Abstract:

【Objective】Explore the reproductive capacity, key stages and morphological changes of eggs in the embryonic development of Bursaphelenchus mucronatus. In addition, time required for the embryonic development and completion of the entire lifecycle were studied to provide a scientific basis for further study of its growth and development.【Method】A total of 180 female adult nematodes in three groups were selected to observe their behavior of egg-laying process at 25 ℃. The cumulative production of the eggs of each group of nematodes was counted every 2 hours until the number of eggs became constant. To observe the entire embryonic development process, the pregnant female nematodes in petri dishes were placed under a Zeiss stereo microscope, and the process of embryonic development was photographed. The time required from egg laying to each key stages of theB. mucronatus embryo was recorded at 25 ℃. Approximately 200 eggs of B. mucronatus were selected and their total hatching rates were calculated every 4 h after 24 hours of development at 25 ℃ until the number of hatchings no longer increased, in 3 sets of replicates. The newly hatched J2 were inoculated onto PDA plates containing the well-grown Botrytis cinerea. Then, on the day 1, 2 and 3 after inoculation of the J2, the nematodes were collected using the Berman funnel method, and the proportions of each age line were calculated to learn the time for nematodes to complete their entire lifecycle.【Result】① B. mucronatus has a good egg-laying ability. The total number of eggs increases rapidly within the first 10 hours. After 16 hours, the numbers of eggs laid gradually reached constant, with an average of 12 eggs/nematode in 28 hours. ② The development process ofB. mucronatus embryos mainly comprised of the following key stages: single-cell stage, 2-cell stage, 3-cell stage, 4-cell stage, 5-cell stage, 8-cell stage, 16-cell stage, blastocyst stage, Lima bean stage, tadpole stage, worm stage, first-stage juveniles (J1) and the stage of second-generation juveniles (J2). ③ In the early stages of embryonic development, two types of depressions, i.e., one-half and one-third, occurred. In addition, two different developmental patterns were observed during the 2-cell stage to 5-cell stage. The first one is that two cells do not move but split directly into three cells; the other one is that the cells move to a form of triangle. It was revealed by observing the development of the first cleavage of 30 eggs and the development of the 2-cell stage of 100 eggs that these development patterns are common. ④ At 25 ℃, the cumulative hatching rate ofB. mucronatus eggs increased with time, reaching 93.31% after 32 h, and then gradually leveled off. ⑤ The time required for the entire embryogenesis of B. mucronatus eggs from single cell to each of the key stages at 25 ℃ took about 28 hours. The J2 of B. mucronatus were able to generate new J2 after 3 days of inoculation with B. cinerea, and the entire life cycle of B. mucronatus completed in just 3 days.【Conclusion】 The entire embryogenesis process of B. mucronatus eggs from single cell to hatching took about 28 hours at 25 ℃. B. mucronatus can complete its entire lifecycle in just 3 days. The 16 hours are the best time to collect a large number of spawning eggs, and the 36 hours are the best time to collect J2. Further studies are worthwhile on the significance of each of the development stages in the early embryonic development ofB. mucronatus.

Key words: Bursaphelenchus mucronatus, spawning, embryo, ontogenesis