Journal o f Chemical Ecology, Vol, 8, No. 6, 1982

P H E R O M O N E A T T R A C T I O N IN T H E SOYBEAN CYST N E M A T O D E Heterodera glycines R A C E 3

J O H N F. R E N D E , P A U L M. T E F F T and L E O N W. B O N E

Department of Physiology Southern Illinois University Carbondale. Illinois 62901 (Received September 29, 1981; revised November 17, 1981) Abstract--Male Heterodera glycines responded to female nematodes during in vitro bioassay. The male's response was dosage-dependent and significant with a pheromone source of more than five females. Male responsiveness was influenced by the pheromone diffusion and response times. Males were most responsive at three days after emergence from the host plant, while females were also most attractive at the same age. Light intensities that ranged from dark to bright had no effect on female location by the male, although bioassay in a nitrogen atmosphere eliminated sexual communication. Mate location was not significant below 25~C and declined slightly at 30 or 33~ Bioassay at pHs from 5 to 8.5 showed a bimodal effect, with maximal attraction around pH 6.

Key Words--Nematode pheromones, nematode, mate location, Heterodera glycines, soybean cyst nematode. INTRODUCTION N o c u r r e n t i n v e s t i g a t i o n s of Heterodera p h e r o m o n e s are e v i d e n t despite the b r o a d e c o n o m i c significance of these n e m a t o d e s an d the i n i t i a t i o n of study over a decade ago. Since insect m a n a g e m e n t t h r o u g h p h e r o m o n e - b a s e d c o n f u s i o n of the male a p p e a r s practical, a similar b i o r a t i o n a l a p p r o a c h to c o n t r o l of injurious n e m a t o d e s would seem feasible after sufficient study. Green (1966) initially r e p o r t e d the a t t r a c t i o n o f male Heterodera rostochiensis and H. schachtii to their females by in vitro bioassay. T h e p h e r o m o n e o f f e m a l e H. schachtii was less labile or m o r e c o n c e n t r a t e d t h a n t h a t o f H. rostochiensis. T h e p h e r o m o n e of H. sehachtii a p p e a r e d n o n v o l a t i l e in a second b i o a s s a y system and thus m o v e d t h r o u g h an a q u e o u s agar m e d i u m ( G r een , 1967). In contrast, Greet et al. (1968) r e p o r t e d that the a t t r a c t a n t s had 981 0098-0331 / 82/0600-0981 $03.00/0 9 1982 Plenum Publishing Corporation

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a volatile component in Heterodera. Regardless of this conflict, the authors knowledgeably standardized their procedures by dose-response analysis of female pheromone activity from H. rostochiensis and H. schachtii. H. rostochiensis males were also slightly attracted to each other in bioassay but were usually more attracted to one of a group of females. Grouped females exhibited increased attractiveness for responding males. However, multiple males that were responding to female pheromone obstructed each other during copulatory attempts (Green et al., 1970). Green and Greet (1972) reported that female 1-1. schachtii and H. rostochiensis produced pheromone throughout their body, and thus concluded the chemical component(s) originated in the hypodermis or pseudocoelom. However, the tail of H. schachtii was more attractive, which suggested that the egg sac may act as a pheromone carrier. Perhaps the most thorough study of nematode pheromone diversity to date was performed by Green and Plumb (1970). Cross-specific pairings often Heterodera species were conducted by in vitro bioassay. Based on the male responses, at least six attractants were postulated with the likelihood of one additional substance. The above investigations suggest that a well-defined system of pheromone communication is present and probably necessary in various species of Heterodera, as one might suspect from the sedentary behavior of the female helminths. Accordingly, we have recently initiated preliminary research on pheromone-mediated reproduction in the soybean cyst nematode Heterodera glycines Race 3. A pheromone-based scheme of biological control of this worm should prove environmentally appealing and would complement the current control practices. M E T H O D S AND M A T E R I A L S

Soybean plants (Union cultivar) were raised in 2 to 3-in. clay pots with a sterile sand-vermiculite mixture in growth chambers at 28~ and a 18:6 light-dark cycle. Hoagland's solution was given about every four days by foliage feeding, and plants were watered daily. When the first pair of trifolate leaves expanded, the soil around the host plant was inoculated with 50-100 manually crushed cysts of Heterodera glycines Race 3. After 13-15 days for nematode development, infected plants were removed and their roots were rinsed. Then the roots were suspended in 250-450 ml of aerated water. Other inoculated plants were left in the original container for maintenance of the life cycle. Initially, males were recovered from the aerated solutions at 1-3 days postemergence for usage as pheromone responders. Females were taken from the roots" at 1-5 days postemergence for bioassay as a pheromone source. Females were removed by clipping a 3-mm segment of root.

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ter

FIG. i. Bioassay chamber for pheromone attraction of H. glycines. C = control chamber for roots only, S = pheromone source chamber for females and roots, R = response chamber for males.

Bioassay devices were modified from Samoiloff et al. (1973) and were lab-fabricated from Ptexiglas. The chambers consisted of three 12-mm circles that were connected at a 45 ~ angle by 2-mm-wide and 5-mm-long channels (Figure 1). The chambers were filled with 1.2 ml of 0.2% sterile nutrient agar (Fisher). Determination of initial dose-response conditions were obtained by placing 1, 3, 5, 10, 12, t5, or 20 females in the left or right chamber and equivalent amounts of roots in the opposite side as a control for any host response. After 8 hr for pheromone production and diffusion, single male H. glycines were placed in the middle chamber. Their bioassay responses were determined after 14, 24, and 36 hr. All bioassays were conducted under uniform light at ambient temperatures (24-25 ~ C) unless otherwise stated. The responses for each dosage of females were obtained by subtracting the males that responded to the root controls from the males that responded to females and dividing by the total trials. Thus, percentages in this study represent net positive responses. Forty replicates were performed for each dosage. Based on initial results, subsequent investigations employed the 24-hr male response to an established 8-hr p h e r o m o n e gradient f r o m females. The influences of nematode age were determined by collecting males on the initial day of emergence with maintenance in tap water for 1,2, 3, 4, or 5 days prior to bioassay of their responses to a dosage of 10 females at one day of age. At least 20 replicates were done for each male age. Pheromone production by females was studied by the recovery of females at 1, 3, 6, 12, or 17 days postemergence. Males were removed daily to reduce mating. Dosages of 10 females were used to obtain the responses of 3-day-old males. Twenty replicates or more were performed for each female age. Other conditions were tested by a standard bioassay with 10 females at one day postemergence as a pheromone source for the response of 3-day-old males. Twenty replicates were done for each of the following treatments.

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Temperature effects on the pheromone system were investigated by bioassay at 16, 20, 25, 30, and 33~ Incandescent light intensities of 7.5, 4.7, 1.9, 0.5, and 0 J o u l e s / m 2 sec were used to examine any influence on pheromone communication. Illumination of the bioassay chambers was determined with a YSI-Kettering radiometer (model 65A) in an enclosed room. No temperature differences were noted.iBioassay of the males' response in a nitrogen atmosphere was conducted in a vacuum vessel after purging with nitrogen. The p H of the bioassay medium was altered to examine any changes in mate location. Agar at several pHs from 5 to 8.5, after adjustment with 0.1 N hydrochloric acid or 0. I N sodium hydroxide, was used to examine any effect o f p H on mate location. Some bioassays were conducted in media which were buffered at p H 6 and 6.5 with sodium phosphate (dibasic) and potassium hydrogen phosphate (monobasic) to prevent changes of the pH by the females. Data were evaluated by linear regression or analysis of variance. The 0.05 probability level was considered significant.

RESULTS

Initial response of male H. glycines after 24 hr to various dosages of female worms is given in Figure 2 for an 8-hr diffusion period. The male's response to increased number of females was linear (r = 0.94). One or three females of H. gtycines elicited only a slight response which was not different from zero (MSE = 10.5). However, the use of over five females caused a significant male response. Maximal male responsiveness (67%) was observed with a 12-female dosage. Bioassay of high number of females revealed reduced attractiveness to the male. Female dosages of 15 and 20 caused 15 and 33% male responses, respectively. This may represent a bioassay artifact that is caused by male habituation or sensory adaptation with extreme pheromone dosages. The influences of time on the male responses were determined after an 8-hr period for pheromone diffusion for selected female dosages (Figure 3). No significant male responses were found to three females until 36 hr of bioassay time (MSE = 3.5). Male responsiveness to a pheromone source of five or more females significantly increased at all examined times (MSE = 3.4). However, no significant difference was found between 24 and 36 hr of response when 12 females were used as a pheromone source. Thus, the male's response in bioassay is dependent on the female dosage and an adequate period for response. Subsequent experimentation was standardized at a 24-hr period for male responses. The bioassay responses to a dosage of 10 females differed with the postemergence age of the tested males (Figure 4). The responses of 1- or

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70-

60-

50Z O

40-

it3

o

30-

.-e 20-

10-

NUMBER OF FEMALES

FIG. 2. Bioassay responses of male H. glycines to various dosages of female nematodes (MSE = 10.53).

2-day-old males did not differ from each other, but were significantly less than older ages. Maximal responses were obtained from males at three days postemergence. Four- or 5-day-old males were identical in their responses, but less responsive than 3-day-old males. Males were most responsive to female H. glycines that were one or 3 days Of age (Figure 5). No difference was found between these ages. However, the male's attraction to 6-day-old females was reduced by 71% while that to older females was not significant (MSE = 8.76). Based on these results, pheromone production by females diminishes with age or as the cyst becomes tanned. Figure 6 shows the response, of males to females at various light intensities. No differences were observed, and all male responses are within the range that was expected from dosage-response analysis (MSE = 2.47). Although H. glycines occupies a subterranean habitat, the pheromone system is functional with bright to moderate illumination as well as dark. Thus, light has no effect on bioassay at the tested levels. However, environmental temperature has a pronounced influence on chemocommunication (Figure 7). Our results indicated that there was no significant male responsiveness to females or no female production of

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70-

=12g 60-

SOZ

0O.

40-

< 30-

20-

10-

14

24 RESPONSE TIME (hrs)

FIG. 3. Bioassay responses of male H. glycines to selected dosages of female nematodes after 14, 24, and 36 hr ( M S E = 2.01, 3.49, and 11.7, respectively). 50-

40-

z 30O gL. r~