As the most severe forestry quarantine disease in several countries, pine wilt disease (PWD) causes substantial economic losses and poses a significant threat to the forest ecosystem. It is necessary to find a rapid and sensitive method for the early diagnosis of the disease to control the development of the disease effectively. This paper investigated the effect of Bursaphelenchus xylophilus（the pinewood nematode; PWN）on the chlorophyll fluorescence kinetic curve (OJIP curve) and the parameters of needles using four-year-old Pinus thunbergii as experimental materials and chlorophyll fluorescence analysis as a technical tool. It was shown by the results in the OJIP curve that the fluorescence intensity of the inoculated plants was significantly increased at points O and I. Additionally, the relative variable fluorescence intensity at points K and J was comparable to that of the control plants. Several chlorophyll fluorescence parameters of the treatment significantly increased or decreased with disease progression. At the same time, the control group had no significant changes in each parameter. Therefore, chlorophyll fluorescence parameters can be used as indicators for the early diagnosis of PWD, among which the DIo/RC parameter was the best. In summary, PWN invasion will produce fluorescence on the PSII of P. thunbergii, and its chlorophyll fluorescence parameters are expected to achieve early PWD diagnosis.

Bacillus pumilus is widely used as a biocontrol agent. To further develop the biological control potential of B. pumilus LYMC-3 against pine blight, a statistical experimental design was used to optimize a liquid medium using low-cost substrates to improve its antagonistic activity against Sphaeropsis sapinea. Through the plate antagonism test and greenhouse control effect test, this study determines the antifungal effect of strain LYMC-3 against S. sapinea and pine blight. Moreover, response surface optimization methodologies were used to systematically optimize the medium composition and culture conditions of the LYMC-3 strain. The plate antagonism test showed that the inhibition rate of LYMC-3 fermentation filtrate (diluted 5-fold) was 66.09%. The greenhouse control effect test showed that the control effect of its fermentation filtrate on shoot blight reached 89.99%. The response surface optimization test ultimately determined that a higher inhibition rate can be achieved under these conditions: the optimal medium components were 7.2 g/L glucose, 15 g/L peptone, and 7.1 g/L magnesium sulfate; the optimal culture conditions were 52% liquid volume, 28 °C culture temperature, an initial pH of 7, and 1% bacterial inoculation volume. Under the optimized system, the five-fold diluted LYMC-3 fermentation filtrate inhibition rate against S. sapinea was 81.23%, which was 15.84% higher than that before optimization. Meanwhile, optimize the selection of lower-cost and more commonly used glucose instead of beef paste as the carbon source for the culture medium, and choose cheaper magnesium sulfate instead of sodium chloride as the nitrogen source.

In previous research, the pine wood nematode Bxadh2 gene significantly increased its expression in pine seedlings inoculated with endophytic Bacillus cereus GD2 and pine wood nematode. To explore pine wood nematode Bxadh2 gene function, we cloned and analyzed its biological information, and we preliminarily studied its physiological function through RNA interference. We found that the Bxadh2 gene’s full CDS length is 1269 bp, which encodes 422 amino acids, and presents a relatively stable hydrophobic protein. The protein encoded by the Bxadh2 gene has no signal peptide or transmembrane structure, and it is an intracellular protein that does not participate in transmembrane movement. The RNAi interference results showed that when the pine wood nematode’s Bxadh2 gene was suppressed, its survival rate and fecundity significantly decreased, indicating that the expression of the Bxadh2 gene was related to the growth and development of pine wood nematodes.