Ashbya gossypii: a model for fungal developmental biology

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.     

The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements

We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.     

Genus-specific host range of Phomopsis amaranthicola (Sphaeropsidales), a bioherbicide agent for Amaranthus spp.

Phomopsis amaranthicola, a newly described species discovered in Florida, is under development as a biological control agent for weeds belonging to the genus Amaranthus. Host-range testing was performed on the fungus, using the centrifugal phylogenetic scheme, with Amaranthus hybridus as the focal plant. Forty-five accessions of 21 species in the genus Amaranthus were tested for susceptibility to infection by this fungus. Fifty-six plant species outside the genus Amaranthus were also tested. The latter included crops in which the fungus might be used as a bioherbicide, members of genera that are closely related to Amaranthus, and selections of species that have been reported as hosts of other species of the genera Phomopsis or Diaporthe. Phomopsis amaranthicola did not infect any of the plants from outside the genus Amaranthus but was highly pathogenic to several species in the genus Amaranthus. All Amaranthus species tested were infected to varying degrees by P. amaranthicola; the resulting mortality levels were also variable. Thus, P. amaranthicola appears to be host-specific to the members of the genus Amaranthus. The generic level of specificity of this pathogen renders it not only a safe bioherbicide agent but also one that could be used against several weedy Amaranthus species.     

PCR-based gene targeting in the filamentous fungus Ashbya gossypii

We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya gossypii. Short guide sequences with 40-46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination. The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3. This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants. Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed. This approach was used for the complete deletion of the open reading frame of the A. gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers. We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers. A gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established. Since short target guide sequences are sufficient to direct homologous integration into the A. gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi. Thus functional analysis of A. gossypii genes is already possible, based on single-pass sequence information.     

Endo-allopolyploidy of autopolyploids and recurrent hybridization—A possible mechanism to explain the unresolved Y-genome donor in polyploid Elymus species (Triticeae: Poaceae)

The genus Elymus L. in the tribe Triticeae (Poaceae) includes economically and ecologically important forage grasses. The genus contains the pivotal St genome from Pseudoroegneria in combination with other genomes in the tribe. Many Elymus species are tetraploids containing the StY genomes. It is thought that polyploidization characterizes the speciation of the genus in which the Y is considered as another key genome. Based on data from cytological, genome in situ hybridization, and molecular studies, we hypothesized an endo-allopolyploidy origin of the StY-genome species from the autotetraploid Pseudoroegneria species. To test this hypothesis, we amplified, cloned, and sequenced five single-copy nuclear genes (i.e., alcohol dehydrogenase 1–3, Adh1–Adh3, RNA polymerase II, Rpb2; and Waxy) from Elymus, Pseudoroegneria, and Hordeum species. The phylogenetic trees constructed based on the sequencing analyses of all genes indicated that diploid and autotetraploid Pseudoroegneria species were closely related, although with considerable genetic variation in tetraploids. In addition, the StY-genome Elymus species tended to have a close relationship with the diploid and autotetraploid Pseudoroegneria species, although different phylogenetic relationships among the gene trees were detected. These results indicated that the StY-genome species may have an autotetraploid origin and experienced recurrent hybridization. The complex St genomes in Pseudoroegneria in the polyploid state may gain more opportunities for within-species differentiation and recurrent hybridization. As a result, series modified versions of St genomes evolved into the StY genomes in some Elymus species.     

多基因联合分析棒束孢属Isaria（Ascomycota,Cordycipitaceae）系统发育关系

对棒束孢属Isaria及近缘属物种开展5基因（nrSSU、nrLSU、tef-1α、rpb1和 rpb2）测序并联合分析,结合GenBank相关类群序列,探讨棒束孢属系统发育关系,最终获得95个菌株、58个明确分类群的2-5基因序列。利用MEGA和MrBayes软件进行多基因聚类分析,结果表明棒束孢属多系起源于虫草菌科中,分3个不同分支。A支主要由Isaria cicadae、I. teniupes、I. coleopterorum、I. fumosorosea和I. cateniannulata等组成;B支包括I. poprawkii、I. locustica、I. javanica、I. amoenerosea和I. cateniobliqua;C支仅有I. farinosa。分支间被Cordyceps militaris、C. ninchukispora、C. pruinosa等隔开。棒束孢在形态上,主要以瓶梗基部膨大、尖端变细及孢子呈链状等特征与其他类群分开,但同时也发现有棒状分生孢子梗和单孢子类型。基于节点的分歧时间预测分析,推测棒束孢属首次分化于70Mya,但棒束孢属主要物种形成却在60-55Mya,且3个分支的棒束孢物种为快速同时形成,而后大多数类群表现遗传稳定。同时发现,与Isaria Clade A较近一支有粉被玛利亚霉Mariannaea pruinosa（C. pruinosa无性型）和蛹草蚧霉Lecanicillium militaris（C. militaris无性型）;与粉棒束孢距离最近一支有Akanthomyces aculeatus（C. tuberculata无性型）和L. attenuatum（C. confragosa无性型）,是两个不同的属征分类群,且相互间遗传距离较近。根据棒束孢属及其近缘种属形态特征的复杂性推测,棒束孢属在快速物种形成中,其近缘类群存在一定程度的丢失和选择性演化。     

木犀科植物叶绿体基因组结构特征和系统发育关系

木犀科11属19个种叶绿体基因组的一般特征和变异特征的比较分析显示, 结果表明, 该科叶绿体基因组大小为154-165 kb, 其差异主要是大单拷贝(LSC)长度的差异所致。Jasminum属3个物种的叶绿体基因组长度与其余物种有较大差异, 该属clpP基因内含子和accD基因丢失。共线性分析表明, Jasminum属3个物种多个基因出现基因重排现象, 倒位可能是重排的主要原因。Jasminum属在IRb/SSC和SSC/IRa边界的基因均与其它物种不同; 重复序列与SSR数量检测结果表明, Jasminum属与其余物种在数量及重复长度上差异较大。基于CDS数据构建的系统发育树表明, Abeliophyllum distichum和Forsythia suspensa为木犀科中较早分化的类群。     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

四种植物精油成分对枸杞棉蚜的生物活性

【目的】研究植物精油4种成分丁香酚(eugenol)、α-松油醇(α-terpineol)、肉桂油(cinnamon oil)和香茅油(citronella oil)对枸杞棉蚜Aphis gossypii的生物活性以及马来酸二乙酯和水杨酸乙酯对这些化合物对枸杞棉蚜毒力的影响,为无公害防治枸杞(Lycium spp.)害虫奠定科学基础。【方法】采用二项选择法测定上述4种精油成分(1%)对枸杞棉蚜无翅成蚜的驱避作用;玻璃管药膜法进行生物测定,评估这4种化合物及其与马来酸二乙酯或水杨酸乙酯的混合液对枸杞棉蚜成蚜的毒力;采用生物化学方法分析这些化合物处理后枸杞棉蚜成蚜乙酰胆碱酯酶(AChE)和三磷酸腺苷酶(ATPase)的离体活性。【结果】α-松油醇(1%)对枸杞棉蚜无翅成蚜的驱避作用明显,1, 2和3 h的驱避率分别为58.1%, 58.6%和55.7%; 3 h时测试的4种化合物对枸杞棉蚜成蚜的驱避率由大到小依次为α-松油醇>香茅油>肉桂油>丁香酚。丁香酚对枸杞棉蚜成蚜的毒杀作用最强,处理4 h时的LC₅₀值为0.48 mL/L,肉桂油、α-松油醇、香茅油对枸杞棉蚜成蚜的LC₅₀值分别为1.23, 6.24和11.97 mL/L。对枸杞棉蚜成蚜的生物测定结果表明,马来酸二乙酯对香茅油、肉桂油、丁香酚和α-松油醇的增效比分别为2.75, 2.86, 1.78和1.56;水杨酸乙酯对香茅油、肉桂油、丁香酚和α-松油醇的增效比分别为3.41, 2.67, 2.00和1.89。毒力比结果表明,所测定的4种化合物对枸杞棉蚜的毒力相差49.9倍,丁香酚与马来酸二乙酯或水杨酸乙酯的混合液对枸杞棉蚜的触杀毒力最高。4种精油成分对枸杞棉蚜成蚜AChE和ATPase活性有不同的作用,丁香酚对AChE活性的抑制作用最强,但对ATPase活性表现为低浓度促进,高浓度抑制。【结论】α-松油醇对枸杞棉蚜驱避作用最好;丁香酚、α-松油醇、肉桂油和香茅油这4种精油成分对枸杞棉蚜的毒力相差很大;丁香酚与马来酸二乙酯或水杨酸乙酯混合后对枸杞棉蚜的毒力提高,具有进一步开发应用的潜力;所测精油成分对枸杞棉蚜离体AChE和ATPase活性有一定抑制作用。     

白粉菌交配型位点基因结构及分子系统学关系

白粉菌是一类植物专性寄生真菌,广泛分布于世界各地,可引起多种植物白粉病。开展白粉菌交配型基因研究可以为全面认识白粉菌的生活史循环和系统演化提供证据。本研究以子囊菌中已报道的10种MAT基因和4种侧翼基因的氨基酸序列为种子序列,采用生物信息学方法在21个白粉菌基因组中进行氨基酸同源性分析,探索MAT基因及其侧翼基因的种类和线性排布。结果表明,21个白粉菌基因组中有10个属于MAT1-1型,包含MAT1-1-1和MAT1-1-3两种基因;10个属于MAT1-2型,仅包含MAT1-2-1基因;在Erysiphe pulchra的基因组中同时存在MAT1-1-1、MAT1-1-3、MAT1-2-1;而4种侧翼基因SLA2、APC5、APN2和CoxVia在21个白粉菌基因组中均存在。21个菌株中有两个菌株Blumeria graminis f. sp. hordei RACE1和Golovinomyces orontii的MAT基因和4种侧翼基因分布在同一个scaffold,其余菌株MAT基因和侧翼基因均不在同一scaffold出现。本研究分别分析了具有MAT1-1和MAT1-2的菌株中MAT基因和4种侧翼基因的排列关系,同时根据9个菌株的MAT基因注释信息,推测了其他菌株的MAT基因结构。结果表明,同属白粉菌的MAT基因结构相同,属间MAT基因结构差异明显。以从白粉菌基因组中获得的MAT1-1-1、MAT1-1-3和MAT1-2-1 3种MAT基因序列分别构建系统发育树,发现布氏白粉菌属Blumeria、白粉菌属Erysiphe和戈洛文白粉菌属Golovinomyces分别形成独立分枝,且白粉菌属与戈洛文白粉菌属形成姊妹群,这与前人对白粉菌系统发育关系的研究结果一致。本研究得到的白粉菌MAT基因种类、分布及其结构的相关结果为进一步实验提供了理论依据,为探究白粉菌的系统发育提供了新的思路。     

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora线粒体基因组的异质性

捕食线虫真菌少孢节丛孢Arthrobotrys oligospora是子囊菌中的一个食肉性丝状真菌,其菌丝体可变态为各种结构精巧的捕食器官用于捕食线虫,在生物防治和进化上极具研究潜力.线粒体基因细胞色素c氧化酶亚基Ⅰ基因(COX1)在真菌物种鉴定中的效果饱受争议.本研究对A.oligospora的不同单孢培养的菌株进行...     

薏苡黑粉菌基因组测序揭示其物种分类及适应性进化

以薏苡黑粉菌病瘿为研究对象,采用固体和液体培养方法对薏苡黑粉菌进行分离纯化培养和形态观察;利用Illumina、Pacbio测序和Hi-C辅助基因组组装技术对黑粉菌进行测序、组装和注释,分析其基因组结构和组成特征。通过与其他6个黑粉菌物种基因组进行比较基因组学研究,分析薏苡黑粉菌的基因家族进化和系统发育关系。在pH 7的固体PDA和液体PDB培养基中实现了薏苡黑粉菌的纯化培养,固体培养菌落表现为白色、表面隆起、具褶皱,较湿润且不透明的形态特征。Illumina、Pacbio和Hi-C组装结果显示,薏苡黑粉菌具有20对染色体(2n=40),基因组大小20.093 417 Mb,GC含量53.81%;共预测和注释到7 476个蛋白编码基因和164个非编码基因,占整个基因组长度的61.02%和0.285 3%;查找到3 674个散在重复序列和8 139个串联重复序列,分别占整个基因组长度的2.754 4%和1.605 4%。同源基因家族聚类显示,7个黑粉菌物种Ustilago coicis、U. bromivora、U. maydis、U. hordei、Sporisorium reilianum、S. scitamineum和S. graminicola的编码基因被聚类为6 999个基因家族,其中共有基因家族5 379个。薏苡黑粉菌包含5 339个单拷贝基因、105个多拷贝基因、89个特异基因、752个其他基因和188个未分类基因。基因家族进化和系统发育聚类表明,薏苡黑粉菌U. coicis在~60.9 MYA分化出来,与雀麦黑粉菌U. bromivova、大麦坚黑粉菌U. hordei聚为一支,表现为更近的亲缘关系;而玉米黑粉菌U. maydis在~66.6 MYA分化出来与3个孢堆黑粉菌物种S. scitamineum、S. graminicola和S. reilianum聚为一支。     

Fossil pinnae,sporangia, and spores of Osmunda from the Eocene of South China and their implications for biogeography and paleoecology

The genus Osmunda L. contains approximately 10 extant species widely distributed in tropical and temperate regions, with the greatest concentration of species in East and Southeast Asia. Osmunda is characterized by dimorphic or commonly hemidimorphic fronds with dimorphic pinnae. Its geological history has been traced back to the Triassic. Most records of the genus are based on rhizomes and rarely on pinnae bearing sporangia and spores. Here, we describe fossil pinnae, sporangia, and spores of Osmunda lignitum (Giebel) Stur recovered from the middle Eocene of the Changchang Formation in the Changchang Basin, Hainan Island and the Youganwo Formation in the Maoming Basin, Guangdong, South China. The fossils closely resemble the extant Osmunda banksiifolia (C. Presl) Kuhn of the subgenus Plenasium on the basis of their morphological and anatomical structures. The present occurrence of O. lignitum indicates subg. Plenasium flourished and extended from the high latitude regions such as Northeast China to the low latitude areas of South China during the middle Eocene. Large numbers of specimens described here also indicate that Osmunda was the dominant understory fern element beneath mixed evergreen broad-leaf angiosperm and gymnosperm forests living in a warm and humid environment.     

篮状菌属分类概述及三个中国新记录种

篮状菌属真菌在空气、土壤中分布广泛,有些种类是重要的工业酶及色素生产菌,有些则可以引起食品的污染变质,还有些种是引起浅层或散播性感染的条件致病菌,如马尔尼菲篮状菌。篮状菌属在传统分类学上是一个有性型属,随着多相分类以及“一种真菌一个名称”概念的广泛普及,该属的概念已经发生了较大的变化,目前全世界已报道177个种。本文基于国内外最新研究结果,对篮状菌属的分类现状、种的范围和形态学区分等进行了概述,同时基于多基因系统发育树以及形态学观察描述了3个中国新记录种Talaromyces amazonensis N. Yilmaz, López-Quint., Vasco-Pal.、T. chloroloma Visagie & K. Jacobs和T. minnesotensis Guevara-Suarez, Cano & Dania García,并详细比较了它们与模式菌株的形态学差别。     

木犀科植物叶绿体基因组结构特征和系统发育关系

木犀科11属19个种叶绿体基因组的一般特征和变异特征的比较分析显示, 结果表明, 该科叶绿体基因组大小为154-165 kb, 其差异主要是大单拷贝(LSC)长度的差异所致。Jasminum属3个物种的叶绿体基因组长度与其余物种有较大差异, 该属clpP基因内含子和accD基因丢失。共线性分析表明, Jasminum属3个物种多个基因出现基因重排现象, 倒位可能是重排的主要原因。Jasminum属在IRb/SSC和SSC/IRa边界的基因均与其它物种不同; 重复序列与SSR数量检测结果表明, Jasminum属与其余物种在数量及重复长度上差异较大。基于CDS数据构建的系统发育树表明, Abeliophyllum distichum和Forsythia suspensa为木犀科中较早分化的类群。     

Screening microbial strain for improving the nutritional value of wheat and corn straws as animal feed

From 18 strains of cellulolytic microorganisms including bacteria and filamentous fungi, one strain of soft rot fungus identified as Chaetomium cellulolyticum was screened with respect to stronger decomposition ability of cellulose and hemicellulose and its ability for protein synthesis. As it grew on raw corn straw in solid layer fermentation (SLF) for 5 days, the amino acid content in the fermentation product attained 19.29% (w/w) from 6.43% while the total cell wall was reduced by 54%. A toxicity test with mice showed that the fermentation product is not poisonous. The two filamentous fungi, Trichoderma pseudokoningii S-38 and Penicillium decumbens JU-A10 produced large amounts of extracellular cellulase and hemicellulase in the SLF process, but their growth was limited and they sporulated profusely with regard to their value as animal feed products.