Stimulation of Armillaria mellea by phenolic fungicides

Phenolic fungicides, which were initially fungicidal to mycelium of Armillaria mellea on the surface of well‐colonised wood billets, eventually stimulated the growth of A. mellea. An extensive growth of rhizomorphs was produced from A. mellea inoculum, which had been exposed to phenolic chemicals for 3 months, compared to few or no rhizomorphs produced from inoculum exposed to water or a suspension of a non‐phenolic fungicide, fenpropidin. Inoculated privet plants grown either in pots or under field conditions were treated with a range of fungicides; fenpropidin, phenyl phenol, cresylic acid or water (control) was applied every 6 months over 21/2 yr. Fenpropidin caused a slightly (but significantly) lower incidence of infection than occurred in untreated plants, but the phenolic fungicides, cresylic acid and phenyl phenol, did not reduce the incidence of infection. The severity of infection (% root circumference colonised at 5 cm depth) was greater following cresylic acid treatments than the other fungicides or water‐treated controls. Use of phenolic fungicides such as cresylic acid for the control of A. mellea may therefore be counter‐productive.     

法国蜜环菌Armillaria gallica菌株遗传多样性的ISSR分析

在中国东北地区共采集到53个法国蜜环菌Armillaria gallica菌株,用ISSR（Inter-Simple Sequence Repeat）标记技术对这些菌株进行遗传多样性分析。用6个ISSR引物扩增所得条带表明,ISSR标记在蜜环菌中存在较高的多态性;亲缘关系树状图表明,有3个菌株遗传分化明显;其余50个分别来自3个不同地理居群的菌株聚成一类,亲缘关系较近,没有表现出地理隔离。     

Specificity and inhibition studies of Armillaria mellea protease.

The action of Armillaria mellea protease has been evaluated on a number of polypeptide substrates. It has been shown to split the Pro7-Lys8 bonds in both native and oxidised lysine-vasopressin and the Ser11-Lys12 bond in glucagon. No other splits were detected in these substrates. The enzyme also caused extensive degradation of S-carboxymethyl lysozyme, S-carcoxymethyl pepsinogen and oxidised ribonuclease. A. In each case the only new amino-terminal residue to appear was lysine. A. mellea protease was inhibited by the chelating agents 1,10-phenanthroline, alpha, alpha'-bipyridine and imidazole. The pK1 values (negative log10 of concentration required for 50% inhibition) for these three inhibitors were 3.9, 3.4 and 1.1, respectively. Lysine, S-2-aminoethylcysteine and short chain aliphatic amines also proved to be relatively good inhibitors of A. mellea protease while arginine was a poor inhibitor.     

A genetic mosaic in the fruiting stage of Armillaria gallica

The basidiome stage of Armillaria gallica can be a genetic mosaic. Ten cells isolated from a single basidiome in 1986 produced nine different genotypes when analyzed for variation at six nuclear loci. Four additional basidiomes collected in 1986 produced mosaic patterns when analyzed for variation at a single nuclear (PCR-RFLP) locus. One basidiome collected in 1993 was not a genetic mosaic because 15 cells isolated from it produced only one genotype when analyzed for six nuclear loci. Two hundred seventy-four samples collected in the field between 1981 and 1998 were analyzed for variation at the PCR-RFLP locus. Samples collected prior to 1988 produced patterns consistent with the existence of mosaicism, but samples collected after 1988 showed no evidence of mosaicism. Genetic mosaicism represents a novel mechanism for partitioning genotypes among the cells of a basidiomycete and has interesting implications for the biology of A. gallica.     

Cleavage by trypsin and by the proteinase from Armillaria mellea at epsilon-N-formyl-lysine residues.

Kinetic studies were made of the hydrolysis by trypsin of alpha-N-acetylglycyl-L-lysine methyl ester and of its neutral analogue alpha-N-acetylglycyl-epsilon-N-formyl-L-lysine methyl ester. The latter substance is a moderately good substrate for trypsin, and this observation is discussed in terms of the substrate specifically of the enzyme. The actions of trypsin and of the lysine-specific proteinase from Armillaria mellea on both a native and a formylated polypeptide substrate were compared. Both enzymes were found to hydrolyse specifically bonds to epsilon-N-formyl-lysine in the formylated substrate.     

Degradation of Raspberry Suberin by Fusarium solani f. sp. Pisi and Armillaria mellea

When submers cultures of Fusarium solani f. sp. pisi and Armillaria mellea were grown in a medium supplemented with 0.5 % suberin isolated from raspberry periderm, hydrolytic enzymes were produced and measured by a spectrophotometric assay using p-nitrophenyl butyrate as substrate. The enzymatic activity in the culture fluids reached its peak after 32 to 44 days of incubation. In a gas-chromatographic assay of the enzymatic degradation of suberin, concentrated culture fluids of suberin-grown fungi were incubated with raspberry suberin. The culture fluids of F. solani and A. mellea catalyzed the release of chloroform-soluble products, which were analyzed by gas-liquid chromatography. Suberin monomers like fatty alcohols and acids with chain-lengths from C₁₆ to C₂₆ as well as C₁₆ and C₁₈ω-hy-droxyacids could be identified as products. The suberin-induced enzymes showed catalytic properties similar to cutin-hydrolyzing enzymes previously isolated from different fungi.     

假蜜环菌黄曲霉毒素氧化酶的基因克隆、表达、纯化及酶学性质分析

【目的】黄曲霉毒素氧化酶(aflatoxin-oxidase,AFO)来源于假蜜环菌(Armillariella tabescens)的细胞内提取物,具有转化黄曲霉毒素B1(Aflatoxin B1,AFB1)的特性。为更进一步了解该酶的性质,我们克隆了AFO的基因,并进行了重组AFO蛋白的表达、纯化和酶学性质分析。【方法】本研究利用基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)获得的AFO短肽序列设计简并引物进行逆转录,再通过cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术获得了AFO基因的全长cDNA序列。构建重组表达载体pPIC9-afo,在毕赤酵母中进行重组AFO(rAFO)的融合分泌表达,用Ni离子螯合层析进行rAFO的纯化,获得有活性的rAFO后,对其进行肽质量指纹(peptide mass fingerprinting,PMF)鉴定和酶学性质分析。【结果】黄曲霉毒素氧化酶(AFO)基因的开放阅读框为2088 bp,编码695个氨基酸;肽质量指纹鉴定结果显示重组AFO的肽片段序列覆盖率为63.2%。活性测定表明纯化后的重组AFO(rAFO)比活力为234 U/mg;对rAFO进行酶学性质分析表明,对于底物黄曲霉毒素B1,rAFO的Km值为3.93±0.20×10-6 mol/L;反应最适温度为30℃,最适pH为6.0;30℃放置90 min后酶活力下降50%;rAFO在pH5.5-7.0之间酶活力较稳定,相对活力维持在51%-65%之间。【结论】本文第一次成功克隆并重组表达了一种具有黄曲霉毒素B1转化功能的酶——黄曲霉毒素氧化酶(aflatoxin-oxidase,AFO),纯化后的重组AFO(rAFO)具有较好的黄曲霉毒素B1转化活性,为进一步研究和应用奠定了基础。     

中国与北美大陆的高卢蜜环菌系统发育分析

在中国和北美大陆分别收集53和15株高卢蜜环菌Armillaria gallica菌株,并用ISSR（inter-simple sequence repeat）分子标记对这些菌株进行了亲缘关系及系统发育分析。结果表明：中国和北美大陆的A. gallica因地理隔离产生明显的遗传分异,在系统发育树上分别形成了各自的进化分支;北美大陆的分支内部遗传分化尚不明显,而中国的进化分支遗传分化程度相对较大且更为古老,中国菌株可能是两个大陆A. gallica的祖先株系。     

Structures of melleolides B-D,three antibacterial sesquiterpenoids from Armillaria mellea

The structures of melleolides B-D, three new protoilludene sesquiterpenoid O-methylorsellinates isolated from a culture of Armillaria mellea, have been elucidated on the basis of chemical and spectral data.     

Infection Foci of Armillaria mellea in First-rotation Hardwoods

Infection foci of Armillaria mellea, often 10–20 m indiameter, were discovered in first-rotation plantings of oakand beech on former arable or heathland sites—they werecommonly associated with thinning stumps containing the fungus.Since the foci were scattered and contained different mycelialtypes of A. mellea it seemed probable that they had arisen asa result of stump infection by basidiospores. There was evidencethat many years had elapsed between stump infection and theproduction of rhizomorphs, and that subsequent growth of thesedepended upon the presence of suitable living roots. Largeroaks seemed little affected whereas many small and medium-sizedtrees had been killed. Armillaria mellea, infection foci in plantations, tree stumps, basidiospores     

Construction of cDNA libraries from cultures of Armillaria mellea

• 1.1. Conditions were established for growth of mycelial cultures of Armillaria mellea such that the production of its lysine-specific proteinase was maximized. Proteinase synthesis was confirmed by immunoprecipitation.
• 2.2. Mycelia grown under these same conditions were used as a source of RNA and this RNA was translatable in a wheat germ translation system to produce proteins with M_r in the range < 10,000–> 90,000
• 3.3. Double-stranded cDNA was prepared and was inserted into the EcoR1 site of λgt10 and λgt11 using an adaptor ligation strategy. Packaging of these materials yielded large cDNA libraries. That from λgt10 contained 2.9 sx 10⁶ pfu/ml with 70% recombinants whereas that from λgt11 contained 2.2 × 10⁶ pfu/ml with 60% recombinants.

     

蜜环菌菌种分离新法——天麻组织分离法

报道了一蜜环菌菌种分离方法——天麻组织分离法,并对此法与常用分离法——菌索分离法进行比较试验。结果发现,天麻组织分离法分离成功率高（78％）,远高于常用的菌索分离法（16％）,且前者操作简便、难度低,所得菌种生活活力、生长形态均优于后法。     

琼脂固体培养基培养蜜环菌菌索总DNA的提取

野外采集的蜜环菌[Armillaria mellea(Vahl.ex Fr.)Quel]在提取DNA前需要分离获得纯化的菌丝体。常规液体培养获得菌丝团的方法感杂率较高,采集固体培养基表面cellophane膜上形成的菌丝则难以获得足量的DNA提取材料。蜜环菌细胞内含有大量多醣类物质,也使得蜜环菌高质量DNA的提取存在一定的困难。本研究通过改进试验,提供一个直接从琼脂固体培养基培养的蜜环菌菌索中提取高质量DNA的方法。其中样品的预先冻融处理方法可以促使蜜环菌菌索与琼脂分离;而在裂解提取缓冲液裂解材料细胞后加入1.25 mol/L KAc溶液,则有利于除去蜜环菌细胞内的多醣类物质以及残留的少量琼脂。通过琼脂糖电泳、紫外分光光度计对DNA浓度及OD值的测定、ISSR引物的PCR扩增以及酶切产物的PCR扩增等方法的检测,结果均表明该方法提取的DNA质量较好,符合进一步进行分子生物学研究的要求。     

The Ecology of Armillaria mellea in Britain Biological Control

The first trial in Britain of a method developed and used inEast Africa to control Armillaria mellea is described. Contraryto experience in East Africa, colonization of hardwood rootsby A. mellea was not reduced either by ringbarking trees beforefelling, or by frill-girdling and poisoning. Five years afterfelling, roots of trees treated in this manner were found tobe fully colonized, while invasion of the roots of untreatedtrees appeared to be still in progress. However, the formerwere far less effective as food bases for A. mellea than thelatter. Further experimentation may-reveal whether the treatmentsdescribed are practical methods for reducing infection in plantationsestablished on infested hardwood sites. Stump poisoning (withno pre-felling treatment) may also prove effective.     

Genetic exchange and recombination in populations of the root-infecting fungus Armillaria gallica

Genetic individuals, or genets, of Armillaria and other root-infecting basidiomycetes are usually found in discrete patches that often include the root systems of several adjacent trees. Each diploid individual is thought to arise in an unique mating event and then grow vegetatively in an expanding territory over a long period of time. Our objective in this study was to describe the population from which such genetic individuals are drawn. In a sample including 274 collections representing 121 genetic individuals of A. gallica (synonym A. bulbosa ) from two sites in each of four regions of eastern North America, genotype frequencies at seven nuclear loci were not significantly different from Hardy-Weinberg expectations. Furthermore, allele frequencies at the seven loci were not significantly different between regions. Additional allelic data from four non-contiguous regions of mitochondrial DNA showed little or no population subdivision over the four regions. Analysis of the distribution of multilocus mtDNA haplotypes revealed some clonal transmission of mtDNAs between genets and nonrandom mating within sites. Despite the sharing of mtDNA types by some individuals, the overall sample contained a high level of genotypic diversity. The apparent linkage equilibrium between some pairs of loci and the high level of phylogenetic inconsistency among all four loci suggest the occurrence heteroplasmy and recombination among mtDNAs of A. gallica in nature. In laboratory matings of two haploid strains with different mtDNA types, a low frequency of recombination in mtDNA was detected.     

Morphological and molecular characterization of the Armillaria cepistipes – A. gallica complex in the Czech Republic and Slovakia

Armillaria cepistipes and A. gallica (Basidiomycota, Physalacriaceae) are morphologically similar species, and they are often nearly indistinguishable using DNA-based methods targeting the ITS region of ribosomal DNA. The aim of this study was to examine morphological and ecological features of A. cepistipes and A. gallica, and to test other DNA-based methods to distinguish the two species. Our results revealed discriminative macro- and micromorphological features between these two species, especially the presence of a distinct central pileus ocella, the shape of the annulus, the character of the velar stipe remnants and the length of the terminal cells of the pileus scales. Ecologically, A. gallica generally prefers warmer areas in lowlands (oak and alluvial forests), while A. cepistipes is more common in hilly and lower montane beech forests in Central Europe. Nevertheless, despite differences in ecological preferences, certain locations between 300 and 500 m a.s.l. are known to sympatrically support both species. The sequences of the translation elongation factor 1-alpha showed high interspecific variability, and this gene is a more appropriate candidate for distinguishing A. gallica from A. cepistipes.