–165 µm); vulva at 44%–52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2–D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required. Key words: DNA sequencing, Georgia, loblolly pine, Longidorus americanum n. sp., molecular data, morphology, new species, needle nematode, phylogenetics, SEM, taxonomy. Needle nematodes ( Longidorus spp.) are an impor- although not as suitable as pine. Small grain crops, in- tant group of plant parasites that cause serious damage cluding sorghum, sorghum-sudan hybrid, wheat, rye, to many plant species (Cohn, 1974). These slender and oats, were poor hosts. Yellow and purple nutsedge, nematodes are readily recognized from most other common weeds at the nursery, and tomato and Cab- Dorylaims by their long (2 mm to 12 mm), rather nar- bage, previously grown at the nursery site, were also row body and their elongated axial spear (odontostyle) poor hosts. plus an extension (odontophore) about half the odon- The objectives of this study were to describe this new tostyle length (Hooper, 1974). Symptoms associated species using light and scanning electron microscopy with Longidorus generally are non-specific and may in- (SEM) observations and to assess the diagnostic value clude plant stunting, root tip galls, and root forking of morphological and molecular characters. (Cohn, 1974). In addition, some Longidorus species are vectors of nepoviruses (Taylor and Brown, 1997). Dur- M aterials and M ethods ing summer 2000, anew species of the needle nema- tode Longidorus was associated with patches of stunted Soil samples (2.5-cm and cholorotic loblolly pine ( Pinus taeda L.) seedlings Morphological characterization: in seedbeds at the Flint River Nursery (Byromville, GA) diam. to a20-cm depth) were collected from areas that (Fraedrich and Cram, 2002). Seedlings from affected contained patches of stunted loblolly pine seedlings. 3 composite areas had poorly developed root systems that lacked Nematodes were extracted from a200-cm soil sample that was thoroughly but gently mixed, using lateral and feeder roots, and the nematode was found the technique of Flegg (1967) with modifications by to damage root systems of loblolly pine in acontrolled Fraedrich and Cram (2002). Nematodes were removed study. Subsequently, various plant species were evalu- from Baermann funnels, and juveniles, males, and fe- ated as potential hosts to this needle nematode (Fraed- males were fixed in warm 3% formaldehyde fixative rich et al., 2003). Slash, loblolly, and longleaf pines and processed to glycerine by the formalin-glycerine were good hosts. Red oak also appeared to be ahost method (Golden, 1990; Hooper, 1970). Light micro- scopic images of fixed nematodes were taken on aLeica Received for publication 5January 2004. WILD MPS48 Leitz DMBR compound microscope fit- 1 Nematology Laboratory, USDA, ARS, Henry A. Wallace Beltsville Agricul- tural Research Center, Bldg. 011A, Room 165B, Beltsville, MD 20705–2350. ted with an ocular micrometer for image measurement. 2 Nematology Laboratory, University of Arkansas, Fayetteville, AR 72701. For SEM, living specimens were fixed in 3% glutaralde- 3 Institute of Parasitology of Russian Academy of Sciences, Leniskii Prospect 33, Moscow 117091, Russia. hyde buffered with 0.05 Mphosphate (pH 6.8), dehy- 4 USDA, Forest Service, 320 Green Street, Athens, GA 30602. drated in agraded series of ethanol, critical-point dried The authors thank Donna M. S. Ellington, Nematology Laboratory, USDA, ARS, Beltsville, Maryland, for technical assistance. Support for S. A. S. from the from liquid CO2 ,and sputter coated with a20to30-nm CLO-Agricultural Research Centre, Merelbeke, Belgium, is gratefully acknowl- layer of gold-palladium. edged. Mention of trade names or commercial products in this publication is solely Molecular characterization: For molecular analysis two for the purpose of providing specific information and does not imply recom- samples were prepared: one sample contained one mendation or endorsement by the U.S. Department of Agriculture. E-mail: [email protected] specimen, and the second one contained five speci- This paper was edited by J. L. Starr. mens of this new longidorid nematode. Nematodes 94 Longidorus americanum n. sp.: Handoo et al. 95 were transferred into 10 µl of double distilled water in S ystematics an Eppendorf tube and crushed with microhomogeni- sator. Eight µl nematode lysis buffer (125 mM Kcl, 25 Longidorus americanum n. sp. mM Tris-Cl, pH 8.3, 3.75mM MgCl2 ,2.5 mM DTT, (Figs. 1 – 5) 1.125% Tween 20) and 2µlofproteinase K(600 µg/ ml) were added. The tubes were incubated at 65 ° C(1 Description hour) and 95 ° C(10 minutes) consecutively. After cen- All measurements are in micrometers unless other- trifugation (1 minute; 16,000g), 4µlofthe DNA sus- wise stated. pension was added to the PCR reaction mixture con- Holotype (female, in glycerine): L=7.4 mm; a=108.0; taining 10 µl 10X Taq incubation buffer, 20 µl 5X Q- b=14.9; c=124.0; c Ј =1.4; J Ј =0.8; V% =46; odonto- solution, 200 µM of each dNTP ( Taq PCR Core Kit, style =143.5; odontophore =83.0; total stylet =226.5; Qiagen, Germany), 1.5 µM of each primer (synthesized anterior end to end of esophagus =500.0; anterior end by Life Technologies, Merelbeke, Belgium), 0.8U Taq to guiding ring =32.5; tail length =60.0; hyaline tail Polymerase ( Taq PCR Core Kit, Qiagen, Germany), and length =17.5; hyaline tail width =22.5; lip width =27.5; double distilled water to a fi nal volume of 100 µl. The mid-body width =69.0; anal-body width =42.5. D2– D3 region of the 28S gene was amplifi ed using the primers D2A (5Ј -ACAAGTACCGTGAGGGAAAGTTG- 3 Ј )and D3B (5Ј -TCGGAAGGAACCAGCTACTA-3Ј ) T ABLE 1. Measurements of paratype females and males of Longidorus americanum n. sp. (Rubtsova et al., 2001). DNA amplifi cation was per- formed using aPTC-100 Programmable Thermal Con- Character Females Males trolled (MJ Research Inc., Watertown, MA) and con- sisted of 4minutes at 94 ° Cand 35 cycles of 1minute at N 27 5 L(mm) 7.00 ±0.80 6.40 ±0.40 94 ° C, 1.5 minutes at 55 ° C, 2minutes at 72 ° C, and 10 (5.40– 9.00) (5.80– 6.80) minutes at 72 ° C. PCR products were purifi ed using a a119.6 ±12.0 106.7 ±11.7 QIAquick PCR Purifi cation Kit (Qiagen Ltd., Crawley, (89.5– 136.6) (94.4– 123.4) West Sussex, UK). DNA fragments were directly se- b9.0 ±4.5 14.5 ±1.6 (4.6– 16.8) (12.2– 15.5) quenced in both directions using PCR primers with a c119.1 ±14.5 113.2 ±5.9 BigDye Terminator Cycle Sequencing Ready Reaction (95.0– 154.6) (102.8– 117.3) Kit (PE Applied Biosystems, UK) according to the c Ј 1.5 ±0.1 1.36 ±0.03 manufacturer’ sinstructions. Sequences were run on an (1.3– 1.7) (1.3– 1.4) J Ј 0.8 ±0.1 — ABI Prism 310 Genetic Analyzer (Applied Biosystems, (0.6– 1.0) Foster City, CA). Sequence for Longidorus americanum n. G1% 0.1 ±0.0 — sp. is deposited at GenBank with accession number (0.0– 0.1) AY494715. G2% 0.1 ±0.0 — (0.0– 0.1) The DNA sequence of Longidorus americanum n. sp. V% 48.8 ±2.2 — was edited with Chromas 1.45. Alignment with other (44.2– 51.7) longidorid sequences (Rubtsova et al., 2001), including Odontostyle 141.9 ±9.7 146.2 ±7.7 the outgroups Xiphinema rivesi and Xiphinema diversicau- (124.0– 165.0) (135.0– 152.0) Odontophore 83.0 ±2.4 52.4 ±7.0 datum (Y. He, unpubl. data), was performed with Clust- (79.2– 91.4) (47.0– 62.9) alX 1.64 with default options (Thompson et al., 1997). Total stylet 224.4 ±9.7 198.7 ±12.1 Equally weighted maximum parsimony was performed (207.1– 249.7) (182.0– 211.1) Anterior end to end of esophagus 602.0 ±104.8 472.7 ±51.4 using PAUP (4.0 beta version) (Swofford, 1998). Gaps (367.6– 777.8) (420.0– 530.0) were treated as missing data. Bootstrap analysis with 100 Guiding ring from anterior end 34.7 ±2.4 34.5 ±0.9 replicates was conducted to assess the degree of sup- (30.5– 42.6) (33.5– 35.5) port for each branch on the tree.