Population dynamics of pinewood nematode and the endoparasitic fungus Esteya vermicola: interactions under experimental conditions

Esteya vermicola (Ophiostomataceae), an endoparasitic fungus, exhibits great potential as a biological control agent against pinewood nematodes (PWNs). The present study reports the interaction between PWNs and E. vermicola at different spore concentrations, number of PWNs and the time of culture. The addition of PWNs enhanced the sporulation of E. vermicola after 10 days of culture. The 5-day-old cultures of E. vermicola prior to addition of PWNs increased the highest amount of sporulation than that of 10- or 15-day-old cultures. The PWNs were completely killed by E. vermicola in the pine tree powder culture medium at the concentrations of 10⁷ and 10⁸ colony-forming-units (CFU) per ml. The interaction of the PWNs and E. vermicola was that PWNs provide nutrition to E. vermicola, however, the PWNs can also feed on mycelium of E. vermicola. The effect of E. vermicola on control of PWNs was determined by the population size, time of pest infection and the duration of co-infection.     

Using the nematophagous fungus Esteya vermicola to control the disastrous pine wilt disease

The present study evaluated the protective effects of the nematophagous fungus Esteya vermicola on the large pine trees of Mt. Wora, Jinju, South Korea for six years. When pine trees were treated with E. vermicola 110 days before artificial normal pinewood nematode (PWN) infection, 30–50% of the trees survived for six years. When pine trees were treated with E. vermicola one week after artificial normal PWN infection, 40% of the trees were saved. In contrast, all of the control trees were killed by pine wilt disease in the first year. Although it has been more than six years since the beginning of this experiment, the existence of E. vermicola inside the treated pine trees was successfully detected using a PCR method with two pairs of specific primers for E. vermicola. These results suggest that E. vermicola possesses great potential as a biocontrol agent to combat the disastrous pine wilt disease. This is the first report of using nematophagous fungi to control pine wilt disease in the field for a duration of over five years.     

Laboratory studies on the development of a conidial formulation of Esteya vermicola

The endoparasitic nematophagous fungus, Esteya vermicola, has potential as a biocontrol agent against pinewood nematode, Bursaphelenchus xylophilus. An E. vermicola conidial formulation was developed to improve conidial resistance to ultraviolet (UV), drought and heat stress. The effective concentration of each protective additive [UV protectant [fulvic acid (FA) and skim milk (SM)]; drought protectant (sorbitol) and heat protectant (calcium chloride)] was determined based on the germination rate of E. vermicola conidia after exposure to the different stressors. A combination of 0.2% FA and 4% SM, 5% sorbitol and 0.05% calcium chloride provided the most effective protection. In addition, the concentrations of spreader–sticker and antibiotic were also decided. The final formulation could be used to improve the resistance of E. vermicola conidia to multiple stressors and to increase nematode mortality compared with unformulated conidia.     

Rapid molecular detection of Esteya vermicola based on specific primers and the FTA-DNA extraction method

Esteya vermicola is an endoparasitic fungus of the pinewood nematode and thus has great biocontrol value. At present, the detection of this fungus is still based on microscopic observations and morphological identification, and the sampling is notably inconvenient and inefficient. In the present study, a pair of specific primers (upstream primer, 5′-GTGCCTCTACCAAGACTCGC-3′; downstream primer, 5′-CGCCAAATGTCAAGATCCGC-3′) was designed to detect E. vermicola. The analysis of the PCR amplification and the agarose gel electrophoresis results led to the establishment of a new method for the detection of E. vermicola through the presence of a 176-bp specific fragment. In addition, the use of a FTA-DNA direct extraction method for the detection of E. vermicola was explored. The results suggest that the proposed method can be effectively used for the rapid detection of E. vermicola and may provide important technical support for follow-up studies of the fungus in field experiments.     

季也蒙假丝酵母对松材线虫病的抑制作用

松材线虫病是重要的森林病害,该病与松材线虫携带的病原菌和松树的内生病原菌密切相关。在室内条件下,初步研究了从人工培养的松材线虫上分离到的菌株C对松材线虫病的抑制作用。在健康的水培马尾松枝上分别接种松材线虫接种液、菌株C接种液、松材线虫与菌株C的混合接种液。处理后松枝的相对重量与相对蒸腾强度均为:接种菌株C的松枝>混合接种的松枝>接种线虫的松枝。处理后15d时,接种线虫的松枝与混合接种的松枝的相对重量有显著性差异(p<0.05)。接种线虫松枝的存活期显著短于其它处理松枝的存活期(p<0.05)。接种菌株C的针叶褐变株数少于接种线虫的松枝,两者有显著性差异(p<0.05)。从接种线虫和混合接种的所有松枝中都分离到松材线虫,且分离出的线虫量没有显著性差异。将8个月生的断根马尾松苗插入菌株C的查彼培养液的滤液中培养,6d后松苗的平均感病指数和感病株率均显著少于对照(p<0.05)。这表明,菌株C对松材线虫病有抑制作用,菌株C培养液中产生的某些代谢物质有利于松苗的抗病和存活。菌株C可能抑制了松树上的内生病原菌和松材线虫携带的病原微生物,或提高了松树的生长力和抗逆能力。经电子显微镜观察并参照AP I 20 C AUX鉴定系统鉴定,菌株C为季也蒙假丝酵母C and id a gu ilierm ond ii。     

Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide

Methyl α- and β-D-galactopyranosides and 4-O-β-D-galactopyranosyl-3,6-anhydro-L-galactose dimethylacetal were sulfated with sulfuric acid and dicyclohexylcarbodi-imide as a condensation reagent. The sulfated sugars were isolated by ion-exchange chromatography, characterized, and assigned by methylation analyses. On the basis of the yield of each sulfated product that was isolated, sulfation on O-6 appeared to be predominant.     

Transmission of the pine wood nematode Bursaphelenchus xylophilus through feeding activity of Monochamus galloprovincialis (Col., Cerambycidae)

Abstract:  Transmission of the pinewood nematode (PWN) Bursaphelenchus xylophilus (Steiner & Bührer) Nickle to Pinus pinaster Aiton branches through feeding wounds of its vector in Portugal, Monochamus galloprovincialis Olivier, was studied under laboratory conditions. All the B. xylophilus -infected beetles transmitted nematodes to branches they fed. The transmission was more frequent during the first 6 weeks after emergence, with transmission peaks during the second and the sixth week. The adult M. galloprovincialis transmitted nematodes for a mean of 5 weeks, independently of the beetle's sex or longevity. No relation was found between beetle feeding intensity and effective transmission of B. xylophilus to the branches. The nematode transmission ceased after the ninth week, even in insects which still had B. xylophilus on their bodies. The longevity of the nematode-free insects (control group) was slightly higher than the B. xylophilus -infected beetles, although with no significant difference. The results emphasize the necessity to control the immature stages of M. galloprovincialis prior to emergence and develop efficient strategies to capture and eliminate the recently emerged beetles, as majority of the nematode infection of healthy pine trees occurs during a short period of few weeks after beetle emergence.     

Highly selective microbial transformation of major ginsenoside Rb(1) to gypenoside LXXV by Esteya vermicola CNU120806

Aims

This study examined the biotransformation pathway of ginsenoside Rb₁ by the fungus Esteya vermicola CNU 120806.

Methods and Results

Ginsenosides Rb₁ and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb₁. The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and gypenoside LXXV. A mole yield of 95·4% gypenoside LXXV was obtained by enzymatic conversion (pH 5·0, temperature 50°C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49·6%) proved a consecutive hydrolyses occurred at the C‐3 position of ginsenoside Rb₁.

Conclusions

Strain CNU 120806 showed a high degree of specific β‐glucosidase activity to convert ginsenosides Rb₁ and Rd to gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50°C and pH 5·0. Compared with its activity against pNPG (100%), the β‐glucosidase exhibited quite lower level of activity against other aryl‐glycosides. Enzymatic hydrolysate, gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C‐3 carbon of ginsenoside Rb₁ and Rd, giving the pathway: ginsenoside Rb₁→ gypenoside XVII → gypenoside LXXV; ginsenoside Rd→F₂→Compound K, but did not hydrolyse the 20‐C, β‐(1‐6)‐glucoside of ginsenoside Rb₁ and Rd.

Significance and Impact of the Study

The results showed an important practical application on the preparation of gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.     

皮下接种松墨天牛卵及幼虫致死松树的研究

为了探明松墨天牛MonochamusalternatusHope直接致死松树的作用 ,作者采用在健康松树皮下接入松墨天牛卵或初孵幼虫方法做了试验和观察。结果表明 ,接入卵或初孵幼虫的松树平均枯死率为41 . 7%。松树枯死取决于松墨天牛幼虫取食时对松树韧皮部的破坏程度。每株接入 5个单位 (1单位指1粒卵或条初孵幼虫 ) ,松树枯死率达 85. 7% ;接入 1个单位 ,枯死率为 1 0 %。初步结论是 ,松墨天牛是引起松树枯死的主要原因之一 ;大力防治松墨天牛是保护我国松林的关键。     

Spatial pattern of pine wilt disease caused byBursaphelenchus xylophilus (Nematoda: Aphelenchoididae) within aPinus thunbergii stand

To understand the mechanism of spread of pine wilt disease caused by the pinewood nematode, Bursaphelenchus xylophilus, which is vectored by a cerambycid, Monochamus alternatus, the spatial distribution of trees weakened by the nematode was examined within a Pinus thunbergii stand from June to October for 4 years. The weakened trees were distributed in a clumped pattern in 1980 and 1981, at an early stage of infestation. In many cases, they showed a double-clumped pattern. The degree of aggregation was higher in June or July than after August. They were uniformly distributed in June or July 1982 and in June 1983 whereas they showed a double-clumped pattern after August. The trees were frequently weakened in June or July when they were near the trees weakened during the previous year. At quadrat sizes of more than 25 m², spatial overlapping was pronounced between trees weakened during June–July of the current year and those weakened in the previous year. The seasonal changes in spatial distribution of weakened trees were explained by the interaction among M. alternatus, B. xylophilus and Pinus trees.     

不同干扰措施对松材线虫入侵松林内物种多样性的影响

经野外调查发现:松材线虫入侵松林后,对原先林型相同的两块染病松林而言,经过人为伐除和保留受害木这两种干扰措施后,任其自然恢复更新,后恢复的林型会大不相同,即对受害木采取不同的干扰措施会对染病松林内林下植被的更新产生不同的影响.基于以上出发点,以松材线虫入侵后采取不同伐除干扰措施的2个马尾松受害群落为研究对象,针对受害木移除和保留两种处理方式,选择α多样性指数函数Rényi指数为测量单位,探讨了不同受害木处理方式下植物多样性的变化规律.结果表明:受害木及时移走的松林内林下灌草多样性比受害木保留的要高.     

不同松林类型节肢动物群落组成和相似性

采用G-test、Sorensen Classic相似性指数和Jaccard Classic相似性指数比较浙江省舟山市受松材线虫Bursaphelenchus xylophilus侵染后形成的湿地松Pinus elliottii杉木Cunninghamia lanceolata混交林、马尾松P.massoniana疏林、马尾松纯林和黑松P.thunbergi马尾松混交林等4种代表性的松林类型中的节肢动物群落的组成,同时采用采用Chao 1指数和ACE指数对4种林型的总物种丰富度进行了估计。研究结果表明,4种不同的松林类型共捕获513种共55141个个体,隶属15目110科。4种松林类型中节肢动物群落的科数、物种数和个体数量都有显著差异(科数G3=9.303,P=0.026;物种数G3=57.362,P=0.000;个体数G3=2 767.568,P=0.000)。马尾松疏林和马尾松纯林中节肢动物群落的相似性最高,湿地松杉木混交林和黑松马尾松混交林这2种针叶混交林内节肢动物群落相似性最低。估计全部节肢动物群落的物种丰富度为586.47(ACE指数)和634.8±31.5(Chao 1±SD)种。     

Effects of mineral salts on the growth,sporulation and virulence of Esteya vermicola,an endoparasitic fungus of the pinewood nematode,Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study, various mineral supplements, such as chloride salts (KCl, CaCl₂, MgCl₂, FeCl₂, and FeCl₃) and calcium salts (CaCl₂, CaCO₃, and CaSO₄) were evaluated for their ability to enhance the growth, sporulation and virulence of E. vermicola. Of the cations tested, CaCl₂ provided the greatest enhancement of growth speed and sporulation. Of the anions tested, CaCO₃ produced the highest proportion of lunate conidia, and CaCl₂ produced the highest adhesive rate and mortality against the nematode, Bursaphelenchus xylophilus. The optimum concentration of CaCl₂ for optimization of sporulation and virulence was 0.4–0.6%. In conclusion, CaCl₂ is highly effective in enhancing growth, sporulation and virulence of Esteya vermicola.     

松材线虫及其关键传媒墨天牛的研究进展

松材线虫Bursaphelenchusxylophilus (Steiner&Buhere,1 93 4Nickle,1 981 )是一种世界多国公认的重大外来生物。其原产地在美国 ,但松材线虫并不严重危害该国松林 ;日本受害最重 ,经过几十年的研究与防治 ,现已基本能控制松材线虫病大发生。自 1 982年我国南京中山陵首次发现松材线虫以来 ,其在我国的扩散趋势越来越明显 ,现已成为我国一种重要入侵病原生物。它不但对我国南方数百万公顷松林构成毁灭性威胁 ,影响我国经济和社会可持续发展 ,而且将破坏我国一些著名风景名胜区、文化遗产地。同时 ,松材线虫正成为一项技术壁垒 ,严重影响我国的进出口贸易。该文简要从松材线虫及其关键传煤墨天牛Monochamusspp .的生物学特性、分布状况 ,松材线虫病的蔓延条件及影响因素等方面概述国内外的研究动态 ,以期为我国对松材线虫的研究与防治工作提供参考     

松墨天牛成虫体内的松材线虫分布和速检研究

为了探索快速测定松墨天牛成虫(下称成虫)的带疫性,用解剖方法和自制便携式显微镜,对成虫携带的松材线虫部位和速检(下称线虫)作了研究,得到下列结果：87．9％的线虫分布在成虫的节间、腹腔和生殖器内;雄成虫阳茎是松材线虫集中分布区,两头平均携带线虫498．5条;交尾时雄成虫把阳茎上的线虫传到雌成虫外生殖器,在雌成虫产卵时再把线虫带入松树形成层组织;在野外检测成虫带疫性平均需时1．57min。     

Effects of pine wilt disease invasion on soil properties and Masson pine forest communities in the Three Gorges reservoir region,China

Pine wilt disease (PWD) has caused significant Masson pine mortality in the Three Gorges reservoir region in central China. In this study, five uniform Masson pine stand types infected by PWD were selected and surveyed on slopes and aspects with similar environmental conditions. In sites that had been infected, soil bulk density was reduced, and the difference among the groups was statistically significant (P < 0.05) at the 0–10 cm and 10–20 cm soil layers, but not at 20–40 cm. Other soil water‐related physical properties, excluding noncapillary porosity, significantly differed among the groups in all soil layers. Additionally, the values of available phosphorus, sodium, potassium, calcium, and magnesium were higher in the invaded stands, but the total nitrogen and organic matter contents were lower. Masson pine does not become reestablished following PWD‐induced mortality but is instead replaced by broad‐leaved tree species. Among the 19 examined environmental variables, five were found to be significantly related with the ordination of plant community structure: Masson pine stumps (MPS), K⁺, capillary water holding capacity (CWHC), capillary porosity (CP), and soil water content (SWC). Among these factors, the plant community structure was principally related to MPS and K⁺. The findings of this study show that the outbreak of PWD has impacted Masson pine forest soil properties and altered forest community composition. The disease is negatively related with the presence of Masson pine and positively associated with that of broad‐leaved tree species.     

化学通讯在松材线虫侵染和扩散中的作用

松材线虫为外来入侵种 ,由其引起的松材线虫病正在我国迅速扩散蔓延 ,造成我国部分地区松林资源的毁灭性破坏。松材线虫病的发生和流行与媒介天牛、寄主植物、共生真菌和细菌密切相关 ,松材线虫 -墨天牛 -松树 -共生微生物之间存在着广泛的化学联系 ,它们通过化学互作 ,调控松材线虫的行为 ,影响松材线虫的侵染和扩散     

Feeding and oviposition preferences of Monochamus galloprovincialis for certain conifers under laboratory conditions

Feeding and oviposition preferences of Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae: Monochamini), the vector of the pine wilt nematode (PWN), Bursaphelenchus xylophilus (Steiner and Bührer) Nickle (Nematoda: Aphelenchoididae), in Portugal, was studied in three laboratory experiments: an adult feeding preference trial between branches of five pines, a no‐choice oviposition experiment on seven conifer bolts, and an oviposition choice trial between maritime pine, Pinus pinaster Aiton, and four other pines, P. pinea L., P. sylvestris L., P. halepensis Miller, or P. radiata D. Don. Scots pine (P. sylvestris) was the pine with the largest bark feeding area, while P. radiata was the least chosen to feed upon. Female M. galloprovincialis laid eggs on P. sylvestris, P. halepensis, P. pinaster, P. radiata, P. pinea, and Pseudotsuga menziesii (Mirbel) Franco, but larvae successfully completed development only on the first four pines. Beetles emerging from maritime pine bolts were slightly bigger than the others. No preference for oviposition was detected when beetles were given the choice between P. pinaster and P. sylvestris or P. halepensis. Inversely, greater oviposition occurred on P. pinaster when compared with P. pinea and P. radiata. Although PWN currently affects only P. pinaster in Portugal, the results on host suitability for M. galloprovincialis suggest that P. sylvestris and P. halepensis are adequate hosts for the insect's feeding and oviposition, and thus may also be PWN hosts in Europe. The systematically high feeding and oviposition attraction for P. sylvestris exhibited by the beetles means that the eventual spread of the PWN to areas where both M. galloprovincialis and P. sylvestris occur may have a significant impact on this widespread and economically important pine, considered to be highly susceptible to B. xylophilus.     

The bimodal adult activity of Monochamus alternatus (Coleoptera: Cerambycidae) caught in pheromone traps in Jeju can be explained by the competitive attractiveness of dying pine trees

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is known to be the primary vector of pinewood nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle that causes pine wilt disease. Adult activity of M. alternatus caught in a pheromone trap on Jeju Island of Korea showed a bimodal form with the first peak in mid to late June and the second peak in mid to late September. The two peaks were separated between mid and late August, showing a valley. Accumulated degree‐days predicted that the emergence of the second generation adults could be possible just before the second peak. But actually no adults of the second generation occurred in the field development experiments of M. alternatus in 2016 and 2017. Pine trees without oleoresin flow (namely dying trees by the infection of pinewood nematode) were abundant during early July to early August. The bimodal adult activity pattern of M. alternatus could be partially explained by the competitive attractiveness of dying trees against pheromone traps, when we accepted the assumption that dying pine trees attract strongly M. alternatus.