Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Enhanced Production of Pinosylvin Stilbene with Aging of Pinus strobus Callus and Nematicidal Activity of Callus Extracts against Pinewood Nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 hr. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Factors Affecting the Ratio of Female to Male of Pinewood Nematodes Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle

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.

Monochamus Saltuarius Endangers Pinus tabuliformis Carr. and Carries Bursaphelenchus xylophilus (Steiner and Buhrer) in China

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.

Investigation of Pinewood Nematodes in Pinus tabuliformis Carr. under Low-Temperature Conditions in Fushun, China

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.