Host Range, Biology, and Pathology of P. punctata: Radice et al. 165 rence of cyst nematodes (Heterodera spp.) in Michigan. 7. Radice, A. D., and P. M. Halisky. 1983. Nema- Plant Disease Reporter 55:399. todes parasitic on turfgrasses in New Jersey. New Jer- 3. Chitwood, B. G. 1949. Cyst formingHeterodera sey Academy Science 28:25 (Abstr.). encountered in soil sampling. Plant Disease Reporter 8. Radice, A. D., R. F. Myers, and P. M. Halisky. 33:130-131. 1983. The grass cyst nematode in New Jersey. Jour- 4. Dunn, R. A. 1969. Extraction of cysts of Het- nal of Nematology 15:4-8 (Abstr.). erodera species from soil by centrifugation in high 9. Spears, J. F. 1956. Occurrence of the grass cyst density solutions. Journal of Nematology 1:7 (Abstr.). nematode, Heterodera punctata, and Heterodera cacti 5. Horne, C. W. 1965. The taxonomic status, group cysts in North Dakota and Minnesota. Plant morphology and biology of a cyst nematode (Nema- Disease Reporter 40:583-584. toda:Heteroderidae) found attacking Poa annua L. 10. Thorne, G. 1928. Heteroder a punctata n. sp. a Ph.D. dissertation, Texas A&M University, College nematode parasite on wheat roots from Saskatche- Station, Texas (Diss. Abstr. 65-12, 287). wan. Scientific Agriculture 8:707-710. 6. Horne, C. W., and W. H. Thames, Jr. 1966. 11. Wheeler, W. H. 1949. Interceptions of the Notes on occurrence and distribution of Heterodera genus Heterodera in foreign soil. Plant Disease Re- punctata. Plant Disease Reporter 50:869-871. porter 33:446. Journal of Nematology 17(2): 165-168. 1985. © The Society of Nematologists 1985. Responses of Anguina agrostis to Detergent and Anesthetic Treatment 1 DONALD L. RIDDLE 2 AND ALAN F. BIRD s Abstract: The infective dauer juvenile (DJ2) of Anguina ag~vstis, a stage capable of surviving desiccation, is up to sixfold more resistant to the detergent sodium dodecyl sulfate than are freshly hatched juveniles or adult males, and twofold more resistant to the anesthetic phenoxypropanol. Thus, the DJ2, like dauer stages of other species, may also be more resistant to various types of environmental stress in its natural habitat. In A. agrostis, however, resistance appears to be acquired gradually during development of the second juvenile stage, rather than during a molt. Key words: Lolium rigidum, physiology, dauer juvenile, rye grass, seed galls, toxicity. Growth and reproduction of Anguina drated, emerge from the seed gall, and agrostis in Australia occur during the spring move through the soil. They are not in the developing inflorescence of its rye thought to undergo further development grass host, Lolium. The nematodes molt until they reach rye grass inflorescences once in the egg, hatch, and grow into the the following spring. Several males and fe- infective dauer juvenile (DJ2) stage, feed- males develop within a single seed, molting ing from cells that line the cavity of a gall three times to the adult stage (8). Associ- developed in place of an ovule (10). De- ation of Corynebacterium rathayi with A. velopment of the DJ2 marks the transition agrostis in the galls kills the nematodes and from a feeding parasitic stage to a dispersal leads to a condition commonly referred to form. The DJ2s are able to enter an an- as annual rye grass toxicity, which can be hydrobiotic state in which they can survive lethal to grazing animals (3,9). the hot, dry summer season. With the ad- The freshly hatched juvenile (FHJ2), vent of autumn rains the DJ2s become hy- 500-600 ~m long, develops into the DJ2, over 800 ~m long, within the gall without Received for publication 16 August 1984. molting (5). Morphological changes asso- Experiments done at the CSIRO Institute of Biological ciated with the transition from FHJ2 to Resources, Division of Horticultural Research in September 1983. Supported by grant HD00367 from the National In- DJ2 include thickening of the cuticle, stitutes of Health, the University of Missouri Alumni Devel- changes in the shape of the lateral alae, opment Fund, and CSIRO. 2 To whom correspondence should be addressed: Division and the accumulation of lipid storage gran- of Biological Sciences, Tucker Hall, University of Missouri, ules. The DJ2 can survive for years in the Columbia, MO 65211. anhydrobiotic state, and hydration of dried CSIRO Division of Horticultural Research, GPO Box 350, Adelaide, South Australia 5001. galls provides a convenient source of active We thank Ms. S. D. Harris for technical assistance. DJ2s for laboratory experiments. In con- 166 Journal of Nematology, Volume 17, No. 2, April 1985 90 90 70 70 50 50' ~ so .50- O9 ~ 2O o~ 20- Cr 0 I0 > >W 5 or" 5- u~ 2- ' I ~ I ' I ' I ' I ' I I I ' I ~ I 0 I 2 3 4 5 6 0 I 2 3 4 5 6 TIME (HRS) TIME (HRS) FIG. 1. Semi-logarithmic plot showing inactiva- FIG. 3. Survival of juvenile and adult Anguina tion of Anguina agrostis in solutions of 1% phenoxy- agrostis in 2% sodium dodecyl sulfate. Adult males (D), propanol. Adult males (vI), freshly hatched juveniles freshly hatched juveniles (FHJ2) from first (less de- (©), and dauer juveniles (Q). veloped) gall (x7), FHJ2 from second gall (O), dauer juveniles (DJ2) from first gall (v), DJ2 from second gall (O), hydrated DJ2 from five galls collected in 19 81 (/x). Bars represent standard error of the mean. trast, adults and FHJ2s are available only in the spring when they can be dissected from seed galls (4). Development on Lolium whether such resistance is acquired grad- multiflorum callus tissue does not proceed ually or abruptly during the transition from beyond the DJ2 stage (5). FHJ2 to DJ2. Previous work has shown that the An- guina tritici DJ2 can survive temperature MATERIALS AND METHODS extremes and dryness (2). Our 0bjectives Anesthesia ofaduhs, FHJ2, and DJ2 sus- were to determine if the A. agrostis DJ2 has pended in 1% aqueous 1-phenoxy-2-pro- resistance to chemical agents such as de- panol (Nipa Laboratories Ltd., N. Pontyp- tergents and anesthetics, and to determine ridd, Glamorgan, Great Britain CF38 2SN) was tested in glass depression plates so that movement could be observed through a dissecting microscope. A nematode was 90 70 judged to be anesthetized when it ceased 50 movement and failed to respond to me- 30 chanical stimulation with an eyelash mounted on a stick. 20 v Survival of FHJ2 and DJ2 stages sus- I0- pended in 2% or 5% (w/w) aqueous solu- tions of SDS (sodium dodecyl sulfate, BDH > cr 5- Chemicals, Port Fairy, Victoria, Australia 3284) was also tested in glass depression plates so that viability could be assessed by 2- direct observation through a dissecting mi- croscope. Death was scored when the ' I--' I ' I ' I ; I ' 1 nematodes ceased movement and failed to 0 I 2 3 4 5 6 respond to mechanical stimulation with a TIME (HRS) platinum wire. When unresponsive ani- mals were removed from the SDS and FIo. 2. Survival ofAng~uina agrostisin two different placed in water, they invariably failed to concentrations of sodium dodecyl sulfate (SDS). Freshly hatched juveniles (FHJ2) in 2% SDS (O), FHJ2 recover. Killed nematodes became straight in 5% SDS (O), dauer juveniles (DJ2) in 2% SDS (/x), and transparent as internal tissues were DJ2 in 5% SDS (A). progressively dissolved by the detergent. A. agrostis Resistance to Chemicals: Riddle, Bird 167 In each experiment, 20 or 50 nematodes to detergent by the DJ2 was studied fur- were used per test population. ther because the response to SDS revealed the greatest differences between FHJ2 and RESULTS DJ2. Solutions of 2% SDS were used in tests Comparative tests with phenoxypropa- of adult males, FHJ2, newly developed (pre- nol were performed on DJ2s hydrated from anhydrobiotic) DJ2 from freshly collected dried galls collected in 1981 at Murray 1983 galls, and additional hydrated DJ2 Bridge, South Australia, and on adults and from galls collected in 1981 (Fig. 3). Hy- their FHJ2 progeny dissected from galls drated DJ2 from five different 1981 galls collected in the same paddock in spring were tested separately and the data aver- 1983. Adult females were not used in these aged. Fifty percent of these DJ2 were killed tests because of their tendency to burst in 220 minutes, in agreement with the pre- when handled. The anesthetic had differ- vious results obtained with a population ent effects on FHJ2, DJ2, and adult males from a single gall (Fig. 2). The similar re- (Fig. 1). In test populations of 20-25 nema- sponse of DJ2 populations from different todes freshly dissected from galls, adult hydrated galls suggests that the degree of males and FHJ2 responded similarly. Fifty resistance exhibited by these populations percent of the males were anesthetized af- of fully developed DJ2 is quite reproduc- ter 50 minutes of exposure and all were ible. However, the slope of the survival inactivated within 90 minutes. The FHJ2 curve is not as steep as that obtained from were perhaps marginally more resistant; other samples. This indicates that although 50% were anesthetized in 70 minutes, and populations from different galls are similar all were anesthetized after 105 minutes. In to each other overall, there is a greater contrast, none of the DJ2 hydrated from variation in the degree of resistance among 1981 galls were anesthetized before 105 individual hydrated DJ2 within each gall minutes, and it took 150 minutes to reach than occurred in. the other samples. 50% inactivation. All the DJ2 were inactive Newly developed DJ2 and FHJ2 were after 210 minutes. Thus, the DJ2 in the taken from two different 1983 galls, each test sample were about twofold more re- containing mixed populations of juveniles.