Pine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a devastating disease in conifer forests in Eurasia. However, information on the effect of PWD on the host microbial community is limited. In this study, the bacterial community structure and potential function in the needles, roots, and soil of diseased pine were studied under field conditions using Illumina MiSeq coupled with Phylogenetic Investigation of Communities by Reconstruction of Unobserved states (PICRUSt) software. The results showed that the community and functional structure of healthy and diseased trees differed only in the roots and needles, respectively (p < 0.05). The needles, roots, and soil formed unique bacterial community and functional structures. The abundant phyla across all samples were Proteobacteria (41.9% of total sequence), Actinobacteria (29.0%), Acidobacteria (12.2%), Bacteroidetes (4.8%), and Planctomycetes (2.1%). The bacterial community in the healthy roots was dominated by Acidobacteria, Planctomycetes, and Rhizobiales, whereas in the diseased roots, Proteobacteria, Firmicutes, and Burkholderiales were dominant. Functionally, groups involved in the cell process and genetic information processing had a higher abundance in the diseased needles, which contributed to the difference in functional structure. The results indicate that PWD can only affect the host bacteria community structure and function in certain anatomical regions of the host tree.

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.

Pine wood nematode disease is a destructive disease to the ecological environment and forest resources. The disease is produced by Bursaphelenchus xylophilus, and the onset rate is extremely fast and the pathogenesis of the disease is not clear. Prevention of this disease is still important in production, so it is of great significance to explore its early diagnosis. In this study, the changes of chlorophyll fluorescence dynamic induction parameters, non-photochemical quenching parameters and organic acid content in needles of 7-year-old-infected Pinus hwangshanensis were studied by chlorophyll fluorescence technology and organic acid mass combined technology. The results showed that in the chlorophyll fluorescence dynamic induction group, Fm/Fo, Fv/Fo, Fm, Psi_o and Pi_Abs showed a significant downward trend, while Phi_Do, ABS/RC, TRO/RC and DIO/RC showed a significant uptrend. In non-photochemical quenching, Fm showed a downward trend, while Qp_L1, Qp_L2, QY_L1, QY_L2 and QY_Lss showed a significant upward trend. In the change of organic acid content, ferulic acid, syringic acid, gallic acid and jasmonic acid showed a significant increasing trend, while benzoic acid and salicylic acid showed a first increasing trend and then decreasing trend. Experimental results show that pine wood nematode had significant effects on photosynthesis and the organic acid content of pine before it showed symptoms of the disease. After comprehensive analysis, chlorophyll fluorescence dynamic induction parameters Pi_Abs, Fm, Phi_Do, TRO/RC, Psi_o, non-photochemical quenching parameters QY_L2, Qp_L1, QY_Lss, QY_L1 and organic acids ferulic acid, syringic acid, benzoic acid and gallic acid contents are ideal indicators for early diagnosis of pine wood nematodiasis.

In order to provide a highly feasible research pathway for the control of larch shoot blight, healthy larch branches and leaves were collected from 13 sampling sites in 8 provinces in China. The antagonistic endophytic bacteria obtained from the screening were used to carry out disease control experiments in potted seedlings. The safety evaluation test was conducted on the antagonistic bacteria. Subsequently, the strains with better preventive effect and high safety were identified by morphological and molecular methods. A total of 391 strains of endophytic bacteria were isolated from healthy larch branches and leaves. Seventy-eight strains of larch endophytic bacteria with antagonistic effect were obtained by primary sieving. Ten strains of endophytic bacteria with obvious antagonism were further obtained by secondary sieving, and all of them had an inhibition rate of more than 57%. Among them, strains YN 2, JL 6, NMG 23, and JL 54 showed the highest inhibition rate of 63.16%–65.08%, which was significantly different from the other treatments. The results of the pot test showed that 14 days after inoculation with the pathogen, strains YN 2 and JL 54 were more effective in the control of larch shoot blight, with the preventive effects reaching 57.7% and 50.0%, respectively. Strains JL 6 and JL 54 were biologically safe in the safety evaluation test. Therefore, strain JL 54 was selected for identification. It was identified as Bacillus amyloliquefaciens through morphological observation, 16S rDNA sequence, gyrB gene sequence and 16S rDNA- gyrB tandem feature sequence analysis. IMPORTANCE: Larch shoot blight is a widely distributed, damaging, and rapidly spreading fungal disease of forest trees that poses a serious threat to larch plantations. Endophytic bacteria have biological effects on host plants against pests and diseases, and they have a growth-promoting effect on plants. In this paper, we investigated for the first time the biocontrol effect of endophytic bacteria on larch shoot blight by screening endophytic bacteria with the function of antagonizing dieback fungi. Bacillus amyloliquefaciens JL 54 has a better prospect of biocontrol against larch shoot blight, which lays the foundation for the application of this bacterium in the future.

Fusarium acuminatum is recognized as the causative agent of root rot in many forestry and agricultural plants. In recent years, root rot and foliage blight caused by F. acuminatum have become widespread and severe in China, particularly affecting Dianthus chinensis. The infection mechanism of F. acuminatum remains a pressing area for research. A crucial approach to elucidating its pathogenic mechanisms involves the genetic modification of candidate genes, which necessitates effective transformation systems. Currently, protoplast-mediated transformation (PMT) serves as a valuable tool for studying plant-pathogen interactions and offers several advantages over conventional transformation methods. In this study, we employed the PMT technique to establish a transformation system for the F. acuminatum strain FDCY-5 due to its benefits such as ease of operation, low cost, high conversion efficiency, and broad applicability. We successfully developed a transformation system capable of producing abundant high-quality protoplasts from F. acuminatum and generating green fluorescent protein (GFP) transformants. To verify whether GFP was constitutively expressed, we utilized fluorescence microscopy alongside PCR technology. The results demonstrated that GFP was effectively transformed into the protoplasts of F. acuminatum and expressed successfully. The established protoplast transformation system for F. acuminatum provides a foundational platform for analyzing functional genes within infected host plants as well as understanding the molecular mechanisms underlying host plant infections by F. acuminatum.