Journal of Nematology 27(2):213-221. 1995. © The Society of Nematologists 1995. Two New Species of Aphelenchoididae (Nemata) from Declining Red Pines in Maryland 1 TAFADZWA R. KAISA 2, AMY L. HARMAN 3, AND DAN M. HARMAN 4 Abstract: Aphelenchoides resinosi n. sp. and Ektaphelenchusjoyceae n. sp. are described and illustrated from red pines of the Allegheny plateau of Maryland, USA. The new species were found in trees infested with Bursaphelenchus xylophilus. Primary diagnostic characters of A. resinosi females are con- striction of the head, basal stylet knobs, tong postnterine sac, two incisures in the lateral field, and conical tail four to five anal body widths long with a simple terminal mucro. Diagnostic characters of the males are two pairs of subventral caudal papillae and spicule shape: Primary diagnostic charac- ters of E. joyceae females are a slight constriction of the head, six similar lips, conical tail, and short postuterine sac. Diagnostic characters of the males are spicule size and shape, a single row of spermatocytes, and one pair of caudal papillae. Within-tree distributions of A. resinosi and E. joyceae are presented. A total of 70% of both red-needled and chlorotic-needled trees in the study were positive for A. resinosi and E. joyceae. Branch hierarchy was related to the percentage of samples positive for A. resinosi. Key words: Aphelenchoides resinosi, Bursaphelenchus xylophilus, Ektaphelenchus joyceae, Maryland, nema- tode, new species, Pinus resinosa, red pine. Aphelenchoides species have been de- from Missouri (13) and the other in red scribed from conifers in the United States. pine from Maryland (9,10). To date, these Massey (19,20,21) described A. rhytium populations have remained unidentified. from eastern white pine (Pinus strobu~ L.), Another nematode, Ektaphelenchus sp., loblolly pine (P. taeda L.), and red pine (P. was collected from the same red pines. resinosa Ait.) in New York; A. conophthori Massey (17"21) described eight Ektaphelen- from eastern white pine in Connecticut; chus species associated with conifers in the and A. hylurgi from eastern white pine in United States, three of which have been Maine. Aphelenchoides pityokteini (21) was synonymized with previous taxa (1). Ek- described from corkbark fir (Abies lasio- taphelenchus josephi and E. riograndensis carpa var. arizonica (Merriam) Lemm.) in were described from limber pine (P. flexilis New Mexico and A. polygraphi (21) from James) and red pine, respectively. Ek- Engelmann spruce (Picea engelmanni taphelenchus obtusus was described from En- Parry) in Arizona. Massey (19) found an gelmann spruce, E. prolobos from corkbark Aphelenchoides sp. of the A. parietinus group fir, and E. sandiaensis from white fir. associated with white fir (A. concolor Gord. The purpose of this paper is to describe & Glend. Lindl.) in New Mexico. In studies two previously undescribed taxa and to of pine wilt disease, two populations of an provide information on their distribution Aphelenchoides sp. were found occurring within dying red pines. with Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 in dying pines MATERIALS AND METHODS (9,10,13). One population was found in as- sociation with Scots pine (P. sylvestris L.) During the summer of 1987, dying red pines infested with Bursaphelenchus xylo- philus were collected from Savage River Received for publication 14 September 1993. State Forest in Allegany County, Mary- i Adapted from the first author's M.S. thesis, Frostburg land. Trees in that stand were 20--26 years State University. 2 P.O. Box 144, Syracuse, NY 13210. old. Although several stages of decadence s Professor of Biology, Frostburg State University, Frost- were noted among the afflicted trees in burg, MD 21532 and to whom correspondence should be red pine forests of Maryland (9,10), only addressed. 4 Associate Professor, University of Maryland, Center for two were chosen for collection: red- Environmental and Estuarine Studies, Appalachian Environ- mental Laboratory, Frostburg State University, Frostburg, needled trees and chlorotic-needled trees. MD 21532. Red-needled trees had uniformly red nee- 213 214 Journal of Nematology, Volume 27, No. 2,June 1995 dles and had died in the fall or winter prior er's method (11). Drawings were done with to the summer of collection. Chlorotic- a microscope fitted with a drawing attach- needled trees were in early stages of dying ment. All measurements are in microme- during midsummer of the year of collec- ters (~m) unless otherwise specified. tion. Ten red-needled trees were collected in May and June, and ten chlorotic- SYSTEMATICS needled trees were collected in July. Aphelenchoides resinosi n. sp. Samples from trunks and branches were (Fig. 1A-E) taken by means of the method of Harman (9,10). The lowest stem sample was re- Measurements moved approximately 20 cm from the Holotype (female in glycerine): L = 506; ground, and subsequent samples were stylet = 12;a-- 27;b = 9;c = 15;c' = taken at 1.5-m intervals. Terminal por- 2.4; V = 72. tions of stems were also collected. Branch Female (n = 40): L = 400-800 (mean = hierarchy (primary, secondary, tertiary) 530; SD = 110); stylet = 10-11 (mean = and branch decadence (three levels) were 10.6; SD = 0.6); ratios-a = 29-53 (mean utilized in sampling. One branch from = 35; SD = 5), b = 7-13 (mean = 9; SD each category on each 1.5-m trunk seg- = 2), c = 12-19 (mean = 15; SD = 2), c' ment was sampled. Samples were collected = 3-4 (mean = 3.4; SD = 0.4), V = 66- from the branches at 1.5-m intervals start- 79 (mean = 69; SD = 2.8). ing at the base of the branch and proceed- Allotype (male in glycerine): L = 484; stylet ing outward. Branch samples consisted of = 11; spicule = 15; a = 26;b = 7; c = 16; cylinders about 10 cm in length. c' = 2.3. Nematodes were extracted by placing Male (n = 26): L = 360-480 (mean = small samples of wood in 1-oz plastic cups 450; SD = 60); stylet = 10 (mean = 10; and soaking in tap water for 24 hours. SD = 0); spicule = 13-15 (mean = 14; SD Specimens were heat-killed and preserved = 0.6); ratios-a = 31-48 (mean = 36; SD in glycerine-formaldehyde. Temporary = 4), b = 6-10 (mean = 8; SD = 1), c = mounts were made in lactophenol cotton 11-19 (mean = 14; SD = 2), c' = 2-3 blue after gently heating the nematodes in (mean = 2.4; SD = 0.1). a cavity slide containing a drop of tap wa- ter. Description Nematodes extracted from each sample Female: Body slender, slightly curved were counted. Final nematode counts were ventrally upon relaxation with heat. Cuti- expressed as number of nematodes per cle with fine, transverse striae. Lateral field gram of dry wood. Sample dry weight was with two incisures. Lip region and tail ter- determined following the method of minus not annulated. Body tapering pos- Brown and Bethel (4). Samples were oven- teriorly to conoid terminus. Terminus with dried at 100 C after nematode extraction small, fine mucro. Head set off by constric- and the weights recorded over a 48-hour tion (Fig. 1C); lips with guiding rods. Stylet period. Drying was stopped when weights delicate, basal knobs small. Stylet nar- remained constant over time, and the con- rowed anteriorly to fine tip. Tip of stylet stant weight was recorded as the dry very faint in preserved and fresh speci- weight of the sample. Eighty-three stem mens. Median bulb spherical with well- samples and 101 branch samples were an- developed, centrally situated valves. Duct alyzed from red-needled trees, and 77 pores into median bulb sometimes visible. stem samples and 97 branch samples from Nerve ring 3/4 of body width posterior chlorotic-needled trees. from base of median bulb, encircling Permanent mounts of nematodes were esophageal gland lobes and anterior end made in anhydrous glycerine after pro- of intestine. Esophageal gland lobes three cessing them slowly to glycerine by Hoop- body widths long. Excretory pore at level New Aphelenchoididae from Red Pine: Kaisa et al. 215 I , A 32 ,u,m B 28,u,m C 18,u,m D E 7/~m Fro. 1. Aphelenchoides resinosi n. sp. A) Full view of female. B) Full view of male. C) Head and neck of female. D) Stylet. E) Spicule. of nerve ring. Vulva slightly protuber- gland lobes. Oocytes in single row. Gonad ant. Vagina narrow, oblique. Ovary out- length 115 ~m. Postuterine sac 49 ~m stretched, not overlapping esophageal long, sometimes collapsed. 216 Journal of Nematology, Volume 27, No. 2,June 1995 Male: Male shorter than female, body ages 450 p~m in length with a conoid, mu- J-shaped. Cuticle finely annulated, lip re- cronate tail bearing two pairs of subventral gion and tail terminus smooth. Head set caudal papillae. Spicules are 14 I~m long, off, lips bearing guiding rods. Excretory arcuate with a prominent condylus. pore, median bulb, and nerve ring same as female. Testis long, outstretched. Tail ta- Relationships pering to conoid terminus with small, In the female body length of 400-800 sharp mucro. Bursa and gubernaculum p,m, the presence of two incisures in the absent. Two pairs of subventral caudal pa- lateral field, and the presence of a simple pillae present: first pair adanal, second mucro, A. resinosi is similar to A. arachidis post-anal, about one body width anterior Bos, 1977 (3), A. fragariae (Ritzema-Bos, to tail tip. Spicules paired, typically aph- 1891) Christie, 1932 (5), and A. helicosoma elenchoid, arcuate and rose-thorn shaped; Maslen, 1979 (16). It differs from A. condylus rounded and prominent, ros- arachidis in the shorter male body (360- trum pointed and less prominent. Point of 480 ~m vs. 560-1040 p~m) and in the num- intersection of dorsal and ventral limbs not ber of caudal papillae (two vs.