Journal of Nematology 20(2):266-287, 1988. © The Society of Nematologists 1988. Tylenchulus graminis n. sp. and T. palustris n. sp. (Tylenchulidae), from Native Flora of Florida, with Notes on T. semipenetrans and T. furcus R. N. INSERRA, 1 N. VOVLAS, ~ J. H. O'BANNON, 1 AND R. P. ESSER1 Abstract: Tylenchulus graminis n. sp. and T. palustris n. sp. are described and illustrated from broomsedge (Andropogon virginicus L.) and pop ash (Fraxinus caroliniana Mill.), respectively. T. gra- minis resembles T. furcus in having a distinct anus, but T. graminis second-stage juveniles (J2) do not have a bifid tail. T. semipenetransdoes not have a perceptible anus. The mature female of T. graminis has a mucronate pointed terminus while T. semipenetrans has a smooth and round terminus. T. graminis males have wider stylet knobs and basal bulb and a longer tail than T. semipenetrans males. T. graminisJ2 have a longer posterior body portion (without large fat globules) than T. semipenetrans J2. T. palustris resembles T. semipenetrans in having an undetectable anus but differs by the short and conoid mature female postvulval section. The male of T. palustris has larger stylet knobs and basal bulb than those of T. semipenetrans and a bluntly rounded tail terminus, which is tapered in T. semipenetrans. T. palustris differs from T. furcus and T. grarainis in having an undetectable anus, by the conoid postvulval section of mature females, by the shorter and rounded tail of males, and the shorter J2 posterior body section without large fat globules. T. graminis and T. palustris are parasites of indigenous flora of Florida. Key words: Andropogon virginicus, broomsedge, citrus, Florida, Fraxinus caroliniana, physiological race, pop ash, taxonomy, Tylenchulusfurcus, Tylenchulus graminis, Tylenchulus palustris, Tylenchulus semipenetrans, scanning electron microscopy. The genus Tylenchulus Cobb, 1913, pres- logical studies, the bush and grass races ently contains two species, Tylenchulus semi- were found to have sufficiently distinctive penetrans Cobb, 1913 and Tylenchulus fur- characters that warranted their elevation cus Van Den Berg & Spaull, 1982 (Fig. 1A, to species rank (10). Described here are B) (2,3,14). T. semipenetrans races attack di- Tylenchulus graminis n. sp. (the grass race) cotyledons, particularly rutaceous species, and Tylenchulus palustris n. sp. (the bush while T. furcus infects monocotyledons, es- race). This study has particular regulatory pecially Saccharum species (12,14). significance in Florida, because land in- Two tylenchulid isolates consistently fested with T. semipenetrans cannot be cer- found in noncuhivated areas of Florida tified for commercial citrus nurseries. previously were identified as races of T. semipenetrans and named the grass and bush MATERIALS AND METHODS races (9,10,13). Schizachyrium rhizomatum Specimens of T. graminis and T. palustris (Swallen) Gould, Andropogon virginicus L. were collected from broomsedge and pop (broomsedge), and Eremochloa ophiuroides ash roots at their respective type localities (Munro) Hack. (centipede grass) were hosts and near Venice and Steinhatchee, Flori- of the grass race (10,13). The only known da. hosts of the bush race were Fraxinus caro- Males and J2 were recovered from soil liniana Mill. (pop ash) and Baccharis ha- by Cobb's sieving and decanting method limifolia L. (saltbush) (10). Neither race or by incubating egg masses (4,8). Entire attacked citrus. In comparative morpho- bodies of T. graminis mature females were removed from broomsedge roots by high pressure water spray and collected on a 35- Received for publication 18 May 1987. ~tm-pore sieve. Long-necked mature fe- Nematologists, Bureau of Nematology, Division of Plant Industry, Florida Department of Agriculture, Gainesville, FL males of T. palustris were removed from 32602. pop ash roots with needles using a stereo- 2 Nematologist, Istituto Nematologia Agraria, CNR, 70126 Bari, Italy. microscope. The authors thank Lykes Pasco Packing Co., Dade City, Specimens for measuring, drawing, and Florida, for funding this project, and Dr. Esther Van Den Berg for providing paratypes of Tylenchulusfurcus. photographing were placed on the surface 266 Tylenchulus graminis and T. palustris n. spp.: Inserra et al. 267 of water agar and covered with a cover slip dian bulb to anterior body--MEBD; (6). Additional specimens were killed and postvulval section cavity--PVSC; postvul- fixed in lactophenol (5) or in hot aqueous val section length--PVSL; postvulval sec- 4% formaldehyde + 1% propionic acid, tion width--PVSW; vulva to excretory dehydrated in ethanol vapor, and mounted pore distance--VEPD; and standard de- in dehydrated glycerin (7). Eggs for mea- viation--SD. Morphological parameters surements were fixed and mounted in 2.5% are listed in the text in alphabetical order. formalin. Adult females with swollen posterior body Specimens for scanning electron mi- portion are indicated in the text with the croscopy (SEM) were killed and fixed in term mature adult females to distinguish formalin-propionic acid 4:1, transferred to them from young, nonswollen adult fe- 1% osmium tetroxide solution for 12 hours, males. All measurements are in microme- infiltrated with Spurr's resin, and mounted ters (#m) unless otherwise stated. on SEM specimen stubs (1). Specimens were coated with gold and observed at 5 kV ac- SYSTEMATICS celerating voltage (1). T. semipenetrans life stages were collected Tylenchulus semipenetrans Cobb, 1913 from a Florida citrus orchard, processed as (Figs. 1A; 6A, B; 7A, B, E; 8A, E; described here, mounted on water agar 10A; 13A; 14A, B) plates, and used for comparison because of Measurements of mature adult females, lack of paratypes. Morphological parame- males, and J2 females of a population from ters of T. semipenetrans, T. graminis, and T. Frostproof, Florida, are in Table 1. palustris were compared statistically by the Student's t-test. Additional comparisons Tylenchulus graminis n. sp. were made with specimens of mature adult (Figs. 3A-C; 4A-E; 5A-D; 6C, D; females, males, and J2 of the citrus and 7C, F; 8B, C, F; 9; 10B) Mediterranean races of T. semipenetrans Holotype (mature adult female in glycerine): collected in Italy and Greece and mounted Basal bulb length 20.0, width 14.2; body on permanent slides or SEM stubs. length 281.5, width 83.6; BWV 25.5; cu- Measurements of distance from genital ticle thickness 3.0; DEGO 3.9; esophagus primordium to the head end and between length 103.0; EPBD 212.0; isthmus length the excretory pore and genital primor- 19.0; MEBD 55.0; metacorpus length 18.3, dium of J2 were obtained only from spec- width 14.2; PVSC 7.1; PVSL 37.7; PVSW imens fixed in lactophenol (5) because of 12.2; stylet knob width 3.5; stylet length the difficulty of observing the genital pri- 12.2; tail length 12.2; VEPD 31.6. Excre- mordium in live specimens. In addition to tory pore from anterior body end as per- the standard morphological characters for centage of total body length 75.3; portion species of the genus Tylenchulus, we include of posterior body swollen as percentage of measurements of the postvulval section of total body length 74.6; vulva from anterior mature adult females as illustrated in Fig- body end as percentage of total body length ure 2. 86.5. Ratios: a = 3.3; b = 2.7; c = 23.4. Because of variation in body cuticle Mature adult female: Measurements of thickness, the cuticle was measured ven- specimens in water agar of two popula- trally and close to the excretory pore. The tions, one from the type locality, Crescent metacorpus position was measured from City, and the other from near Venice, Flor- the head end to the anterior end of the ida, are in Table 2. Body translucent white, metacorpus. about % of posterior portion variable, sac- The following abbreviations are used in cate. Body swollen 66.0-85.0% of total the text: body width at vulva--BWV; dor- body length, 59.0-73.0% in one popula- sal esophageal gland opening--DEGO; ex- tion from south Florida (Table 2). Entire cretory pore to anterior body--EPBD; me- body hook shaped, curved ventrally, widest QO TABLE 1. Measurements of Tylenchulus semipenetrans from Florida. Mature adult females'(N = 25) Males (N = 20) J2 females (N = 20) Standard Standard Standard Morphological characters Range Mean deviation Range Mean deviation Range Mean deviation Measurements in #m Basal bulb length 17.3-27.5 22.0 3.3 12.2-25.5 19.2 3.6 18.3-21.4 19.6 0.9 Basal bulb width 9.1-16.3 12.2 1.7 5.1-8.0 6.4"H: 0.8 7.6-9.1 8.3 0.5 Body length 312.1-465.1 389.2 43.0 346.8-380.4 362.1 9.5 333.5-384.5 363.0 14.8 Body width 61.2-I 14.2 85.7 12.5 10.2-12.2 11.2 0.4 12.7-13.7 13.1 0.2 BWV 21.4-30.6 25.7 2.6 Cuticle thickness 2.9-5.6 3.7~ 0.7 % DEGO 4.2-6.1 5.2t 0.3 4.5-6.1 5.1 0.3 3.0-4.0 3.7 0.3 t,a Esophagus length 104.0-159.1 119.8 11.9 96.9-130.5 104.6 7.7 102.0-115.2 108.3 3.2 EPBD 264.9-400.4 326.9 36.3 196.8-237.6 206.8 9.4 182.5-212.1 197.7 9.6 Genital primordium anterior body end distance 184.7-211.1 197.8 9.3 Genital primordium excretory pore distance 6.1-24.4 16.5"[" 5.0 Gubernaculum length 3.1-4.0 3.8 0.4 Isthmus length 11.2-30.6 19.4 4.0 22.4-40.8 30.6 4.9 24.4-31.6 27.3 1.8 MEBD 49.9-71.4 59.4 6.0 37.7-48.9 42.5 3.4 40.8-49.