Abstract:

【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.

Key words: Pinus massoniana, Bursaphelenchus xylophilus, embryogenic cell lines, cell viability, nematode reproduction, resistance evaluation