Abstract:

Objective: This study aims to elucidate the relationships among the reproduction and dispersal of Bursaphelenchus xylophilus, symptom manifestation, and therapeutic efficacy in infected pine trees, and to explore effective trunk injection techniques for disease control. Method: This study targeted four-year-old Pinus massoniana, eight-year-old Pinus thunbergii, and twenty-year-old naturally infected pine trees, and artificial inoculation and natural infection experiments were conducted. Artificial inoculation was performed using the highly virulent AMA3 strain of B. xylophilus, with doses of 8 000 nematodes per tree for P. massoniana and 5 000 nematodes per tree for P. thunbergii. Stratified sampling was conducted at 5 hours, 1, 3, 5, 10, 20, and 30 days post-inoculation. Quantitative analysis was performed to determine nematode populations in inoculated twigs, main trunks, and adjacent branches, while external symptom development on P. massoniana was simultaneously observed and recorded. In naturally infected forest stands, the symptom severity was graded into five stages: early 1 (beetle feeding marks present, needles not discolored), early 2 (slight discoloration of infected branches), middle 1 (single withered branch), middle 2 (<50% overall wilting), and late (≥50% overall wilting). Trunk injection treatments were administered using 2% emamectin benzoate emulsion (for young trees) and 20% emamectin benzoate soluble liquid (for mature trees), and the treatment efficacy was periodically monitored. Result: 1) Four-year-old P. massoniana: during the asymptomatic phase (5 days post-inoculation), B. xylophilus rapidly disseminated within the tree, invading the main trunk by day 3 and spreading throughout the entire plant by day 5. At this stage, the nematode populations were small, and the survival rate reached 60% following chemical treatment. As the disease progressed to the early symptomatic phase (10 days post-inoculation), nematode numbers increased exponentially, with chlorosis appearing in needles of inoculated branches, and the survival rate declined to 20%. By the full symptomatic phase (20 days post-inoculation), nematode populations peaked, and treatments were ineffective. 2) Eight-year-old P. thunbergii: during the asymptomatic phase (fully green needles), B. xylophilus aggregated in the inoculated branches with a diffusion rate of 2.5 cm·d^?1. In early stage 1 (needle chlorosis), nematodes proliferated within inoculated branches but exhibited reduced diffusion speed (1.7 cm·d^?1). At early stage 2 (needle yellowing), nematodes accelerated their migration toward the main trunk (4.0 cm·d^?1). During the middle stage (red-brown needles accompanied by chlorosis in adjacent branches), nematodes spread bidirectionally along the main trunk, reaching peak population density (4.0 cm·d^?1). In the late stage (whole plant wilting), nematode populations within the tree declined (4.0 cm·d^?1). 3) Twenty-year-old naturally infected pine trees: during early stage 1 (beetle feeding wounds on twigs, no needle discoloration) and early stage 2 (slight discoloration of infected branches), the survival rate reached 100% following chemical treatment. In middle stage 1 (wilting of a single infected branch), the survival rate decreased to 75%. By middle stage 2 (<50% overall needle wilting), the survival rate was only 20%. At the late stage (≥50% overall needle wilting), the treatment was completely ineffective. Conclusion: Based on the symptoms initially manifested in the earliest infected twigs, trunk injection therapy in pine trees aged eight years old or older is theoretically and practically feasible. Moreover, treatment efficacy has a positive correlation with tree size. This injection technique fills a critical technical gap in the management of pine wilt disease.

Key words: pine wilt disease, distribution of Bursaphelenchus xylophilus, symptom manifestation, trunk injection therapy, early diagnosis