【Objective】Systematic investigation of the start and end dates of pine wood nematode (PWN, Bursaphelenchus xylophilu) transmission following the emergence of long-horned beetle(Monochamus alternatus), the number and percentage of PWN transmitted, the temporal dynamics of PWN transmission, and the relationships between the number of PWN carried by adult beetle, the food intake and the longevity of M. alternatus, the duration of PWN transmission by M. alternatus and the number of PWN transmission. 【Method】Dead specimens of Pinus massoniana were collected in epidemic area of pine wilt disease before the emergence of M. alternatus. The specimens were cut into logs (1.0-1.5 m in length), and maintained in a large field cage (2.0 m × 2.0 m × 2.0 m) and kept outdoors. The M. alternatus adults were collected daily during the period of adult beetle emergence from the logs and fed individually with 1-year-old fresh twigs (diameter: 1-2 cm. length: 25-30 cm) of P. massoniana in insect-rearing cages (0.5 m × 0.5 m × 0.5 m). Twigs were replaced every 3 days and measured the amount of food consumed by each M. alternatus. The PWNs were extracted by the Baermann funnel method and record the number of PWNs transmitted by M. alternatus. The M. alternatus were dissected after they died. The PWNs remaining within the adult insects were extracted by the Baermann funnel method and counted under a microscope. Record the longevity and PWN carrying capacity of M. alternatus.【Result】 Only 16.7% ofM. alternatus transmit PWN in the early stage of their emergence, and 77.8% of M. alternatus transmit PWNs 7-12 days after their emergence. In addition, 88.8% of M. alternatus ended the transmission of PWNs 16-30 days after their emergence. The number of PWN transmitted was small in the early stage of the emergence of M. alternatus. However, the number of PWN transmitted increased rapidly one week after the emergence of M. alternatus. Two peaks of transmission occurred at 12 days and 24 days post the emergence of M. alternatus. The transmission of PWN continued at a low level one month after the emergence of M. alternatus until the end of transmission. On average, each of the M. alternatus carried (4 303.3 ± 7 399) and transmitted (490.7 ± 611.6) PWN. The 94.4% of M. alternatus could not transmit all PWNs to logs, and the proportion of PWN transmitted was 29.8%. The average duration of PWN transmission by was (15.4 ± 7.8) days. The mean longevity of M. alternatus was (37.8 ± 5.6) days. 【Conclusion】The date starting to transmit PWN was not significant between male and female M. alternatus after emergence, but there was a significant difference in the end of PWN transmission. The start date of the transmission of PWNs by M. alternatus was concentrated, but the end date of the transmission of PWNs was dispersed. The characteristics of the start-stop time of PWN transmission by M. alternatus may be related to the physiological changes of the PWN in adults beetle, which needs to be further studied. There was no significant difference in the number of PWN carried and transmitted between male and female M. alternatus. There was no significant difference between male and female M. alternatus in the temporal dynamics of transmission of PWN. The longer the duration of PWN transmission and larger the number of PWN carried by eachM. alternatus, the more PWN was transmitted. The duration of PWN transmission was little affected by the longevity of the M. alternatus or the number of PWN carried. There was no relationship between the longevity of M. alternatus and number of PWN transmitted; but PWN had a weak and not significant negative effect on the lifespan of the adult beetle. After emergence, the amount of food consumed by M. alternatus maintained at a high level throughout the adult stage without obvious regularity; there was no relationship detected between the amount of food consumed and the number of PWN transmitted.
【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.
【Objective】 The pine wood nematode, Bursaphelenchus xylophilus, is an invasive species that damages pine trees, with significant ecological and financial consequences around the world. In China, the species has recently breached its optimal temperature line and has now spread to several districts in Liaoning Province. In this paper, we outline the reasons for the rapid spread of B. xylophilus to northern China by compiling evidence about its temperature adaptability. We compare two isolates, from southern and northern China, to understand differences in pathogenicity and reproductive ability. 【Method】 We compared the morphological index values of the southern B. xylophilus strain AMA3 with the northern B. xylophilus strain FCBX. We tested the virulence and reproductive ability of both strains of B. xylophilus in Pinus thunbergii material to assess its adaptability. We used a culture of Botrytis cinereal, a fungus which contributes to pine disease, to simulate field pathogenicity conditions, under different temperature conditions.【Result】There were no significant differences in the morphological index values of AMA3 and FCBX, all of which were typical of Umbellifera nematodes. Both strains were cultured with B. cinereal in the temperature range of 15-35 ℃, and we found that increases in temperature correlated with increases in reproduction. Reproduction of FCBX strains was generally higher than that of AMA3 strains, although this was reversed at -30 ℃. At low temperatures (15-20 ℃), FCBX produced significantly more replicates than AMA3 did. After vaccination against AMA3 and FCBX, P. thunbergii specimens had different susceptibility to disease, although FCBX maintained a higher relative pathogenicity than AMA3. Thirty-five days after inoculation, P. thunbergii specimens exhibited a mortality of 80% when infected with FCBX, and the disease index was 85; samples infected with AMA3 had a mortality of 60%, and the disease index was 73. Over this period, FCBX reproduced more than AMA3 strains in the P. thunbergii samples, but this difference was not significant. 【Conclusion】 Compared with southern strains (AMA3), northernB. xylophilus strains (FCBX) had slightly larger morphological indicators, higher reproductive capacity in B. cinereal, activity ability, higher reproduction in P. thunbergii, and greater pathogenicity. As B. xylophilus has moved northward, it has been subject to low temperature conditions, and we suggest it has acclimatized to these conditions. It has now broken through its optimal temperature line, and northern strains exhibit strong reproductive capacity, high pathogenicity and adaptability to low temperature conditions.
【Objective】This study is focused on the genetic variations and population structures among different Bursaphelenchus xylophilus strains isolated from Guangdong Province using single nucleotide polymorphisms (SNPs) as markers. This work provided fundamental information for the establishment of a B. xylophilus tracking system in China.【Method】Thirty strains ofB. xylophilus from different regions of the Guangdong Province were collected and sequenced. Genome-wide SNPs were analyzed using bioinformatics, and population splitting events were discovered based on previous SNP data.【Result】Statistical analysis of SNPs across 30 B. xylophilus strains showed that the number of SNPs and homozygotes in seven of the 30 strains (GD01, GD09, GD12, GD20, GD22, GD24 and GD25) were less than the remaining 23 strains. The genotypes that occurred with a higher frequency in these seven strains were A->G, C->G, G->C, T->C, while the other 23 strains contained genotypes that were A->G, C->T, G->A, T->C. The strains from Guangdong Province could be divided into 3 populations based on PCA and hierarchical clustering.【Conclusion】The genetic diversity ofB. xylophilus population in Guangdong Province is relatively high; thus, it should have different sources through which genetic variation is introduced.
