【Objective】 To explore whether the interaction of pine families with varying levels of disease resistance in the same plot with pine wood nematode (Bursaphelenchus xylophilus) under natural conditions affects the pathogenicity and reproduction of B. xylophilus, and to determine if the expression level of cytochrome P450 i.e. CYP family-related genes of B. xylophilus strains with noticeable differences in pathogenicity different after interaction with pine trees, the following study was conducted.【Method】This study monitored the gene pools of several families of Pinus densiflora and P. thunbergii, which were introduced from Japan previously in the lab. Field monitoring was conducted over several years, revealing significant differences in disease resistance among different pine families. Dead wood samples from each family in the pine tree gene pool were collected, and automated molecular detection was used to identify B. xylophilus. Nematode samples from five P. densiflora families and five P. thunbergii families, confirmed to have died from B. xylophilus, were isolated. A total of 10 B. xylophilus strains were obtained through monoheterotic in vivo culture, and their pathogenicity and reproduction were tested. The relative expression of two cytochrome P450 genes in four B. xylophilus strains with significantly different pathogenicity was detected by RT-qPCR. 【Result】The results indicated that in the same tree species within the same plot, the stronger the disease resistance of the pine families, the lower the reproduction rate of B. xylophilus strains obtained under monoheterotic living culture. Conversely, weaker disease resistance in pine families corresponded with higher reproduction rates of B. xylophilus strains. Inoculating each strain with 2-year-old P. thunbergii revealed that the pathogenicity test results of nematode strains obtained from diseased and dead trees of five P. thunbergii families were positively correlated with the resistance of pine families. The B. xylophilus strain JYK-34, isolated from the most resistant family (34), showed the highest pathogenicity. However, for P. densiflora families, there was no positive correlation between resistance and the pathogenicity of B. xylophilus. The B. xylophilus JYK-18, isolated from a highly resistant family (18), exhibited weak pathogenicity. Compared with the control B. xylophilus, the increased expression of two cytochrome P450 genes, BxCYP33C4 and BxCYP33C9, was positively correlated with the pathogenicity of the four B. xylophilus strains, namely JYK-34>JYK-17>JYK-18>JYK-31, after interacting with the P. thunbergii seedlings for 30 days. 【Conclusion】The differentiation in the reproduction of B. xylophilus was consistent among different disease-resistant families of P. densiflora and P. thunbergii. The pathogenicity of B. xylophilus isolated from pine trees with different resistance levels within the same plot in the resistant pine tree gene pool was not uniform, with different strains exhibiting varying pathogenicity. The cytochrome P450 genes BxCYP33C4 and BxCYP33C9 in B. xylophilus are involved in the interaction between B. xylophilus and pines and are also important genes related to the pathogenicity of B. xylophilus.
【Objective】To clone the glutathione S-transferases (GST) genes of Monochamus alternatus (Coleoptera: Cerambycidae) and to determine the expression levels of these genes under heat stress in adult males and females, as well as at different developmental stages of M. alternatus, we used molecular biology and bioinformatics methods. 【Method】 Three GST cDNA sequences were cloned, and their structural characteristics were analyzed using DNAMAN 9.0 and I-TASSER software. qRT-PCR was employed to measure the relative expression levels of GST genes in adult and fourth instar larvae of M. alternatus under varying temperatures and treatment durations. The role of these GST genes in protecting the organism from oxidative stress was assessed through disc diffusion assays. 【Result】Three GST cDNA sequences were cloned and named MaltGSTe1, MaltGSTe2, and MaltGSTt1. MaltGSTe1 and MaltGSTe2 belong to the Epsilon family, while MaltGSTt1 belongs to the Theta family. Analysis of three-dimensional protein structures indicated that these GST are cytoplasmic. The relative expression levels of MaltGSTe1, MaltGSTe2 and MaltGSTt1 in fourth instar larvae significantly changed under heat stress, with MaltGSTe2 showing the greatest change. In males, the expression level of MaltGSTt1 was significantly down-regulated. Escherichia coli expressing the GST genes demonstrated enhanced antioxidant capacity, with MaltGSTe2 exhibiting the strongest activity. 【Conclusion】We successfully cloned three GST genes and investigated their expression characteristics under high temperature stress in M. alternatus. High temperature stress was found to induce up-regulation of GST gene expression. Disc diffusion assays confirmed that heterogeneously expressed GST proteins have antioxidant capabilities. These findings suggest that GST genes play a role in the response mechanism to high temperature stress by protecting against oxidative stress, providing a theoretical foundation for exploring the heat resistance mechanisms of M. alternatus in subtropical regions.
【Objective】Pinus massoniana, a predominant tree species in Chinese forests, is highly susceptible to pine wood nematode (PWN), Bursaphelenchus xylophilus, which causes pine wilt disease and can lead to severe economic and ecological damage. This study aimed to develop an evaluation system for assessing the resistance of P. massoniana embryogenic callus to PWN and to screen for cell lines that exhibit enhanced resistance.【Method】The embryogenic callus induced from immature embryos of P. massoniana, sourced from Guangyun Forest Farm in Pingle County, Guilin City, Guangxi Zhuang Autonomous Region, China, was used as the experimental material, and the embryogenic callus of P. thunbergii and P. elliottii was used as the control group. The strongly virulent PWN strain AMA3c28, maintained at Nanjing Forestry University, China, was utilized for inoculation. Bacterial-free PWNs were obtained by sterilizing nematode eggs with 15% H2O2 for 50 min followed by rinsing in sterile water three times. The sterilized eggs were then inoculated onto pine callus, where they hatched and proliferated. The bacterial-free PWNs were collected and each callus piece from different cell lines was inoculated with 50 μL of a nematode suspension containing approximately 500 nematodes. Control groups were inoculated with an equal amount of sterile water. Post-inoculation, the cultures were incubated at 25 ℃ in the dark, and morphological changes were observed after 10 d, followed by the microscopic examination of cellular morphology. The cell viability was assessed using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. The population dynamics of PWN within the callus were evaluated by re-isolating the nematodes using the Baermann funnel technique. 【Result】The study revealed a marked variation in PWN resistance among the P. massoniana cell lines. The cell lines GX19-1-2 and GX20-3-3 exhibited the highest resistance and minimal morphological changes, maintaining an cell structural integrity after inoculation with bacterial-free PWNs for 10 days. In contrast, cell lines GX20-4-4, GX20-1-10, GX20-3-5, GX20-1-1 and GX20-1-7 exhibited severe browning, tortured cell structure, and significant growth inhibition, indicating weaker resistance. TTC staining confirmed these observations, with resistant cell lines showing vibrant red staining similar to the control groups, while susceptible lines turned pink and white, indicating reduced cell viability. Microscopic examination of cell structures post-inoculation further validated the resistance profiles, with resistant lines maintaining clear embryonic head-stem structures, and susceptible lines showing disrupted cellular integrity and a leakage of cellular contents. The population dynamics of PWN within the callus varied significantly among cell lines. Notably, the P. thunbergii cell line 36-2 exhibited the highest reproduction of PWN (107 333 ± 9 333), indicating a high susceptibility. In contrast, the P. massoniana cell lines GX19-1-2 and GX20-3-3 showed lower PWN levels, which were significantly lower than that of the two P. elliottii cell lines 1907-9 and 1927-1, suggesting a strong inhibitory effect on nematode reproduction and strong resistance. Cell line GX20-3-8 had similar resistance compared to that of P. elliotti cell lines. 【Conclusion】This study successfully developed an in vitro evaluation system for assessing the resistance of P. massoniana to PWN, revealing that cell lines GX19-1-2 and GX20-3-3 exhibit promising levels of resistance. The results act as a basis for future research, contribute to the development of resistant P. massoniana varieties, and can be employed to establish a new research platform for enhancing our understanding of the interactions between host pine trees and the PWN.
