A method for obtaining abundant virgin females of Bursaphelenchus xylophilus and B. mucronatus (Nematoda: Aphelenchoididae) by prolonged chilling

Abstract

The pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, is the causative agent of pine wilt, which has been causing devastating damage to pine forests in East Asia (Mamiya and Kiyohara, 1972; Evans et al., 1996). The nematode is believed to have been introduced into Japan from North America (Tares et al., 1992; Beckenbach et al., 1992) and then spread to East Asia and Portugal (Mota et al., 1999). Bursaphelenchus mucronatus Mamiya et Enda, which is widely distributed in Eurasia, has a morphology and life history similar to that of B. xylophilus; both species reproduce in dying or newly dead trees of the family Pinacea and are transmitted by cerambycid beetles in the genus Monochamus, although B. mucronatus has little or no virulence to susceptible pine species (Mamiya and Enda, 1979; Burgermeister et al., 2005). Consequently, inter-specific and inter-isolate hybridization between B. xylophilus and B. mucronatus have been performed to determine their taxonomic status (Mamiya, 1986; de Guiran and Bruguier, 1989; Riga et al., 1992; Bolla and Boschert, 1993) and to clarify the effect of inter-specific hybridization between the two Bursaphelenchus species on the epidemic of pine wilt disease in Japan (Terashita, 1981). Furthermore, to determine if the disease development of pine wilt requires the reproduction of B. xylophilus, Kiyohara and Yamada (1995) inoculated Pinus thunbergii seedlings with 2,000 or 5,000 virgin females.Experiments for cross hybridization and analysis of virulence require virgin females. Previous studies have reported some methods for obtaining virgin females. Terashita (1981) and Mamiya (1986) transferred juveniles individually to Botrytis cinerea Persoon mycelial mats on small PDA medium blocks and picked up virgin adult females after 14 days of culture. Bolla and Boschert (1993) used third-stage female juveniles identified by the development of the genital anlagen. De Guiran and Bruguier (1989) obtained female juveniles immediately before molting to adults by rearing the fourth-stage juveniles, whose development had been arrested by the lack of food, on a B. cinerea mycelial mat,within 24 hr. The female juveniles were distinguished by a hyaline spot at the location of the future vagina. Riga et al. (1992) used fourth-stage female juveniles obtained from the lids of Petri dishes where the nematode populations were reared on B. cinerea on 1 % PDA. All virgin females so far used were obtained from populations cultured at 25 or 29 degrees C. Tomminen et al. (1991) showed that a long duration of chilling at 3 degrees C caused the percentage of third-stage dispersal juveniles in B. xylophilus populations to increase from 50 to 80 %. As the juveniles resume their development when returned to a favorable temperature condition, it may be possible to use chilling to obtain abundant virgin females simultaneously. If the proportion of virgin females that can be distinguished easily from others increases with increasing the duration of chilling, chilled populations will become an important source of virgin females. Therefore, we examined the effect of chilling duration on the age distribution, including the proportion of final-stage female juveniles immediately before molting to adults, by using laboratory populations of B. xylophilus and B. mucronatus.