Vertical Migration of the Rice White-Tip Nematode, Aphelenchoides besseyi ~

J. A. ADAMO, C. P. MADAMBA and T. A. CHEN 2

•4bstract: In the laboratory, the vertical migration of Aphelenchoides besseyi was favored by rough surfaces and an inverse water gradient. In relation to gravity, the nematode migrated down or tap equally, a circumstance suggesting that a geotaxis was not involved. Stems of rice seedlings were effective surfaces for vertical migration of nematodes only when the stems were continuously supplied with moisture. Key Words: behavior, geolaxis.

The literature dealing with the rice it was an ectoparasite on rice. It has been white-tip disease and its causal organism, suggested that tile nematode does not over- Aphelenchoides besseyi Christie, was re- winter in soil (5) and is disseminated pri- cently reviewed by Ou (11) and Ichinohe marily through infested seed (7). Fukano (10). Although no work on vertical migra- (7) indicated that irrigation water contain- tion has been reported, a number of obser- ing infested seeds was a secondary means of vations indicate the importance of the nem- spread. A chemical attraction for A. besseyi atode's migratory behavior to survival, dis- to certain cultivars of young growing rice semination, and its general life pattern. plants or aqueous extracts of germinating Several factors which affect orientation seed has been demonstrated (9). The nem- and vertical migration of above-ground, atode migrates upward in moisture that plant-parasitic nematodes have been in- forms on the walls of flasks when it is reared vestigated (1, 2, 3, 9, 18). Some parasitolo- on fungi growing on steamed rice (16). We gists have studied the nature of plant sur- have also observed that worms aggregate in faces as related to the movement of animal- droplets on both covers of Petri plates and parasitic nematodes and the abiotic ecolog- on walls of culture tubes. ical factors involved in vertical migration Since A. besseyi must migrate vertically (4, 6, 12, 13). Studies on the vertical migra- to reach developing seed, we initiated in- tion of plant-parasitic forms have concen- vestigations to determine the nature of trated primarily on Aphelenchoides ritz- vertical migration and the factors that in- emabosi (Schwartz) Steiner and Buhrer and fluence this behavior. Experiments were un- Ditylenchus dipsaci (Kiihn) Filipjev. dertaken to determine the effects of mois- In the case of A. besseyi, the literature ture, texture, surface area, and gravity on clearly suggests a relationship between mi- migration of A. besseyi. gratory behavior and the overall biology of the animal. Apparently, infection of rice MATERIALS AND METHODS plants by the nematode from soil is min- Nematodes used were from cultures imal, whereas the organism was found to maintained in the laboratory on Alternaria spread via irrigation water (19). Tamara brassicae (Berk.) Sacc. grown in potato- and Kegasawa (14) reported that A. besseyi dextrose-agar (PDA). The nematodes were did not attack the young roots of rice seed- originally isolated from rice grown in lings. However, once on the rice plant, the animal was found to move from one part to Potatan, Iloilo, the Philippines. Studies another (8). Todd (15) could not find the were conducted during December, January, nematode in any tissues and concluded that and February when the normal laboratory telnperatures were 25-30 C. All experiments were conducted in the dark. Tile test struc- Received for publication 25 October 1974. 'Conducted at the Nematology Research Laboratory, Uni- tures were removed after 18-24 11 of incuba- versity of the Philippines, Los Bafios, while the senior tion, soaked in water, and the water ex- author was on sabbatical leave and on a Fulbright-Hays grant. amined for numbers of nematodes present. -~Associate Professor. Department of S~ience. Ocean County Upward migration in a moist environ- College, Toms River, New Jersey 08753; Associate Profes- sor and Chairman, Department of Zoology, University of ment: Upward migration was determined the Philippines. Los Bahos; and Professor, Department of by using capillary tubing (0.8 mm diam), Plant Biology, Rutgers, the State University of New Jersey, New Brunswick 08903. glass microscope slides (13 x 38 mm),

146 Vertical Migration/Aphelenchoides besseyi: Adamo et al. 147 wooden match sticks (2 x 2 mm), and rice the same length were used (Fig. I-F). Two seedling (Oryza sativa Linn., 'Taichung were of the same width (14-15 mm), but Native I') stems about 4 mm wide. All the one had a rough surface and the other was materials were 38 mm long. Each structure smooth. The third glass slide had a rough was placed individually and separately in surface and a narrowed width (7-8 ram). A an upright position in a Syracuse dish previ- film of moisture was provided for each ously filled with melted paraffin wax (Fig. slide by a string drawing water from a ! A-C). One ml of sterile distilled water was well. The slides were removed from the placed around each structure, and 10 hand- culture dishes, and the worms were counted picked, freshly isolated, active worms were 18 h after initiation. The experiment was added. Each treatment was replicated 10 replicated 5 times. times. The Syracuse dishes were placed in Downward migration in a film of mois- enamel pans which in turn were kept in ture: The descending behavior of the nem- large moistened plastic bags. After 20 h, the atode was determined by using a set-up structures were removed and soaked in similar to that previously described. Two water. Rice stems and match sticks were cotton strings were connected to each of teased into small pieces and soaked over- five dishes of minced culture and extended night. Nematodes isolated from each struc- downward into small wells below (Fig. 2-A). ture were counted. One well of each pair of strings was pro- Upward migration in a firm of moisture: vided with water. Each string was removed In addition to the vertical structures uti- after 24 h and soaked before the nematodes lized in the first experiment, three-ply cot- were counted. ton string (0.7 mm diam) was also included Vertical migration in relation to grav- in the next series of tests. A film of moisture i~: A pair of wells with accompanying was provided to each surface by a string cotton strings was attached directly to each serving as a wick drawing water from a culture dish containing the nematodes. One small well. A pair of capillary tubes, match well of each pair was placed above the cul- sticks, rice stems, or strings was placed in ture dish, and the other was placed below an upright position in a dish containing (Fig. 2-C). In the first treatment, only the minced nematode-fungus culture. Treat- upper wells were filled with water, whereas ments were replicated l0 times. Each struc- in the second treatment, the lower wells ture was individually provided with a small were filled. In the third treatment, both the well (Fig. l-E, 2-B). One of the wells of upper and lower wells were supplied with each pair contained 3 ml of water, and the water. No water was supplied in any well in other was empty. the last treatment. Instead, water was ap- Two other structures, glass slides and plied on the culture dishes. All treatments capillary tubes without wells, were also in- were replicated 5 times. The wells and cluded in the second experiment. Three strings were removed after 20 h and the 13 x 38 mm glass slides, provided with wells, numbers of nematodes present were de- were placed in a dish of minced culture. termined. Two of these slides had smooth surfaces, and the other was roughened with a dia- RESULTS mond pencil (Fig. l-D). One of the smooth slides was allowed to remain dry. A pair of Upward migration in a moist environ- capillary tubes, without wells, was placed in ment: Tile upward migration of A. besseyi a dish of culture. One of the tubes was half- on vertical structures from a "pool" of filled with water, and the other was dry. water and in a moist environment was Each structure was removed after 24 h, found to vary with surface conditions. The soaked, and the number of nematodes on ascent on nontextured, nonabsorbent, sur- each was determined as previously de- faces of capillary tubes or glass slides was scribed. nil. The rate of climb on rough, absorbent Surface area: A third experiment was match sticks was high although it was not conducted to determine the relationship of so on rice seedling stems exhibiting tex- surface area to numbers of nematodes tured surfaces. climbing glass slides. Three glass slides of Upward migration in a film of moisture: 148 Journal of Nematology, Volume 8, No. 2, April 1976

i Vertical Migration/Aphelenchoides besseyi: Adamo et al. 149

FIG. 2 (A-C). Arrangement of strings, wells, and cultures for vertical migratory studies on the nem- alode, Aphelenchoides besseyi. A) Set-up for descending studies. B) Position of material for ascending studies, t:) Arrangement of materials for tests involving vertical migration in relation to gravity.

The vertical ascent of nematodes in a film Surface area: The number of nematodes of moisture was found to be texture de- ascending glass slides was found to vary pendent (Table 1). The upward climb of with the area of the structure (Table 2). A. besseyi on smooth surfaces, regardless of The results show that nematodes climbed moisture conditions, was almost nil. The rough-surfaced glass slides o£ wider width number of worms climbing wet and dry more effectively than smooth surfaces. More capillary tubes did not differ. Likewise, no worms climbed roughened 14.5-mm wide differences were found in the numbers of slides than those with a 7.5 mm width. nematodes moving up wet and dry smooth- Downward migration: The descending surfaced glass slides. All textured surfaces movement of nematodes on string was ob- supplied with a continuous film of mois- served when moisture was initiated from a ture (from a source located above the dish) source located below the culture dish. As in encouraged an upward movement of nem- the earlier experiment, strings connected to atodes. A few worms were observed to move dry wells became wet as a result of capillary upward on strings connected to dry wells. action. The string became wet when moisture was Vertical migration in relation to grav- imbibed from the culture dish by capillary ity: The vertical migration of A. besseyi action. was not directly affected by gravity (Table

///// \\\\\ FIG. 1 (A-F). Structures and arrangements of materials used in ascending migratory studies on the nematode, Aphelenchoides besseyi. A-C) Capillary tube, match stick, and "cut" glass slide set-up in wax- lilled dishes with "pools" of water. D) Rough and smooth slides. E) Match sticks--showing the arrangement of vertical structures, strings and wells for migration studies in a film of moisture. F~ Modified glass slides-- showing the position of three different slides (rough thin, smooth wide, and rough wide), strings, and wells for upward migratory studies. 150 Journal o[ Nematology, Volume 8, No. 2, April 1976

TABLE l. Upward migration of Aphelenchoides TABLE 2. Upward migration of Aphelenchoides besseyi in a film of moisture on various structures. besseyi as affected by the surface area of moistened glass slides.

Structure No. and Surface nematodes No. nematodes treatment texture x ascending y Treatment ascending*

Capillary tube without well Smooth slide with ca. A dry surface (control) NT 0 a area of 145 mm-"/cm (control) 2 a B wet surface NT 0a Rough slide with ca. Capillary tube with well area of 75 mmZ/cm 75 b A dry well (control) NT 0 a B wet well NT 0 a Rough slide with ca. area of 145 mm2/cm 165 c (;lass slide with well A dry well (control) NT 0 a 'Mean of five replications each. Treatment means B wet well NT 6 a followed by the same letter do not differ from one C wet well T 111 b another according to Duncan's multiple range test Match stick with well (P = .05). A dry well (control) T 1 a B wet well T 86 b parently came from tile fungi in the culture Rice seedling with well dish. A dry well (control) T 6 a B wet well T 90 b DISCUSSION String with well A dry well (control)' T 4 a The results of the present work showed B wet well T 113 b that A. besseyi climbed any roughened sur- face continuously supplied with moisture xT = Textured; NT = uontextured. or which had the ability to hold moisture. rMean of 10 replications each. Treatment means Effectiveness of surfaces for vertical migra- followed by the same letter do not differ from one tion was positively related to structure another according to Duncan's muhiple range test (P = .05). width. Aphelenchoides besseyi descended 'Strings attached to dry wells became wet due to under conditions of a water gradient initi- capillary action. ated from below, and in most cases the animal migrated against an opposite gentle S). There was significant upward movement TABLE 3. Vertical migration of Aphelenchoides of the nematode when only the wells above besseyi in a fihn of moisture on string. the culture initially contained water. On the other hand, the direction of movement shifted downward markedly when only the No. nematodes Treatment (position migrating wells below had water. In cases where both of moisture supply) (ascending/descending)" upper and lower wells were provided with water, migration in both directions was ob- Above culture dish b 212/3"* served. However, the mean number of nem- atodes moving upward was greater than Below culture dish 3/210"* that migrating downward. The flow of Both above and water from the wells in this treatment was below culture dish 400/99* appreciably influenced by gravity. After Neither above nor incubation, the upper wells were nearly below culture dish empty, but the lower wells were almost full. (in culture dish only) 20/60* When all wells were initially dry, migration was comparatively less, but there was signif- "Mean of five replications each. Mean numbers of icant downward movement of the nema- nematodes ascending and descending were analyzed for each treatment by the paired t-test, (*, ** = tode. Like those in all previous tests, strings significant at 5 and 1% levels, respectively). attached to dry wells were wet upon re- bln all cases strings attached to dry wells became moval and tinted with pigment which ap- wet due to capillary action. Vertical Migration/.4phelenchoides besseyi: .4damo et al. 151

flow of water. The data also indicated that LITERATURE CITED the movement was not directional with re- 1. BARRACLOUGH, R. M., and N. FRENCH. spect to gravity; ,4. besseyi migrated up or 1965. Observations on the orientation of down equally well, a circumstance suggest- Aphelenchoides ritzemabosi (Schwartz). Nem- ing that a geotaxis was not involved. atologica 11:199-206. 2. BLAKE, C. D. 1962. Some observations on the Stems of rice seedlings were found to be nrientatiou of Ditylenchus dipsaci and inva- relatively ineffective structures for nema- sion of oat seedlings. Nematologica 8:177-192. tode migration when they were wetted from ,'4. BLOOM, J. R. 1964. Photonegative reaction of below. However, when moisture was con- the chrysanthemum foliar nematode (Aphe- tinuously supplied, A. besseyi could readily lenchoides ritzemabosi). Phytopathology 54: 118-119. migrate on rice stems. 4. BUCKLEY, J. J. c. 1940. Observations on the Most of the data obtained were in gen- vertical migration of infective larvae of cer- eral agreement with the concept of greater tain bursate nematodes. J. Helminthol. 18: ntigration toward the wetter end of a water 173-182. 5. CRALLEY, E. M. 1952. Control of white tip of gradient. In two experiments conducted, rice. Arkansas Farm Res. 1 (I):6. the effect of gravity on the flow of water in- 6. CROFTEN, H. D. 1954. The vertical migration fluenced nematode migration. When mois- of infective larvae of strongyloid nematodes. ture was applied to the surface of the cul- J. Hehninthol. 28:35-52. tures but not supplied in the wells, it was 7. FUKANO, H. 1962. Ecological studies on white- tip disease of rice plant caused by Aphelen- found that water migrated up and down choides besseyi Christie and its control. strings as indicated by its physical presence. B. Fnkuoka Agric. Stn. 18:108. In this case, more water and more worms 8. GOTO, K., and R. FUKATSU. 1952. Studies on were found below, although generally low white tip of rice plant caused by Aphelen- choides oryzae Yokoo. II. Number and distri- numbers of nematodes were encountered. bution of the nematode on the affected These events can be explained by way of a plants. Annu. Phytopathol. Soc. Japan 16: water gradient hypothesis and gravity. How- 57-60. ever, when upper and lower wells were ini- 9. GOTO, K., and R. FUKATSU. 1956. Studies on tially wet and the cultures relatively dry, it the white tip of rice plant. Ill. Analysis of varietal resistance and its nature. B. Natl. was found that the upper wells were nearly Inst. Agric. Sci. 6:123-149. dry while the lower wells were full and 10. ICHINOHE, M. 1972. Nematode diseases of pigmented at the end of the experiment. rice. Pages 127-143 in J. M. Webster, ed. These results suggested that water initially Economic nematology. Academic Press, Lon- don and New York. 563 p. and continuously migrated down from up- 11. OU, S. H. 1972. Rice diseases. Commonwealth per wells, while at some point the upward Mycological Institute Kew, Surrey, England. migration of water from the lower wells 368 p. stopped and reversed its direction or trend 12. REES, G. 1950. Observations on the vertical of movement. If a gradient was the operat- migration of the third-stage larvae of Haemonchus contortus (Rud.) on experi- ing factor, equal or higher numbers of nem- mental plots of Lolium pereune S 24, in rela- atodes should have been found descending. tion to meteorological and micro-meteoro- In this case, more worms migrated upward. logical factors. Parasitology 40:127-143. The data suggested that the worms were af- 13. ROGERS, W. P. 1940. The effects of environ- fected by another factor. mental conditions on the accessibility of third stage trichostrongyle larvae to grazing ani- There is little evidence for rheotactic mals. Parasitology 32:208-225. responses of plant-parasitic nematodes. The 14. TAMURA, I., and K. KEGASAWA. 1958. Studies findings of Wallace (17), and of Barraclough on the ecology of the rice nematode Aphelen- and French (1) showed this was not the case choides besseyi Christie. lI. On the parasitic with .4. ritzemabosi. A form of flow orienta- ability of the rice nematode and their move- ment into hills. Jpn. J. Ecol. 8(1):37-42. tion, perhaps as a tactile stimulation, would 15. TODD, E, H. 1952. Further studies on the help to explain the present observation and white tip diseases of rice. Proc. Assoc. 5th would be in agreement with the data. Agric. Workers 49:141 (Abstr.). Whether this observation was a result of 16. TODD, E. H., and J. G. ATKINS, JR, 1952. the nature of the experiment or a form of Laboratory culture of the rice white tip nem- atode, and inoculation studies. Phytopatb- flow orientation remains to be shown. ology 42:21 (Abstr.). 17. WALLACE, H. R. 1959. Movement of eel- worms. V. Observations on Aphelenchoides 152 Journal of Nematology, Volume 8, No. 2, April 1976

rilzemabosi (Scltwarlz, 1912) Steiner, 1932 lolngica 6:222-236. on florists' chrvsauthenttnns. Annu. Appl. 19. YOSHll, H., atttl S. YAMAMOTO. 1950. A rice Biol. 47(2):350-360. nematode disease IIl. Infection course of the 18. WALLACE, H. R. 1961. The orientation of present disease. J. Fac. Agriculture, Kyushu Ditylenchus dipsaci to physical stimuli. Nema- Univ. 9:287-292.

Adenosine Triphosphate Quantification as Related to Cryptobiosis, Nematode Eggs, and Larvae~ HARVEY W. SPURR, JR. 2

Abstract: Sonification was the most effective method used for disintegrating nematode eggs and larvae for adenosine triphosphate (ATP) determinations. Sensitivity of the assay was sufficient to measure ATP in one larva. Second-stage larvae of Anguina tritici averaged 1 x 10s femto- grams (fg) ATP and Meloidogyne incognita eggs, 0.8 x 105 fg ATP. Larvae of Panagrellus redivivus, a saprobe, averaged 12.2 x 105 fg ATP, a measurement which was considerably higher than the ATP levels in plant parasites. Endophytic bacteria and fungi from wheat galls were detected as background organisms associated with A. tritiei activated by hydration. Also, bacteria in suspensitms of eggs from M. incognita prepared with NaCIO were measured by the use of butanol extraction and ATP determination. Second-stage A. tritici larvae increased in ATP content within 40 mln after being activated from cryptobiosis by hydration. In the cryptobiotic state, larvae had 50% less ATI' than when active. ATP concentrations were similar in galls of different ages. Apparently, ATP concentrations do not change during cryptobiosis. Starvation results in a decline in ATP concentration/larva. Subjecting A. tritici larvae to the lethal tem- perature of 60 C resulted in a three-fold increase in the decay rate o[ ATP over that of larvae snnified, then heated at 60 C. These results suggest an association between ATP decay and the mechanism that causes death of larvae at elevated temperatures. Key Words: Anguina tritici, Meloidogyne incognita, Panagrellus redivivus, starvation, thermal inactivation.

Living organisms depend on adenosine bacterial ATP and populations (1, 2), I de- triphosphate (ATP) and similar com- signed these experiments to: (i) extract and pounds to energize vital metabolic activ- quantify nematode ATP, (ii) compare ATP ities, and nematodes in various states of in different larvae and eggs, and (iii) de- hypobiosis must preserve adequate ATP, or termine ATP in cryptobiotic larvae before a mechanism for synthesizing it, to survive. and during hydration and activation. The measurement of ATP in nematodes has been hampered by the lack of suitable MATERIALS AND METHODS techniques. When a sensitive instrument was developed for the quantification of Sources and species of nematodes: To obtain active larvae of Anguina tritici Received for publication 26 August 1975. (Steinbuch) Filip., wheat galls ("cockles") l Cooperative investigations of the Southern Region, Agri- were soaked in water, opened with a teas- cultural Research Service, United States Department of Agriculture and the North Carolina State University Agri- ing needle, and the second-stage larvae cultural Experiment Station, Raleigh. Paper No. 4830 of squeezed out with forceps into water in a the North Carolina Agricultural Experiment Station, Ra- leigh. Mention of a trademark or proprietary product watch glass. Hydration caused the larvae to does not constitute a guarantee or warranty of the prod- take shape and become active. When galls uct by the U.S. Department of Agriculture or North Caro- lina State University, and does not imply its approval to were soaked 1 h or less before excision, the exclusion of other products that may be suitable. larval movement began 8-24 h after excision 2Research Plant Pathologist, Southern Region, Agricul- tural Research Service, U. S. Department of Agriculture, and fewer viable larvae were obtained than Oxford Tobacco Research Laboratory, Oxford, North Caro- when galls were soaked for longer periods lina 27565 and Professor of Plant Pathology, North Caro- lina State University, Raleigh. The author appreciates the before excision. When galls were soaked for technical assistance of Eleanor T. Howard and thanks Dr. 1-3 days prior to excision, high numbers of T. T. Hebert for providing wheat galls and W. M. Wil- liams for the photographs. active larvae were observed after 45-60 rain.