【Objective】 This study aimed to determine the expression characteristics and biological functions of Bx-sex-1, a sex determination gene in Bursaphelenchus xylophilus. 【Method】 The phylogenetic analysis was performed using the neighbor joining (NJ) method, based on the Jones-Taylor-Thornton (JTT) model in the molecular evolutionary genetic analysis (Mega 6). After collecting pine wood nematodes at different developmental stages using synchronous culture, cDNA was synthesized from the extracted RNA, and the relative expression level of Bx-sex-1 was studied using real-time quantitative PCR (qRT-PCR), where the copy number of Bx-sex-1 in the female and male genomes was analyzed through the absolute quantitative analysis. B. xylophilus adults were socked with double-strand RNA (dsRNA), and the hatching rate and the female to male sex ratio of the progeny were observed. 【Result】 The phylogenetic tree showed that the pine wood nematode Bx-sex-1 was a clade with two other plant parasitic nematodes, and had evolved into a different clade from the free and animal parasitic nematodes, respectively. The Bx-sex-1 gene exhibited the highest relative expression level in the egg period, with the lowest expression in the L2 period that increased gradually up to the adult period. Both female and male genomes had a single copy of the gene. Compared with untreated B. xylophilus, the hatching rate of the eggs and the sex ratio decreased in the experimental samples. 【Conclusion】 The Bx-sex-1 gene of B. xylophilus may play an important role in egg development and sex determination.
【Objective】 Jiangsu, Zhejiang, Anhui and Shandong, these four provinces in east China, are recognized as the initial occurrence of pine wilt disease in China. And east China is also the most suitable growth area for Bursaphelenchus xylophilus. Studying the genetic differentiation of the B. xylophilus population in those regions will provide important basic genetic information to establish a B. xylophilus epidemic source tracing system in China. 【Method】 Samples of B. xylophilus were collected from pine trees affected by pine wilt disease in east China, and strains were isolated and purified. The genomic deoxyribonucleic acid (DNA) of strains was extracted and sequenced. The number and types of single nucleotide polymorphism (SNP) loci and genotypes in each region were analyzed using bioinformatics software. Cluster analysis was used to compare the genetic differentiation among different strains based on SNP markers, while Treemix was used to detect gene penetration routes. 【Result】 A total of 67 B. xylophilus strains were isolated and collected in 60 county-level administrative regions of Anhui (AH), Fujian (FJ), Jiangsu (JS), Jiangxi (JX), Shandong (SD) and Zhejiang Province (ZJ) in East China. All strains were sequenced through the whole genome; genome calling analysis indicated 12 genotypes, of the 67 strains, where six types (i.e., A→G, C→G, C→T, G→A, G→C and T→C) occurred at a higher frequency than others. A total of 6 531 684 SNP loci were obtained and the number of SNP was different between strains. The number of SNP counts, homozygotes, missing SNPs, and private SNPs showed no significant regional differences among the six provinces, and there was no significant correlation with invasion time. Principal component analysis showed that 67 strains of B. xylophilus may be divided into three groups; each group had a specific correlation with a geographic origin. Most strains belonged to group 1, which included all strains from Zhejiang and Jiangxi, as well as most of strains from the other four provinces. Group 2 included 14 strains from Jiangsu, Anhui, Shandong and Fujian, and group 3 included only seven strains from Anhui, Fujian and Shandong provinces. Two gene migration routes detected by Treemix: ① group 2 of Jiangsu to group 1 of Anhui; and ② group 3 of Shandong to group 2 of Anhui. 【Conclusion】 There were abundant SNP loci in B. xylophilus in east China, where the SNP characteristics of different strains were relatively disparate, and there was no clear regularity between SNP diversity and invasion time. Overall, the B. xylophilus strains in east China may be divided into three groups, and there was a specific correlation between different groups and geographic areas. In the area where the 1-AH and 2-AH groups were located, there may have been re-invasion by strains from groups in other areas.
【Objective】The pine wilt disease is one of the serious forestry diseases in China, and causes rapid death in pine trees. To provide a theoretical support for the prevention and control of pine wood disease in Huangshan City, an investigation on the species of longicorn beetles and related nematodes was carried out in Pinus massoniana forest. 【Method】Longicorn beetles were collected indoors and outdoors from the Bocun forest farm. The beetles were fixed and identified, and their species and number were recorded. To confirm the presence of nematode vectors, Baermann funnel technique was used to extract the nematodes from beetles, which are harmful to the vitality of pine trees. After soaking the beetles for 12 h, a 15 mL centrifuge tube was used to place the soaking solution at the end of the funnel, which was then observed under a microscope to determine the presence or absence of nematodes. The ratio of beetles that carry nematodes was calculated by determining the ratio of nematode-containing beetles to the total number. To identify the species of nematodes carried by beetles, they were first cultured with fungi and preliminarily identified by observing their morphological characteristics. The molecular identification technique of Bursaphelenchus xylophilus was used to determine if the nematode was B. xylophilus. For the other parasitic nematodes, the 28S rDNA gene of nematodes was amplified, sequenced, blasted, and the phylogenetic tree was constructed for identification. Besides, the ratio and the number of nematodes carried by beetles at different times were counted. 【Result】 Total 14 longicorn beetles belonging to 12 genera and four subfamilies were collected. Two species of beetles Monochamus alternatus and Acanthocinus griseus were associated with nematodes, and their overall nematode carrying rates were 45.8% and 13.6%, respectively. M. alternatus and A. griseus both carry two types of dispersal nematodes with different morphological characteristics, one with a pointed tail and the other with a rounded tail. Results of the morphological and molecular analysis revealed the nematode with the pointed tail as B. xylophilus, while the other as Contortylenchus sp. During the period from June 19 to July 23, the weekly nematode isolation test results showed that the nematodes carried by female adults of M. alternatus were 76.5%, 66.0%, 52.9%, 34.7% and 10.0%, respectively; the proportion of nematodes carried by male adults of M. alternatus was 76.5%, 60.0%, 50.0%, 30.8% and 14.7%. The average number of nematodes carried by per individual female adult of M. alternatus was 1 018.0, 530.1, 202.5, 200.4 and 80.0, and the average number of nematodes carried by per individual male adult was 495.1, 68.3, 74.9, 8.2 and 3.8. 【Conclusion】 A total of 14 longicorn beetles were recorded in the masson pine forest in Bocun forest farm, Huangshan City. Among them, M. alternatus and A. griseus can carry B. xylophilus and Convertylenchus sp., the former being the main strain. The number and proportion of M. alternatus carrying nematodes gradually decreased with time.
【Objective】 In this study, we aimed to screen out saprophytic nematode strains that were easy to colonize and could accelerate the extinction rate of the Bursaphelenchus xylophilus population to substantially reduce the quantity of B. xylophilus in infected trees before the emergence of vector insects, thus effectively controlling the spread of the disease. 【Method】 Eight species of saprophytic nematodes were isolated from the infected trees, which were collected from epidemic areas in Anhui, Sichuan and Jiangsu Province. Eight species of saprophytic nematodes were inoculated into the infected wood blocks with an inoculation quantity of one, two and four times the number of B. xylophilus in the wood blocks. Wood blocks inoculated with sterile water were used as controls, which were cultured at 15 and 25 ℃ for 15 and 30 days, respectively. The quantity of B. xylophilus and saprophytic nematodes in the infected wood blocks were isolated and calculated. The effects of inoculation with saprophytic nematodes on the quantity of B. xylophilus were analyzed under the different inoculation amounts and different ambient temperatures. In the field, eight species of saprophytic nematodes were inoculated into the segments of the plague woods with the inoculation amount of 2.0×105~2.5×105 individuls, and segments inoculated with sterile water were used as the control. After 120 days, the wood samples were drilled. After 150 days, the Monochamus alternatus were captured and the wood samples around the pupae room were collected. The quantity of B. xylophilus collected was separated and counted. The changes in the quantity of B. xylophilus in the infected trees were compared before and after inoculation, and the effects of saprophytic nematodes on the quantity of B. xylophilus in the whole infected trees, M. alternatus and around the pupa rooms were analyzed.【Result】 The results from the indoor inoculation experiment showed that the number of B. xylophilus in the strain JHS2 and JHS4 treatment groups with four times and 1 times the inoculation amount on the 30th day could be substantially reduced when cultured at 15 ℃. On the 15th day of culture at 25 ℃, the reduction rate of the quantity of B. xylophilus in the strain JHS4 treatment group with three different inoculation doses were significantly higher than that of the control group. After 30 days of culture, the reduction rate of B. xylophilus in the treatment group which inoculated four times the inoculation amount of strain JHS4 was 2.9 times higher than that of the control group. Field inoculation results showed that strain JHS4 could accelerate the extinction rate of B. xylophilus in wood segments. Compared with the control group, the reduction rate of B. xylophilus in wood segments was 96%, which was significantly higher than that of the control group (74%) over the same period. Compared with CK, the quantity of B. xylophilus carried by wood samples around the pupa chambers and emerged M. alternatus in all treatment groups was often lower than that of the control group. The quantity of B. xylophilus carried by wood samples around the pupae chambers and emerged M. alternatus in strain JHS4 treatment group was the lowest, and the quantity of B. xylophilus carried by wood samples in the pupae chamber was 20 individuls per gram, and the average quantity of B. xylophilus carried by M. alternatus was 132 individuls per head.【Conclusion】 The saprophytic nematodes can accelerate the extinction rate of the quantity of B. xylophilus in infected trees and the pupae chamber, and M. alternatus. It is clear that the saprophytic nematodes have biological control potential for the pine wilt disease, and their application in the control of B. xylophilus has technical feasibility and broad prospects, which is worthy of further development and research.
【Objective】The pine wilt disease is the most serious forest disease in China. Despite considerable investment in manpower, material and financial resources, the prevention and control situation of the pine wilt disease continues to be a serious issue. Due to the limitations of the large-scale application of chemical nematicides, developing high efficiency botanical nematicides to control the pine wilt disease epidemic will be of great importance. 【Method】 The extracts of 30 plants including Alternanthera philoxeroides, Cayratia japonica and Rosmarinus officinalis were prepared using the ethanol impregnation method; their poisonous activity to the pine wilt disease was then determined. Petroleum ether, ethyl acetate, n-butanol and water were used to extract the active ingredients from two plants via the liquid-liquid extraction method. The concentrations of the solutions were 10.000, 5.000, 2.500, 1.250 and 0.625 g/L, respectively, and insecticidal activities against pine wood nematodes (Bursaphelenchus xylophilus) were determined. The egg suspension was cultured in a constant temperature and moisture incubator at 25 ℃, and observation began after 24 h and ended after 32 h. Microscopic examination was used to count the number of nematode eggs, and the corresponding cumulative hatching and hatching inhibition rates of nematode eggs were calculated. 【Result】 (1) The pine wood nematode was treated with ethanol extracts of A. philoxeroides and C. japonica at a mass concentration of 10 g/L; after 72 h, the corrected mortality of nematodes was 100%. (2) The ethyl acetate extract of A. philoxeroides and the aqueous extract of C. japonica were the key nematocidal substances. The Lethal Concentration 50 (LC50) of pine wood nematodes was 0.588 8 and 0.969 3 g/L after 72 h of the treatment. (3) The in vitro egg incubation test demonstrated that the extract from A. philoxeroides in ethyl acetate exhibited the greatest rate of nematode incubation inhibition of 79.77%, followed by the aqueous extract from C. japonica which had an incubation inhibition rate of 46.51%. 【Conclusion】 A. philoxeroides and C. japonica demonstrated the strong inhibitory biological activity for nematode adults and eggs, which may be developed and utilized as new nematicides for plants.
【Objective】 Bacillus cereus NJSZ-13 strain is an endophytic bacterium in pine which can kill pine wood nematodes (Bursaphelenchus xylophilus). This study investigated whether this strain could induce the resistance of masson pine(Pinus massoniana) to pine wood nematodes which provides a theoretical basis for further development and utilization of this strain. 【Method】 The disease development of different treatments were observed and studied by inoculating with pine wood nematodes at four days after applying the bacterial suspension and inactivated bacterial suspension of NJSZ-13 strain on the roots of two years old masson pine seedlings. The changes of defense-related enzyme activities in masson pine were determined within 4 days after applying the bacteria and within 28 days after inoculating with pine wood nematode AMA3 strain.【Result】 The results showed that at 42 days after inoculated with pine wood nematodes, the effects of the bacterial suspension and inactivated bacterial suspension which induce the resistance of masson pine to pine wood nematode were 54% and 33%, and the disease occurrence time was delayed. After inoculation of NJSZ-13 strain, the content of MDA in the masson pine decreased, the activity of CAT decreased, and POD,SOD,PAL,PPO activities were significantly higher than those of the control. After inoculation with the pine wood nematode, the activity of the five enzymes of P. massoniana treated by the suspension and the inactivated suspension was significantly higher than that of the control, and the suspension showed higher changes in the index of massion pine than that of the inactivated suspension.【Conclusion】 Bacillus cereus NJSZ-13 strain could induce the resistance of masson pine to pine wood nematodes and the cell suspension is better.
【Objective】 The transcriptional activity of Bx-HSF-1 and the expression of its target genes at the seven life stages of Bursaphelenchus xylophilus were studied to reveal its role in the growth and development of B. xylophilus. 【Method】 The CDS (coding sequence) of Bx-hsf-1 gene was constructed into plasmid pGBKT7 to obtain the yeast (Saccharomyces cerevisiae) bait expression vector pGBKT7-Bx-hsf-1. The self-activation of pGBKT7-Bx-hsf-1 was detected. RNA interference and transcriptome sequencing techniques were used to identify the differentially expressed genes of B. xylophilus after Bx-hsf-1 gene was knocked down. The gene interaction analysis of differentially expressed genes was analyzed, and the target genes that were directly regulated by Bx-HSF-1 were screened out. RT-qPCR (quantitative real-time PCR) was used to identify the expression levels of five genes in J2, DJ3, DJ4, J3, J4, female, and male stages of B. xylophilus. 【Result】 The pGBKT7-Bx-hsf-1 yeast turned blue on SD/-Trp/-His/-Ade+X-α-gal medium and could grow on SD/-Trp and SD/-Trp/-His/-Ade mediums. A yeast transactivation activity assay suggested that Bx-HSF-1 had transcriptional activity. RT-qPCR results revealed the significant suppression of Bx-hsf-1 after RNAi treatment for 12 h. The total 110 differentially expressed genes were identified using a transcriptome analysis, of which 86 were down regulated. Total five target genes (Bx-daf-21, Bx-hsp-1, Bx-hsp-70, Bx-sti and Bx-cdc) were filtered using a gene interaction analysis. The expression of Bx-daf-21 was consistent with the expression of Bx-hsf-1 in the development of seven life stages of B. xylophilus, whereas the expression of Bx-cdc in DJ3 and the expression of Bx-hsp-70 were inconsistent with the expression of Bx-hsf-1. The expression of Bx-hsp-1 and Bx-sti was not correlated with the expression of Bx-hsf-1 in the seven life stages. 【Conclusion】 Bx-HSF-1 had a transcriptional activation function and participated in the development process of B. xylophilus through transcriptional regulations.
