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.

Promoting Effect of Choline-Phosphate Cytidylyltransferase Gene (pcyt-1) on Departure of Pinewood Nematode from Monochamus alternatus

In order to study the key gene in internal causes of pinewood nematode (PWN), Bursaphelenchus xylophilus, a departure from its vector beetle, Monochamus alternatus, we collected PWNs extracted from newly emerged M. alternatus and beetles 7 days after emergence. The total RNAs of the two groups of PWNs were extracted, transcriptomes sequencing was performed, and gene expression differences between the two groups of PWN were analyzed. It was found that the expression of the choline-phosphate cytidylyltransferase gene (pcyt-1) was markedly up-regulated. After inhibition of pcyt-1 expression by RNA interference, the rate of lipid degradation in PWN decreased significantly, and the motility of PWN also decreased significantly. The analysis identified that phosphatidylcholine could promote the emulsification and degradation of neutral lipid granules in PWN, which provides sufficient energy for PWN departure from M. alternatus. The up-regulation of the gene pcyt-1 is an important internal factor for PWN departure from its vector.

The Potential of Esteya spp. for the Biocontrol of the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD) and a quarantine organism in many countries. Managing PWD involves strict regulations and heavy contingency plans, and present climate change scenarios predict a spread of the disease. The urgent need for sustainable management strategies has led to an increasing interest in promising biocontrol agents capable of suppressing the PWN, like endoparasitic nematophagous fungi of the Esteya genus. Here, we review different aspects of the biology and ecology of these nematophagous fungi and provide future prospects.

Effect of storage conditions on the shelf-life extension of fungus-colonized substrates based on Metarhizium anisopliae using modified atmosphere packaging

Metarhizium anisopliae is a promising alternative to chemical pesticides against pine wilt disease caused by Bursaphelenchus xylophilus. Herein, we investigated the efficacy of modified atmosphere packaging (MAP) to prolong the shelf-life of the M. anisopliae conidia. The effects of various conditions on its stability were also examined. M. anisopliae-inoculated millet grains were treated in a MAP system with different packaging materials (polypropylene, PP; polyethylene terephthalate, PET; ethylene vinyl alcohol, EVOH), gas compositions (high CO2 atmosphere, ≈ 90%; high O2 atmosphere, > 95%; high N2 atmosphere, > 95%; 30% CO2 + 70% N2; 50% CO2 + 50% N2; 70% CO2 + 30% N2), and storage temperatures (4 and 25 °C). Results revealed EVOH film as the best for the preservation of gases at all concentrations for 28 days. MAP treatment in the high-barrier EVOH film under an atmosphere of 30% CO2 + 70% N2 achieved 80.5% viability of dried conidia (7.4% moisture content), with 44.2–64.9% viability recorded with the other treatments. Cold storage for technical concentrates formulation promoted extension of shelf-life of MAP-treated conidia. These results imply that MAP under optimized conditions could enhance the shelf-life of fungus-based biopesticides in fungus-colonized substrates formulations.

Surveillance of pine wilt disease by high resolution satellite

Pine wilt disease caused by the pinewood nematode Bursaphelenchus xylophilus has led to the death of a large number of pine trees in China. This destructive disease has the characteristics of bring wide-spread, fast onset, and long incubation time. Most importantly, in China, the fatality rate in pines is as high as 100%. The key to reducing this mortality is how to quickly find the infected trees. We proposed a method of automatically identifying infected trees by a convolution neural network and bounding box tool. This method rapidly locates the infected area by classifying and recognizing remote sensing images obtained by high resolution earth observation Satellite. The recognition accuracy of the test data set was 99.4%, and the remote sensing image combined with convolution neural network algorithm can identify and determine the distribution of the infected trees. It can provide strong technical support for the prevention and control of pine wilt disease.

Phytonematodes as a limiting biotic factor of agricultural production in the world (review)

Purpose. To analyze domestic and foreign scientific literature on the species composition and harmfulness of the world's most dangerous parasitic species of phytonematodes in crops. Results. Today, according to the available literature, the most dangerous species of phytonematodes include: gall nematode (Meloidogyne spp.), cyst-forming nematode (Heterodera spp. and Globodera spp.), root lesion nematode (Pratylenchus spp.), banana drill nematode (Radoholus similis), stem nematode (Ditylenchus dipsaci), pine stem nematode (Bursaphelenchus xylophilus), reniform nematode (Rotylenchulus reniformis), xiphinema index (Xiphinema index), false head nematode (Nacobbus aberrans), and rice leaf nematode (Aphelenchoides besseyi). Conclusions. The results of research on the prevalence and harmfulness of parasitic nematode species in crops convince us of the need for a more detailed study of this group of microorganisms. Due to the rapid development of molecular genetic methods in the last decade, scientists have been able to expand and improve their knowledge of identifying species, races and pathotypes of phytonematodes, their biological and environmental characteristics, and most importantly, to discover and understand extremely complex mechanisms of parasite and host plants. Nematologists are confident that further research in these and other areas in the future will create a basis for developing a new strategy for long-term and environmentally safe control of these dangerous plant parasites.