Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

Effect of an ectomycorrhizal fungus,Laccaria laccata,on Fusarium damping-off in Pinus banksiana seedlings

Damping-off caused by Fusarium oxysporum Schlecht in Pinus banksiana Lamb, was reduced significantly when inoculated with an ectomycorrhizal fungus, Laccaria laccata (Scop. ex Fr.) Berk. and Br. In paired culture, growth of F. oxysporum was significantly reduced by L. laccata. The number of colony forming units of F. oxysporum was reduced significantly in the rhizosphere of P. banksiana seedlings when inoculated with L. laccata. Spore germination and germ tube length of F. oxysporum was inhibited strongly by culture filtrate of L. laccata and root exudate of P. banksiana inoculated with L. laccata. Mycorrhizal seedlings had significantly higher amount of total soluble phenols than nonmycorrhizal ones.     

松材线虫Bxpel2基因的克隆及RNA干扰载体构建

[目的]为了构建松材线虫(Bursaphelenchus xylophilus)果胶酶Bxpel2基因干扰载体.[方法]通过Trizol 法提取松材线虫总RNA,反转录合成cDNA,设计带T₇启动子的果胶酶Bxpel2基因引物,以cDNA为模板扩增出果胶酶Bxpel2基因片段,连接到RNA干扰载体,再以干扰载体为模板,PCR扩增出目的片段后进行测序鉴定,合成果胶酶Bxpel2基因双链RNA(dsRNA),采用RT-PCR检测松材线虫Bxpel2基因干扰后的表达情况.[结果]表明:1)提取的松材线虫总RNA完整性好,无降解;2)成功克隆出松材线虫果胶酶Bxpel2基因片段(790 bp)并将其连接至pMD19-T载体;3)以RNA干扰载体为模板合成dsRNA,浓度分别为1.313 mg·mL^-1和1.152 mg·mL^-1;4)RT-PCR结果显示,松材线虫经过dsRNA干扰后,Bxpel2基因表达基本受到抑制.[结论]成功构建松材线虫果胶酶Bxpel2基因干扰载体,为进一步研究Bxpel2基因在松材线虫致病过程中的作用和功能奠定了良好的基础.     

Efficacy of spinosad and methoprene,applied alone or in combination,against six stored-product insect species

Efficacy of the insecticides spinosad and methoprene, applied alone or in combination to wheat, was evaluated against six stored-product insect species, Rhyzopertha dominica, Sitophilus oryzae, S. granarius, Cryptolestes ferrugineus, Oryzaephilus surinamensis, and Liposcelis bostrychophila. The concentrations of the insecticides were 0.1 and 0.5 ppm for spinosad and 1 and 5 ppm for methoprene. Parental mortality of R. dominica was 100% on wheat treated with either rate of spinosad, with no progeny production in any combination treatment. Parental mortality of S. oryzae did not exceed 62%, and progeny production was not reduced by any insecticide treatment. All S. granarius adults exposed on wheat treated with spinosad alone at 0.5 ppm were killed, but parental mortality was reduced when methoprene was added. Progeny production was reduced in treatments containing spinosad. Mortality of adult C. ferrugineus was 97% or greater in treatments containing spinosad, but adult mortality of O. surinamensis and L. bostrychophila was not reduced by insecticide treatments. The specific combinations of spinosad and methoprene evaluated in our study would have no benefit over spinosad used alone for control of any of the six species tested.     

大花蕙兰转齿兰环斑病毒外壳蛋白基因及检测

[目的]通过植物转基因技术获得抗病毒大花蕙兰种质资源,优化转化体系和鉴定方法.[方法]本研究克隆了齿兰环斑病毒外壳蛋白基因,并构建了该基因的pBI121表达载体,用根癌农杆菌介导法转化大花蕙兰,尝试以巢式PCR方法检测转基因再生植株.[结果]优化了大花蕙兰遗传转化体系,建立了利用巢式PCR技术检测转基因大花蕙兰植株的方法,获得了32株转基因株(系).[结论]优化了以类原球茎为外植体的农杆菌介导转化大花蕙兰的方法,确定以5%~10%类原球茎存活时的抗生素(卡那霉素)浓度为筛选浓度;获得了转ORSV CP基因大花蕙兰植株;对大花蕙兰转基因植株检测时,巢式PCR较普通PCR更灵敏、准确.     

Interaction between Epicoccum purpurascens and xylophagous basidiomycetes on wood blocks

Epicoccum purpurascens, a component of the bark micromycota, was evaluated as a potential biocontrol agent for three decay‐causing hymenomycetes frequently found in Buenos Aires city trees, Ganoderma platense, Inonotus rickii and Rigidoporus ulmarius. Treatments consisted of dual cultures of E. purpurascens and xylophagous fungi on box elder (Acer negundo) wood blocks. Two inoculation sequences were tested: (i) the biocontrol agent as the first microorganism inoculated followed by the pathogen; and (ii) the xylophagous fungus as the first microorganism inoculated followed by the biocontrol agent. After 3 months of incubation, percentage dry weight loss was calculated. When E. purpurascens was allowed to colonize wood blocks before the introduction of the xylophagous fungus, dry weight loss was significantly reduced in all instances. However, as a secondary colonizer, E. purpurascens mildly reduced the weight loss caused by G. platense, had no effect on degradation by I. rickii and increased wood weight loss by R. ulmarius.     

Production of hybrid seed in a seed orchard of two larch species,Larix sibirica and Larix decidua

The larch seed orchard studied is intended to produce interspecific hybrid seed between Larix sibirica and Larix decidua. Only the L. sibirica clone (half of the grafts) in the orchard was intended to act as the seed parent, and the six L. decidua clones are pollinators. The proportion of hybrid seed produced was determined using Mdh3 locus as a genetic marker. The mean percentage of hybrid seed produced by the grafts of L. decidua was 93, but that of the hybrid seed produced by L. sibirica was only 18. The results can be explained by differences in flowering abundance. The grafts of L. sibirica produced several times more pollen than those of L. decidua. Contrary to the original plans, L. decidua should be used as the seed parent and L. sibirica only as the pollen parent in the studied seed orchard.     

Screening method for wood extractives: direct cellulose thin-layer chromatography plate

A method for screening wood extractives was developed using cellulose thin-layer chromatography plate (Cell-TLC) separation and was directly applied to bioassays. Wood meal (60 mesh) from nangka (Artocarpus heterophyllus Lamk) heartwood was extracted with hot methanol. The crude extract was separated using a Cell-TLC plate (50 × 50mm). Two broad bands with R_f values of 0.46 and 0.96 were found, and the bands showed completely different effects against the pest termite Coptotermes formosanus Shiraki and the decay fungus Fomitopsis palustris. The band with R_f 0.46 was preferentially consumed by workers of C. formosanus, and it did not show any growth inhibition against F. palustris when the Cell-TLC plate was directly exposed to the organism. In contrast, the band with R_f 0.96 appeared to repel strongly the feeding by C. formosanus and inhibited the growth of F. palustris. It was concluded that the Cell-TLC system was applicable for screening wood extracts consisting of many compounds.     

Effect of laminate configuration on the modulus of elasticity of glulam evaluated using a strain gauge method

The purpose of this study was to measure the strain of glulam laminae by affixing a strain gauge at the central axis of the lateral face in order to determine the effect of the configuration of the glulam. Japanese cedar and southern pine were used in the study. The strain and stress of the laminae during the bending test were recorded, and the modulus of elasticity (MOE) was calculated. The influence of the MOE of the adhesive layers was also considered. Results showed that the MOE of the laminated elements increased as its specific gravity increased, although the specific gravity was not the only factor evaluated. The MOE of glulam (E _bsg) measured by the strain gauge method was very close to the observed MOE of glulam (E _b) and a significant linear relationship was identified. The MOE (e) of laminae measured by the strain gauge method was very close to that determined (E) under the “free condition” before bonding. The difference between the modified MOE (E _bsa) and E _bsg was not significant. The MOE of glulams made of heterogeneously graded lumbers were about 23% and 31% larger than the average MOE of their laminae for Japanese cedar and southern pine, respectively. The actual neutral axis was just below the longitudinal center line. It shifted slightly within the proportional limit range and moved downward when the load increased.     

Effects of surface and light conditions of fallen logs on the emergence and survival of coniferous seedlings and saplings

We surveyed the germination number (N _cs) of 2-year and a 1-year survival of Abies sachalinensis and Picea jezoensis seedlings and saplings on 29 fallen logs from 2004 to 2005 in a natural coniferous forest in Hokkaido, northern Japan, in relation to the surface and light conditions of fallen logs. Moss height (H _moss), log hardness (Hardness), and the area of fallen log (Area) were measured as the surface conditions by each 1-m block from bottom to top of all fallen logs. The relative photosynthetic photon flux density (rPPFD) 10 cm above the tallest seedling in each block was measured as the light condition. In addition, the height of the tallest seedling or sapling in each block (H _max), the difference between a height of each seedling and sapling and the H _max (Shading), and a height of seedlings and saplings in 2004 (H _ini) were considered. N _cs of A. sachalinensis was affected by Hardness and Area, whereas N _cs of P. jezoensis was affected by H _moss, Hardness, Area, H _max, and rPPFD. The survival of seedlings (height < 5 cm) and saplings (5 cm ≤ height < 50 cm) were affected by H _ini, rPPFD, and shading for both species. However, the survival of P. jezoensis saplings was more sensitive to decrease in rPPFD and increase of shading than that of A. sachalinensis. Therefore, seedling emergence was influenced by surface conditions, whereas survival was affected by light conditions. Furthermore, P. jezoensis emergence and survival were more sensitive to surface and light conditions than that of A. sachalinensis.     

Fungal decay of dead elms

The pattern of decay developing in Ulmns procera killed by Ceratocystis ulmi was examined. Flammulina velutipes had caused some decay and discolouration in most trees which had been dead for more than two years. Decay caused by Pleurotus sapidus was less common. Pleurotus palmatus was frequently isolated from wood in the upper trunk showing no visible deterioration. Trees should be felled within two years of death in order to retain the value of their timber.     

平榛ChWRKY28基因克隆及表达模式分析

[目的]研究平榛ChWRKY28基因序列特征及其在不同非生物胁迫下的表达规律.[方法]以平榛为试材,采用RACE-PCR方法进行基因克隆;利用实时荧光定量PCR方法检测基因在不同组织及不同非生物胁迫下的表达模式.[结果]表明:克隆得到的WRKY基因,全长1 342 bp,基因内部包含1个长963 bp的完整开放阅读框,编码320个氨基酸残基,命名为ChWRKY28.构建的系统发育树表明:该序列与拟南芥AtWRKY28及杨树PtrWRKY93的关系最近,相似性分别为49%和60%.基因表达分析表明:ChWRKY28在雄花序、雌花芽及茎中均有表达,但在茎部(皮)中的表达量高于雄花序和雌花芽中的表达量,具有组织表达特异性;低温、干旱及盐胁迫均能诱导ChWRKY28基因的表达,但受诱导程度存在差异.亚细胞定位分析结果表明:ChWRKY28蛋白分布在细胞核内,是一个核蛋白.[结论]推测ChWRKY28基因可能参与植物响应非生物胁迫的信号转导过程.     

麻疯树BRI1基因的鉴定及其在不同发育时期花蕾中的表达分析

[目的]研究油菜素内酯受体BRI1的同源基因(JcBRI1)在麻疯树花发育过程中的作用。[方法]利用RT-PCR技术克隆JcBRI1基因的CDS,以pET-30a(+)质粒为框架构建原核表达载体转化至大肠杆菌进行诱导表达,随后利用LC-MS/MS对表达产物进行质谱鉴定,并以氨基酸序列为基础分析JcBRI1蛋白质结构。利用荧光定量PCR分析麻疯树JcBRI1基因在雌雄花发育的关键时期的表达水平,初步确定其在麻疯树花发育过程中的表达模式。[结果]克隆获得了JcBRI1基因的CDS,长度为3 591 bp。SDS-PAGE检测结果表明:JcBRI1基因在大肠杆菌BL21(DE3)和Rosetta(DE3)中均能表达,但在低温环境的表达量更高。原核表达产物的LC-MS/MS鉴定结果表明:JcBRI1氨基酸序列与预期的一致,JcBRI1基因的表达产物为麻疯树BRI1蛋白。对JcBRI1在麻疯树花器官不同发育时期的表达水平进行检测分析,结果表明:JcBRI1基因的表达量在雌花发育的第一个时期即大孢子母细胞时期就达到了最高值,在随后几个时期持续下调;然而,其在雄花各个时期的表达量并无明显差异,表明其很有可能参与了麻疯树雌花大孢子母细胞发育过程。[结论]麻疯树JcBRI1基因为LRR-RLKs家族成员BRI1的同源基因,很有可能参与麻疯树雌花大孢子母细胞的发育过程,至于JcBRI1在麻疯树大孢子母细胞发育过程中的具体作用机制还需进一步研究。     

基于cpDNA rps16序列分析兰考泡桐与白花泡桐和毛泡桐的遗传关系

【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。     

Fungi associated with Cyclaneusma needle cast in Scots pine in the west of Poland

Needles of Pinus sylvestris with and without symptoms of Cyclaneusma needle cast, from the west of Poland, were examined for abundance and diversity of fungi using Illumina sequencing. Fungal communities were dominated by Ascomycota (93.6%–98.6% of OTUs). Basidiomycota and non‐culturable fungi were less frequent. Needles were colonized by 260 taxa. Ascomycota, Basidiomycota and Glomeromycota were represented by 149, 39 and 1 taxa. Abundance of fungi was least in 1.5‐year‐old needles attached to twigs and greatest in fallen 2‐year‐old needles. Fungal communities had least diversity in 1.5‐year‐old needles and most diversity in 0.5‐year‐old needles of current growth. It was found that (a) the most common fungi were the needle pathogens Cyclaneusma minus, Lophodermium spp. and Sydowia polyspora; (b) less common potential pathogens were Cenangium ferruginosum, Coniothyrium complex, Desmazierella acicola, Neocatenulostroma germanicum and species in the genera Neodidymelliopsis, Pestalotiopsis, Phoma, Pleurophoma and Pyrenochaeta; (c) common primary or secondary saprotrophs included species of Alternaria, Aureobasidium, Beauveria, Cladophialophora, Cladosporium, Epicoccum, Exophiala, Lecanicillium, Penicillium, Cryptococcus and Kwoniella; (d) Lophodermium was represented mostly by Lophodermium pinastri which occurred 4–72 times more frequently than Lophodermium seditiosum; (e) frequencies of C. minus and C. ferruginosum were lower in the 0.5‐year‐old symptomless needles, increased in the symptomatic and symptomless 1.5‐year‐old needles and decreased after needle fall; (f) frequency of L. seditiosum was highest in 0.5‐year‐old needles; (g) frequency of L. pinastri increased with needle age whereas S. polyspora increased after needle fall; (h) lower frequency of L. pinastrii was associated with higher frequency of S. polyspora. It was concluded that Cyclaneusa needle cast in Poland may be caused by C. minus accompanied by C. ferruginosum, L. seditiosum, L. pinastrii and S. polyspora. Participation of Coniothyrium spp., Epicoccum nigrum, Pestalotiopsis spp. and Phoma spp. in the disease progress cannot be excluded.     

Biocontrol of Cytospora canker of poplar in north‐east China with Trichoderma longibrachiatum

Trichoderma isolate T05, collected and isolated from a poplar (Populus simonii × Populus nigra) stand in the province of Heilongjiang, China, was tested for its efficacy in controlling Cytospora chrysosperma in vitro and Cytospora canker of poplar in the field. Trichoderma isolate T05 was identified as Trichoderma longibrachiatum in sect. Longibrachiatum based on both morphological characteristics and phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) oligonucleotide sequences and the short intron of tef1. During dual culturing, the inhibitory effect of T. longibrachiatum T05 on C. chrysosperma increased over time, and inhibition up to 81% was observed 120 h after inoculation. Hyphae of T. longibrachiatum T05 grew alongside, coiled and sporulated around the pathogen hyphae, which were generally observed to be wrinkled, collapsed, disrupted and degraded. Culture filtrates of T. longibrachiatum T05 that were sterilized by either bacterial filtration or autoclaving suppressed the growth of the pathogen colony. Filtrate sterilized by filtration showed greater inhibitory activity than the autoclaved filtrate, which still had some inhibitory activity, demonstrating that some inhibitory substances were heat unstable and some heat stable. Cytospora chrysosperma C01 colony growth was also reduced by volatile antibiotics produced by T. longibrachiatum T05, with inhibition of 54% observed after 96 h. In a field trial, Cytospora canker on poplar trunks was controlled with efficacy similar to that of routine chemical control by applying a T. longibrachiatum preparation.     

Transfer of the P-type of Heterobasidion annosum from old-growth stumps of Picea abies to Picea abies and Larix×eurolepis

The infection of Picea abies and Larix x eurolepis by Heterobasidion annosum was studied in felled trees in Sweden. Thinnings were carried out in two stands of L. x eurolepis, 15 and 20 years old, and in a 25-year-old stand of P. abies, established on a site heavily infected by H. annosum. The 15- and 20-year-old L. x eurolepis stands exhibited the greatest incidence of butt rot with infections amounting to 38 and 57%, respectively, of removed trees. The incidence of butt rot in the P. abies stand was only 5%. Heterobasidion annosum was the main butt rot causing fungus. All isolates of H. annosum were of the P-intersterility group. Transfer of H. annosum from old-growth P. abies stumps to trees felled in the thinning of the next generation was demonstrated by somatic incompatibility tests. However, the majority of infections could not be connected with decay already established in the previous generation. In L. c eurolepis, rot columns were frequent, including also several genotypes in the same stem, and typically positioned in the inner sapwood. The few infections of P. abies were situated in the heartwood.     

Effect of extractions on dynamic mechanical properties of white mulberry (Morus alba)

Vibrational properties of wood are affected by several parameters, of which extractives can be one of the most important ones. Wood for European musical instruments has been often studied, but traditional Middle Eastern ones had been left unnoticed. In this study white mulberry (Morus alba L.), the main material for long-necked lutes in Iran, was extracted by five solvents of various polarities (water included). Free-free bar forced vibrations were used to measure longitudinal (L) loss tangent (tanδ), storage (elastic) modulus (E′) and specific modulus (E′/γ) in the acoustic range. Their anisotropy between the 3 axes of orthotropy was determined by dynamic mechanical analysis. Native wood had a quite low E _L′/γ but its tanδ was smaller than expected, and the anisotropy of tanδ and E′/γ was very low. Removal of extractives caused tanδ to increase and moduli to decrease. Acetone, the most effective solvent on damping despite a moderate extraction yield, increased tanδ _L by at least 20% but did not modify E′/γ as much. When used successively, its effects masked those of solvents used afterwards. Anisotropy of E′/γ was nearly unchanged after extraction in methanol or hot water, while tanδ was much more increased in R than in T direction. Results suggest that in white mulberry, damping is governed more by nature and localization of extractives rather than by their crud abundance.     

Effect of fertilization on growth and ectomycorrhizal development of container-grown and bare-root nursery conifer seedlings

The effect of three levels of fertilizer on thegrowth of three species of containerized-grownconifer seedlings (Pinus contorta, Picea glauca, and Picea mariana) and twospecies of bare-root conifer seedlings (Pinus sylvestris and Larix sibirica),and on the colonization of these seedlings bysix species of ectomycorrhizal fungi (Hebeloma longicaudum, Laccaria bicolor,Paxillus involutus, Pisolithustinctorius, Rhizopogon vinicolor andSuillus tomentosus), was studied. Thegrowth of the seedlings in both container-grownand bare-root nurseries increased as the levelsof fertilizer increased. For better seedlinggrowth and environmental quality it may be possible to reduce the level of fertilizers in commercial nurseries upto 33% by using selected mycorrhizal fungi.Ectomycorrhizal colonization in all seedlingswas not affected by fertilizer levels. Hebeloma longicaudum, L. bicolor, P.involutus, and P. tinctorius formedwell-developed ectomycorrhizae, whereasectomycorrhizal development by R.vinicolor and S. tomentosus was poor.Native mycorrhizal fungi colonizednon-inoculated control seedlings; however,their colonization was always lower than withinoculated fungi.