牛巴氏杆菌病多重PCR检测技术的建立与应用

【背景】牛巴氏杆菌病是由血清型(A、B、E)多杀性巴氏杆菌(Pasteurella multocida,Pm)引起的一种严重危害养牛业的重要传染病,病原学聚合酶链反应(polymerase chain reaction,PCR)方法是诊断并防控该病的有效手段。【目的】建立检测血清型(A、B、E)多杀性巴氏杆菌的多重PCR方法,为临床诊断牛巴氏杆菌病和病原分型提供技术支撑。【方法】参考多杀性巴氏杆菌hyaD-hyaC基因、bcbD基因和ecbJ基因特异区域,设计3对特异性引物,以温度梯度PCR法确定适宜退火温度(T_m);采用棋盘试验优化引物浓度并初步建立多重PCR方法;采用重组质粒标准品及阳性菌株菌液确定其敏感性(最小检测量);以8种常见牛感染病原体[溶血性曼氏杆菌(Mannheimia hemolytica)C1655、大肠埃希氏菌(Escherichia coli)C237、产单核细胞李氏杆菌(Listeria monocytogenes)C1597、金黄色葡萄球菌(Staphylococcus aureus)C3053、都柏林沙门氏菌(Salmonella dublin)C79351、副结核分枝杆菌(Mycobacterium paratuberculosis)C1625、牛传染性鼻气管炎病毒(bovine infectious rhinotracheitis virus)CAV1546和牛支原体分离株(Mycoplasma bovis)C65-1]核酸样本确定其特异性;制备3批诊断试剂,对敏感性和特异性样品进行批间和批内试验,确定其重复性;运用建立的方法使用3种不同型号的PCR仪检测敏感性和特异性样品,确定其适用性;通过检测临床样本及人工模拟感染样本评价临床应用效果。【结果】在T_m为55℃时,3对引物浓度分别为0.25、0.30和0.20μmol/L条件下建立多重PCR方法较优,可以同时检测多杀性巴氏杆菌血清A型(821bp)、血清B型(203bp)和血清E型(363bp);该方法敏感性高,对重组质粒标准品pMD-A、pMD-B和pMD-E检测限分别为43.080、3.710和4.350copies/μL,对阳性菌液最低检出限均为10²CFU细菌;其特异性强,仅对血清型(A、B、E)多杀性巴氏杆菌有特异性扩增条带,同时对其他病原体均无扩增条带;该方法重复性良好,批间与批内试验均一致;临床样本及人工模拟感染样本检测结果显示与病原分离鉴定符合率为100%。【结论】成功建立了一种可鉴定不同血清型的牛多杀性巴氏杆菌多重PCR检测方法。     

猪肠病毒G (EV-G)荧光定量PCR检测方法的建立与四川地区分子流行病学调查

【背景】猪肠病毒G (Enterovirus G,EV-G)在猪群中普遍存在,可引起猪的皮肤损伤、肌肉麻痹、肺炎、发热、腹泻等,也存在无症状感染。【目的】建立检测EV-G的荧光定量PCR (real-time quantitative PCR,RT-qPCR)方法并开展四川地区分子流行病学调查。【方法】根据EV-G的5ʹUTR基因保守序列设计特异性引物,建立可检测EV-G各基因型的RT-qPCR,并评估其灵敏性、特异性和重复性,开展四川地区EV-G的流行调查。【结果】标准品在1.89×10²-1.89×10⁸ copies/μL浓度范围内线性关系良好;在特异性试验中,其他9种猪病毒(猪德尔塔冠状病毒、猪流行性腹泻病毒、传染性胃肠炎病毒、日本乙型脑炎病毒、猪萨佩罗病毒、猪细小病毒、猪圆环病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒)检测均为阴性;最低检测浓度为1.89×10¹ copies/μL;组内和组间变异系数分别小于1%和2%。检测2013-2021年四川省431份猪腹泻样品,EV-G总体阳性率为31.1%,表明该病毒在四川地区的感染较普遍。随机选取9份四川的EV-G阳性样品扩增VP1基因并测序分析,发现四川EV-G流行的基因型包括G1、G3、G4和G9,但以EV-G1为优势基因型。【结论】本研究建立了一种检测EV-G各基因型的RT-qPCR方法,并初步掌握了四川EV-G的流行现状,为后续深入开展该病毒的研究奠定了基础。     

大肠杆菌Zn2+敏感突变株构建及其功能验证

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用,Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中,zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株,并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株,利用λ Red重组系统,通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上,利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因,获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E.coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件,同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠定了基础。     

检测蜜蜂急性麻痹病毒TaqMan实时荧光RT-PCR方法的建立和初步应用

[背景] 蜜蜂急性麻痹病毒（Acute Bee Paralysis Virus,ABPV）是一种高毒力的蜜蜂病毒,可以引起蜜蜂的大批死亡和蜂群衰竭。[目的] 建立一种快速、灵敏的ABPV实时荧光RT-PCR检测方法。[方法] 根据ABPV衣壳蛋白基因保守序列设计引物和探针,通过对引物、探针浓度和退火温度等反应条件进行优化,建立基于TaqMan探针检测ABPV的实时荧光RT-PCR方法,并对方法的灵敏性、特异性和稳定性进行验证。[结果] ABPV实时荧光RT-PCR检测方法在9.8×10¹-9.8×10⁸ copies/μL之间呈现良好的线性关系,线性相关系数R²为0.998,扩增效率为103.8%。该方法的检测灵敏度为9.8 copies/μL;对其他蜜蜂病毒不发生交叉反应,具有良好的特异性;重复性试验结果显示组内和组间的变异系数分别为0.19%-0.80%和0.57%-1.07%,重复性良好。对2018年-2019年在福建地区采集的70份蜜蜂样品进行ABPV检测,阳性率为2.86%。[结论] 建立的ABPV实时荧光RT-PCR检测方法能用于该病的实验室检测、流行病学调查和疫情监测。     

猪链球菌2型硫氧还蛋白还原酶介导细菌应激与致病作用

【背景】硫氧还蛋白还原酶（thioredoxin reductase,TRR）是硫氧还蛋白系统关键组成部分,对病原菌应对体内外氧化应激、调节细菌稳态和介导致病过程具有重要作用。【目的】探究硫氧还蛋白还原酶TRR在人畜共患猪链球菌2型感染过程中参与的生物学效应。【方法】同源重组法构建猪链球菌2型硫氧还蛋白还原酶trr基因缺失株（Δtrr）及回补株（cΔtrr）,通过细菌染色、点板计数、体外细胞和动物感染模型等试验比较分析trr基因对细菌形态、抗应激反应及致病过程的影响。【结果】缺失trr对猪链球菌2型形态与生长特性的影响不大,但可增强细菌抗热应激、氧化应激和酸应激能力,缺失株对上皮细胞黏附力下降,侵袭进入脑血管内皮细胞作用显著降低,易于被吞噬细胞吞噬清除,对小鼠模型致病效应显著减弱。【结论】猪链球菌2型TRR因子参与细菌应激反应,介导细菌黏附、侵袭等致病过程,是猪链球菌2型新的潜在毒力因子。     

来源于中国南海的微生物新种乙醇速生杆菌(Celeribacter ethanolicus)多相分类研究

【目的】研究来源于南海海水的一株速生杆菌属菌株NH195^T 的多相分类。【方法】采用表型、基因型和化学分类方法,并综合系统发育关系结果,分析菌株NH195^T 的分类学地位。【结果】菌株NH195^T 是一株革兰氏阴性、好氧、杆状、无运动性细菌;能积累Poly-β-hydroxybutyrate (PHB);能在0.5%-10.0% (质量体积比) NaCl 浓度,pH 5.0-9.0 和20-40 ℃ 条件下生长,最适NaCl 生长浓度为1.0%-3.0%;氧化酶、触酶和脲酶反应结果阳性。菌株NH195^T 主要呼吸醌为Q-10,主要脂肪酸为C_18:1ω7c、C_18:1ω6c 和11 methyl C_18:1ω7c,主要极性脂为磷脂酰胆碱、磷脂酰甘油、双磷脂酰甘油、一个未知的氨基脂和两个未知脂。基因组G+C 含量为61.3 mol%。基于16S rRNA 基因的系统发育结果显示,菌株NH195^T 隶属于速生杆菌属;其与速生杆菌属标准菌株的16S rRNA 基因相似性范围为94.4%-97.7%。菌株NH195^T 与速生杆菌属标准菌株C. halophilus ZXM137^T 和C. indicus P73^T 的平均核苷酸一致性(ANI)分别为78.6%和78.0%;基于基因组数据计算所得的DNA 杂交同源率分别为26.1%和23.0%。【结论】基于表型和基因型结果,菌株NH195^T 代表了速生杆菌属一个新物种,命名为Celeribacter ethanolicus,标准菌株为NH195^T (CGMCC 1.15406^T=JCM 31095^T)。     

多杀性巴氏杆菌aroA基因缺失突变株的构建及鉴定

【目的】构建多杀性巴氏杆菌aroA基因缺失突变株,并验证其致病性。【方法】采用正向筛选同源重组技术构建多杀性巴氏杆菌aroA基因缺失突变株,利用PCR对突变株进行鉴定,分析其遗传稳定性、生长特性和致病性。【结果】成功构建多杀性巴氏杆菌aroA基因缺失突变株,连续传代20代,遗传稳定;突变株体外生长曲线表明,在前6h生长速度稍慢于亲本菌,随后两者生长速度一致。对小鼠的致病性试验表明:经腹腔注射aroA基因缺失突变株在1.0×106 CFU对小鼠无致死性,而亲本菌株在1.0×102 CFU对小鼠是致死性的。【结论】本研究获得多杀性巴氏杆菌aroA基因缺失突变株,对小鼠的致病性是减弱的。多杀性巴氏杆菌突变株的构建有助于研究其致病机理。     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

基于双重芯片式数字PCR快速鉴定KPC型碳青霉烯耐药肺炎克雷伯菌的方法

【背景】由于碳青霉烯类药物的泛用和滥用,致使肺炎克雷伯菌碳青霉烯耐药株与日俱增,产碳青霉烯酶是肺炎克雷伯菌对碳青霉烯类药物耐药的主要原因。目前对肺炎克雷伯菌碳青霉烯耐药株的检测方法存在费时费力、特异性差、灵敏度低等问题。【目的】建立一种能同时检测肺炎克雷伯菌和碳青霉烯酶基因bla_KPC的双重芯片式数字PCR方法。【方法】依据肺炎克雷伯菌的特有基因yhaI和碳青霉烯耐药基因bla_KPC保守序列设计特异性引物和探针,确定双重芯片式数字PCR同时对yhaI和bla_KPC两个基因核酸浓度绝对定量的检测范围、检出限和最佳实验体系,并进行方法特异性、灵敏度、重复性分析及临床菌株的检测。【结果】双重芯片式数字PCR检测灵敏度比双重实时荧光定量PCR提高了约1.5个数量级,在两基因同时检出的情况下,最低检出限分别为3.74 copies/μL (yhaI基因)和1.93 copies/μL (bla_KPC基因);优化后的双重芯片式数字PCR对参考菌株检测特异性的结果与双重实时荧光定量PCR结果一致;利用优化后的双重芯片式数字PCR方法共检测58株临床菌株,其中肺炎克雷伯菌43株,属肺炎克雷伯菌且含有bla_KPC基因的菌株13株,这与质谱及耐药谱检测结果一致。【结论】利用双重芯片式数字PCR技术建立了产KPC型碳青霉烯酶肺炎克雷伯菌的绝对定量检测方法。该方法特异性强、灵敏度高、准确度好,可用于检测具有碳青霉烯酶基因bla_KPC的肺炎克雷伯菌的核酸检测和定量分析,也为产其他类型碳青霉烯酶的病原菌检测提供了新的技术参考。     

四川地区猪源艰难梭菌分子分型调查

[背景] 艰难梭菌是一种重要的人畜共患肠道病原菌,可引起人和多种动物抗生素相关性腹泻或假膜性肠炎。四川作为我国主要的生猪产区,还未有猪源艰难梭菌流行病学调查的相关报道,对猪源艰难梭菌的防控及保障猪肉安全带来挑战。[目的] 调查四川地区猪源艰难梭菌的感染、流行情况,并对分离出的艰难梭菌进行分子分型研究。[方法] 收集来自四川生猪主要产区6个养殖场中猪的粪便标本（n=110）,采用厌氧培养技术在艰难梭菌鉴别培养基上进行分离培养;采用PCR方法扩增艰难梭菌4个毒素基因（tcdA、tcdB、cdtA、cdtB）和7个管家基因（adk、atpA、dxr、glyA、recA、sodA、tpi）,对分离株进行毒素基因分型和多位点序列分型。[结果] 从110份样品中,经革兰氏染色镜检及PCR鉴定,共分离出20株艰难梭菌,分离率高达18.18%;毒素基因分型结果显示共获得3种毒素基因型,包括tcdA⁺tcdB⁺cdtA/cdtB⁺（n=3）、tcdA⁺tcdB⁺cdtA/cdtB^—（n=6）、tcdA^—tcdB^—cdtA/cdtB^—（n=11）;多位点序列分型结果显示获得5个ST型,包括ST11（n=3）、ST3（n=1）、ST35（n=2）、ST36（n=4）、ST109（n=10）;进化树结果显示,所有分离株聚集为2个大群,分别为3个分支和17个分支。[结论] 四川主要生猪产区猪群存在艰难梭菌感染,分离株的分子分型呈多样性,主要流行型为ST11、ST3、ST35、ST36、ST109型,并且存在ST11型高毒力菌株流行的风险。     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

New species of Phaeoramularia, Pseudocercospora and Stenella from Western Ghats of India

Four new species of the hyphomycete genera Phaeoramularia viz. Ph. caesalpinae, Pseudocercospora viz., Ps. tiliacearum, Stenella, viz. S. argyreiae and S. grewiae occurring on Caesalpinia bonducella Fleming (Caesalpiniaceae), Grewia sp. (Tiliaceae), Argyrea sp. Lour (Convolvulaceae) and Grewia sp. L. (Tiliaceae), respectively are described and illustrated here. All these fungi were collected from Western Ghats of India.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

A polysaccharide from Lichina pygmaea and L. confinis supports the recognition of Lichinomycetes

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the development of thalli with cyanobacteria, has recently been recognized as a separate class of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccharide from Lichina pygmaea and L. confinis was purified and characterized to investigate whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan comprising a repeating unit consisting of an α-(1→6)-mannan backbone, mainly substituted by single α-galactofuranose residues at the O-2- or the O-2,4- positions linked to a small mannan core. With the exception of the trisubstituted mannopyranose residues previously described in polysaccharides from other lichens belonging to orders now placed in Lecanoromycetes, the structure of this galactomannan most closely resembles those found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data support molecular studies showing that Lichina species are remote from Lecanoromycetes as the galactofuranose residues are in the α-configuration. That the Lichinomycetes were part of an ancestral lichenized group can not be established from the present data because the extracted polysaccharide does not have the galactofuranose residue in the β configuration; however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan and was part of an early radiation in the ascomycetes.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.