Xingyao Zhang

The pine wood nematode (Bursaphelenchus xylophilus), the pathogen of pine wilt disease (PWD), has caused enormous economic losses in Asian forests. Whether venom allergen proteins (VAPs) are involved in the accumulation of key defense substances in pine trees during the interaction between B. xylophilus and host trees, and their specific function as putative effectors secreted through stylets, has not been fully elucidated. In this study, the role of the BxVAP2 effector protein in the infection process was analyzed through bioinformatics and phylogenetic tree construction. The expression profile of BxVAP2 during infection was analyzed using qRT-PCR, and its expression under the stress of Pinus massoniana metabolites was examined. Toxicity assays were conducted through the Agrobacterium transient expression of BxVAP2 in Nicotiana benthamiana, and its subcellular localization was investigated. The results showed that BxVAP2 contains a CAP domain and shares close evolutionary relationships with venom allergen proteins from related species, such as Bursaphelenchus mucronatus, Aphelenchoides besseyi, Aphelenchoides fujianensis, and Meloidogyne graminicola. BxVAP2 was upregulated during the infection of P. massoniana, indicating that BxVAP2 is a key effector in the infection and colonization process of B. xylophilus and may play an important role during the rapid population growth phase. BxVAP2 responds to P. massoniana metabolites, where different concentrations of α-pinene suppressed its expression, while high concentrations of β-pinene promoted its expression. Subcellular localization revealed that BxVAP2 localizes to the cell membrane and nucleus. The transient expression of BxVAP2 in N. benthamiana induced programmed cell death and regulated pattern-triggered immunity marker genes. These findings suggest that BxVAP2 plays an important role in the interaction between B. xylophilus and its host, responding to terpene stress and triggering plant defense.

Pinewood nematode, Bursaphelenchus xylophilus, a pine-parasitic nematode, poses a serious threat to pine trees globally, causing pine wilt disease. When dispersal-stage juvenile 4 (dauer, J_IV, a durable stage) of B. xylophilus enters the new pine, it transforms into a propagative adult (dauer moulting) and reproduces quickly. Our previous studies have found that pine-volatile β-pinene promotes dauer moulting of B. xylophilus; however, this mechanism is not clear. Here, this study is attempting to unravel the molecular process underlying dauer moulting of B. xylophilus through signal chemical tests and transcriptome analysis. The results showed that β-pinene could promote dauer moulting of B. xylophilus, while other common dauer moulting signals, such as dafachronic acid (DA), part of the TGF/insulin signal pathway, were inoperative. Moreover, the J_IV soaked in 1% β-pinene for only 6 h could transform into adults at a significant rate. Therefore, the transcriptomes of J_IV soaked in 1% β-pinene for 6 h were sequenced. It was found that 15,556 genes were expressed; however, only 156 genes were expressed differentially and enriched in the metabolism of xenobiotics, peroxisome, fatty acid metabolism, and carbon metabolism, indicating that energy metabolism was active at the early stage of dauer moulting. With a stricter parameter, the number of differential genes fell to 19, including 4 sterol hydroxylase, 5 dehydrogenase, 2 glucuronosyltransferase, 5 nuclear-related factor, 1 calcium-binding protein, 1 nitrogen metabolic regulation protein, and 1 cystathionine gamma-lyase. These results indicated that dauer moulting of B. xylophilus into adults might not be regulated by the TGF-β/insulin signal pathway but by another new signal pathway related to the 19 differential genes which need more exploration. Our results contribute to the understanding of the molecular mechanisms behind dauer moulting and may be useful in reducing pine wilt disease by suppressing this moulting to cut the life cycle of B. xylophilus.

Pinus armandii (P. armandii) is extensively abundant in western China and, as a pioneer tree, and prominently influences local ecology. However, pine forests in this region have been significantly damaged by Dendroctonus armandi (D. armandi) infestations, in close association with ophiostomatoid fungi. This study aimed to identify the diversity of ophiostomatoid fungi associated with D. armandi infesting P. armandii in western China. A total of 695 ophiostomatoid fungal strains were isolated from 1040 tissue pieces from D. armandi galleries and 89 adult beetles at four sites. In this study, based on multiloci DNA sequence data, as well as morphological and physiological characteristics, seven species belonging to five genera were identified including three known species, Esteyea vermicola, Graphium pseudormiticum and L. wushanense, two novel taxa, Graphilbum parakesiyea and Ophiostoma shennongense, and an unidentified Ophiostoma sp. 1. A neotype of Leptographium qinlingense. Ophiostoma shennongense was the dominant taxon (78.99%) in the ophiostomatoid community. This study provides a valuable scientific theoretical basis for the occurrence and management of D. armandi in the future.

Nematodes can enter cryptobiosis by dehydration as an adaptation to low-temperature environments and recover from cryptobiosis by rehydration after environmental improvement. In this work, the survival of Bursaphelenchusxylophilus third-stage dispersal juveniles was studied in response to low-temperature treatment. The average survival rates were 1.7% after −80 °C treatment for 30 d and 82.2% after −20 °C treatment for 30 d. The changes of water content and inorganic salt ions that occur in pine trees during winter gradually alter the osmotic pressure in the liquid environment to dehydrate B. xylophilus juveniles, resulting in improved survival after low-temperature treatment. The survival rate at −20 °C improved to 92.1% when the juveniles entered cryptobiosis by osmotic regulation. The results of this study demonstrate that B. xylophilus third-stage dispersal juveniles can resist low-temperature stress through cryptobiosis, providing the theoretical basis for the identification of areas potentially vulnerable to B. xylophilus in the mid-temperature and cold temperature zones of China.

Diplodia tip blight caused by Sphaeropsis sapinea (Fr.) Dyko and B. Sutton is a serious threat to the health of natural secondary Japanese red pine (Pinus densiflora Sieb. et Zucc.) forests. To explore the effect of plant diversity on Diplodia tip blight disease occurrence, the correlation between Diplodia tip blight in P. densiflora and plant diversity in various stand types and vertical structure layers were analyzed by Pearson correlation coefficients and generalized linear model while keeping environmental factors relatively consistent. Disease index of Diplodia tip blight in P. densiflora was positively correlated with the plant diversity indices in the tree layers of P. densiflora–conifer mixed forest. In contrast, it was negatively correlated with the plant diversity indices in the tree layers of P. densiflora–hardwood mixed forest and P. densiflora pure forest. In shrub–herb layers of all stands except the P. densiflora pure forest herb layer, the disease index was positively correlated with the plant diversity indices. The prediction models for the disease index of Diplodia tip blight in the P. densiflora–hardwood mixed forest, P. densiflora–conifer mixed forest, and P. densiflora pure forest were also established. The foregoing results suggest that in the Kunyu Mountains of China, the differences in plant diversity among various stand types and vertical structure layers have different effects on the occurrence of Diplodia tip blight in P. densiflora. Plant diversity largely reflects the occurrence of Diplodia tip blight disease. However, if the effect of plant diversity on Diplodia tip blight is fully reflected, the plant species characteristics, especially tree genetic relationships, should be considered. In this way, plant species diversity structure in natural secondary Japanese red pine forests may be modified through forest management to lower the incidence of Diplodia tip blight.

The pinewood nematode Bursaphelenchus xylophilus (B. xylophilus) is responsible for a devastating disease of pine forests. Its pathogenicity is closely related to the numbers of individual B. xylophilus. The ratio of female to male (sex ratio) is key to understanding population change in this species. The sex ratio of B. xylophilus varies widely, but it is unclear how it is affected by environmental changes. The sex ratios of nematodes, isolated from different samples in the wild, varied between 0.93 and 2.20. Under laboratory conditions, maternal age and the population did not affect the sex ratio of progeny. A change from good to poor nutritional status was associated with a reduction of the sex ratio of progeny from 1.85 to 1.41, which was speculated to result from a change in the primary sex ratio. Thus, B. xylophilus effectively maintains the sex ratio with maternal age and population changes but adjusts the sex ratio of progeny on the basis of the changes in nutrition.

The pinewood nematode (Bursaphelenchus xylophilus) can cause fatal damage to trees and is transmitted by the vector species of the Monochamus genus. In December 2017, pinewood nematodes were found to be harming a large plot of pine trees, Pinus tabuliformis, in a new region with an average annual temperature of 6.7 °C in China. However, the vector insects were unknown and urgently needed to be identified. Hence, in April 2018, we collected wood sections of P. tabuliformis trees that had died from pine wilt disease. All 127 longhorn beetles that emerged from the P. tabuliformis samples were identified as Monochamus saltuarius, and the nematodes they carried were fourth-stage dispersal juveniles of the pinewood nematode. The carrier rate of pinewood nematodes in the M. saltuarius population was 58.3%, and the average carrying capacity was 642.4 ± 89.3. In the same P. tabuliformis plot, 742 M. saltuarius beetles were collected from hormone traps. Our results revealed that M. saltuarius can damage P. tabuliformis and is a new vector species of the pinewood nematode in China. These findings can inform the prevention and control of pinewood nematode damage to pine forests.

In recent years, the pinewood nematode has continuously adapted to low-temperature environments and expanded from the South to the North of China. In December 2018, a large area of pinewood nematode was suspected to be harmful to Pinus tabuliformis under natural conditions in Fushun City, Liaoning Province. In order to clarify the low-temperature environment and population characteristics of pinewood nematodes in this new epidemic area, we analyzed the difference in temperature between the inside and outside of P. tabuliformis in low-temperature environments, conducted the morphological and molecular identification of pinewood nematodes in P. tabuliformis, summarized the distribution characteristics of the wintering of pinewood nematodes and explored the population structure of pinewood nematodes under different low-temperature conditions. The results indicated that the diurnal variation of temperature in dead P. tabuliformis was significantly less than the environment temperature. The lowest temperature in P. tabuliformis was 3.2 °C higher than the lowest temperature in the environment in one day; the pathogen of a large area of dead P. tabuliformis in Fushun was pinewood nematode (Bursaphelenchus xylophilus); 84.9% of the average populations of pinewood nematodes were third-stage dispersal juveniles, which mainly gathered in 5 cm within the pupal chamber of Monochamus saltuarius Gebler. At −40 ℃, most of the third-stage dispersal juveniles of pinewood nematode in dead pine can still survive. Our study laid a foundation for the understanding of the low-temperature adaptation mechanism of pinewood nematode and contributed to the monitoring of pine wilt disease in the mid-temperate zone.

Poplar canker, mainly caused by Botryosphaeria species, is a serious disease that has resulted in the reduced productivity and death of poplar worldwide. Different Populus species have varied resistance levels to poplar canker; however, whether phenolic compounds in poplar are involved in this resistance remains uncertain. Here, we determined the concentrations of phenolic compounds and their antifungal activities in canker-resistant P. tomentosa and canker-susceptible P. beijingensis. We identified 29 phenolic compounds with significantly different concentrations between the two species. Salicylic acid (SA), tremuloidin, salicin, and poplin were dominant in P. tomentosa, while benzoic acid (BA) and catechol were dominant in P. beijingensis. These six phenolic compounds were further tested for antifungal activities. SA, BA, and catechol showed significant antifungal activities against Botryosphaeria dothidea, while the other three compounds showed no activity. As BA or catechol can be converted in plants to SA, which is a remote signaling molecule that plays an important role in plant defenses, we presumed that the resistance of P. tomentosa was initiated by SA, while the susceptibility of P. beijingensis resulted from the absence of SA. Further studies are required to confirm this hypothesis. These results provide a foundation for future research on the disease-resistance mechanisms of poplar.

Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.