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.

Enhanced accumulation of pinosylvin stilbenes and related gene expression in Pinus strobus after infection of pine wood nematode

Pine wood nematodes (PWNs: Bursaphelenchus xylophilus) infect pine trees and cause serious pine wilt disease. Eastern white pine (Pinus strobus) has resistance to PWN. However, the detailed defense mechanisms of P. strobus against PWN are not well known. When P. strobus plants were infected with PWNs, the accumulation of stilbenoids, dihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME), were increased remarkably. DPME and PME had the high nematicidal activity. Interestingly, the nematicidal activity of the two compounds was resulted in a developmental stage-dependent manner. PME was more toxic to adult PWNs than juveniles, whereas DPME was found more toxic to juvenile PWNs than the adults. The genes involved in PME and DPME biosynthesis such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), pinosylvin synthase (STS), and pinosylvin O-methyltransferase (PMT) were isolated using de novo sequencing of the transcriptome in P. strobus. In addition, transcription factors (bHLH, MYB and WRKY) related to stilbene biosynthesis were isolated. qPCR analyses of the selected genes (PAL, 4CL, STS, and PMT) including transcription factors (bHLH, MYB and WRKY) revealed that the expression level of the selected genes highly enhanced after PWN infection. Our results suggest that pinosylvin-type stilbenoid biosynthesis is highly responsive to PWN infection and plays an important role in PWN resistance of P. strobus trees.

Аутфітосозологічне оцінювання дендроекзотів відділу Pinophyta ботанічного саду Чернівецького національного університету імені Юрія Федьковича

Здійснено інтегральне аутфітосозологічне оцінювання 41 виду деревних інтродуцентів відділу Pinophyta, які ростуть у ботанічному саду Чернівецького національного університету ім. Юрія Федьковича в умовах відкритого ґрунту. Відповідно до значень вирахуваного для кожного виду аутфітосозологічного індексу досліджувані дендроекзоти віднесено до чотирьох аутфітосозологічних класів (II-V); найбільше видів (27) увійшли до ІІІ класу. Для рослин кожного із аутфітосозологічних класів охарактеризовано ознаки, які найістотніше впливають на фітосозологічну цінність видів: категорія раритетності у Червоному списку МСОП, фітогеографічне та історичне значення, регіональна репрезентативність та кількість штучних природоохоронних об'єктів у межах Чернівецької області, де росте той чи інший вид. Зокрема, найчастіше у дендропарках, парках-пам'ятках садово-паркового мистецтва та у складі пам'яток природи Чернівецької області трапляються Thuja occidentalis L. (30 місцезростань), Pinus strobus L. (22), Platycladus orientalis (L.) Franco (19 місцезростань), і в межах 12-14 природоохоронних об'єктів – Pinus nigra J. F. Arnold, Ginkgo biloba L., Chamaecyparis pisifera (Siebold & Zucc.) Endl. Відзначено, що 51,2 % із досліджуваних видів трапляються, окрім ботанічного саду Чернівецького національного університету, також у дендропарках, парках-пам'ятках садово-паркового мистецтва та пам'ятках природи місцевого значення Чернівецької області. Раритетні види деревних інтродуцентів відділу Pinophyta цінні як резерв генетичного матеріалу. Тому особливої охорони потребують ті з них, які перебувають під загрозою зникнення (категорія раритетності у Червоному списку МСОП – CR, EN та VU), а також ті, що увійшли до ІІ аутфітосозологічного класу (Taxodium distichum (L.) Rich., Sequoiadendron giganteum (Lindl.) J. Buchholz, Metasequoia glyptostroboides Hu & W. C. Cheng, Picea koyamae Shiras.), та частина видів, віднесених до ІІІ класу (зокрема, ті з них, які представлені 1-2 особинами лише у ботанічному саду Чернівецького національного університету, і не здатні розмножуватися насіннєвим шляхом). Під час розроблення рекомендацій з ефективної охорони досліджуваних деревних інтродуцентів варто звертати увагу на екологічні вимоги видів, оскільки від цього значною мірою залежить успішність росту і розвитку інтродуцентів.

White pine and Scots pine tree-ring chronologies as indicators of climate-related non-native and native tree growth patterns in Estonia

Three tree-ring chronologies of white pine (Pinus strobus), a species which are non-native to Europe, were constructed for Suuremõisa, Jädivere and Järvselja sites in Estonia. These chronologies were related to instrumental climate records and Scots pine (P. sylvestris) chronologies from nearby sites. Growth rates of P. strobus exceeded those of P. sylvestris. The chronologies of the non-native and native pine species relatively well correlated with each other. Moreover, tree-ring growth of both species correlated positively with late-winter and spring (February–May) temperatures and negatively with spring (April) precipitation. While P. strobus growth was positively associated with summer precipitation, the growth of P. sylvestris remained positively related to the growing season temperatures. Both species exhibited a negative growth anomaly from 1939 to 1942. Keywords: Pinus strobus, Pinus sylvestris, dendroclimatology, Estonia