泊沙康唑对临床来源接合菌的体外抗菌活性研究

目的研究泊沙康唑对临床来源的接合菌体外抗菌活性。方法对临床来源的43株接合菌采用ITS区序列分析进行准确鉴定,采用E-test纸片法研究泊沙康唑对43株菌的体外药物敏感性,观察其MIC值。结果经ITS序列分析鉴定,43株临床来源接合菌中最为常见的是小孢根霉和不规则毛霉(各12株),其次为米根霉(10株),ramosa(3株)。其他菌种分别为ornata、总状共头霉、微小根毛霉、卷曲毛霉、印度毛霉和冻土毛霉各1株。E-test纸片法测得泊沙康唑对毛霉属、根霉属和Lichtheimia spp.的MIC值范围分别为0.19～>32μg/mL、0.19～3μg/mL、0.002～0.38μg/mL,对总状共头霉和微小根毛霉的MIC值分别为1μg/mL和0.19μg/mL。结论泊沙康唑对大部分接合菌有效,不同种属MIC值范围不同,毛霉属真菌的MIC值差异较大。     

抗多重耐药病原菌的越南槐内生菌TRPH-35的抗菌活性及其鉴定

以大肠杆ATCC25922、金黄色葡萄球菌ATCC25923及其临床多重耐药分离株ETEC-AD9、MRSA-DF12共4株人体病原细菌为靶标菌,采用琼脂块法从越南槐内生真菌中筛选抗菌活性菌株,并以改良的微量肉汤稀释法测定其代谢产物的最小抑菌浓度;根据形态和分子特征鉴定拮抗菌株的分类学地位。结果表明,菌株TRPH-35的活菌块对各病原细菌的抑菌圈直径与阳性对照相当;其代谢产物对各病原菌的最小抑菌浓度为20~40μg/mL;初步鉴定该菌株为刺盘孢。本研究结果表明,菌株TRPH-35鉴定为刺盘孢,对参试病原细菌均显示强的抗菌活性,具有较大的开发应用潜力。     

Rhizoctonia spp. Causing Root and Hypocotyl Rot in Phaseolus vulgaris in Cuba

Sixty isolates of Rhizoctonia spp. were obtained from Cuban bean fields during the period 2004–2007. Isolates were characterized with different techniques, including nuclei staining, pectic zymogram, PCR–RFLP analysis of the rDNA–ITS region and sequencing of the rDNA–ITS region. The majority of the isolates were identified as multinucleate Rhizoctonia solani isolates, representing two different anastomosis groups (AGs), AG 2‐2 WB and AG 4 HGI; the remaining isolates were binucleate Rhizoctonia isolates and belonged to AG F and AG A. AG 4 HGI isolates were equally distributed in all soil types; AG 2‐2 isolates were more frequently isolated from cambisols, whereas AG F isolates were related to calcisols. Pathogenicity experiments in vitro and in the greenhouse, revealed that binucleate isolates only caused root rot, whereas R. solani isolates were able to cause root rot and hypocotyl rot. Furthermore, differences in virulence level were observed between R. solani and binucleate isolates and among different AGs. Isolates of R. solani AG 4 HGI and R. solani AG 2‐2 WB were the most aggressive, binucleate isolates of AG F were intermediate aggressive, whereas a binucleate isolate of AG A was weakly aggressive. In contrast with other reports about R. solani in bean, web blight symptoms were never observed during this study.     

莴苣叶疫病和茎腐病的病原

2021年3–6月,从甘肃省的生菜(叶用莴苣)叶片和青海省的莴笋(茎用莴苣)茎秆罹病样本上分离得到腐霉属卵菌。通过Koch’s法则明确了分出菌株的致病性。依据形态学和分子生物学特征,将3个菌株鉴定为嗜导管腐霉Pythium tracheiphilum。在核糖体DNA内转录间隔区(rDNA-ITS)、细胞色素氧化酶亚基1(cox1)和核糖体DNA28S大亚基(rDNA-LSU)基因联合系统发育树中,甘肃菌株(LPy-B)和青海菌株(LPy-C和LPy-D)被聚在P. tracheiphilum的不同亚群里,不同菌株的适宜生长温度和产孢特性存在差异。孢子囊顶生、间生或侧生,球形,17.13–53.73μm,或近球形至葫芦状,24.58–56.72×18.62–53.73μm;休止孢球形,6.70–9.68μm;藏卵器光滑,顶生或间生,球形,15.64–23.09μm;每个藏卵器有雄器1–2个,雄器与藏卵器同丝或异丝生;卵孢子满器或近满器,球形,直径13.41–20.11μm,卵孢子壁厚0.74–2.23μm。致病性测定结果表明,除莴苣外,嗜导管腐霉还可侵染菊科的华蒲公英和刺儿菜、十字花科的...     

Isolation and characterization of thermotolerant yeasts for the production of second-generation bioethanol

The purpose of this study was to isolate, identify, and characterize the thermotolerant yeasts for use in high-temperature ethanol fermentation. Thermotolerant yeasts were isolated and screened from soil samples collected from the Mekong Delta, Vietnam, using the enrichment method. Classification and identification of the selected thermotolerant yeasts were performed using matrix-assisted laser desorption ionization/time-of-fight mass spectrometry (MALDI-TOF/MS) and nucleotide sequencing of the D1/D2 domain of the 26S rDNA and the internal transcribed spacer (ITS) 1 and 2 regions. The ethanol production by the selected thermotolerant yeast was carried out using pineapple waste hydrolysate (PWH) as feedstock. A total of 174 yeast isolates were obtained from 80 soil samples collected from 13 provinces in the Mekong Delta, Vietnam. Using MALDI-TOF/MS and nucleotide sequencing of the D1/D2 domain and the ITS 1 and 2 regions, six different yeast species were identified, including Meyerozyma caribbica, Saccharomyces cerevisiae, Candida tropicalis, Torulaspora globosa, Pichia manshurica, and Pichia kudriavzevii. Among the isolated thermotolerant yeasts, P. kudriavzevii CM4.2 displayed great potential for high-temperature ethanol fermentation. The maximum ethanol concentration (36.91 g/L) and volumetric ethanol productivity (4.10 g/L h) produced at 45 °C by P. kudriavzevii CM4.2 were achieved using PWH containing 103.08 g/L of total sugars as a feedstock. These findings clearly demonstrate that the newly isolated thermotolerant yeast P. kudriavzevii CM4.2 has a high potential for second-generation bioethanol production at high temperature.     

烟曲霉再鉴定、标准化CSP分型及体外药物敏感性

目的 了解烟曲霉复合体临床株菌种分布,经典烟曲霉临床株CSP基因型及对常见抗真菌药物敏感性状况.方法 菌株来源:北京大学真菌和真菌病研究中心保存分离自125名患者的162株烟曲霉复合体菌株.通过形态学,最高生长温度及分子生物学测序分步鉴定;对CSP基因进行扩增、测序,采用国际化命名体系进行CSP分型;采用微量液基稀释法测定经典烟曲霉对伊曲康唑(ITC)、两性霉素B(AMB)、伏立康唑(VRC)及卡泊芬净(CAS)的敏感性.结果 所有烟曲霉复合体菌株均为经典烟曲霉;共分为16个CSP基因型,最常见为t04A、t03和t01;分离自4名患者的13株菌对ITC的MICs≥4 μg/mL,其中2株菌AMB和VRC的MICa分别为4μg/mL和16 μg/mL.CAS的MECs最高为4μg/mL,仅1株.结论 未检出烟曲霉相关新种;经典烟曲霉临床株共16个CSP基因型,分布与国际研究结果基本一致,其中5个为新型.我国经典烟曲霉临床株ITC耐药率为3.2％,个别菌株AMB,VRC和CAS耐药.     

Genetic Characterization and Pathogenicity of Rhizoctonia solani Associated with Common Bean Web Blight in the Main Bean Growing area of Argentina

Common bean web blight (WB), caused by the fungus Rhizoctonia solani (teleomorph Thanatephorus cucumeris), is among the endemic fungal diseases of major impact in north‐western Argentina (NWA). This study aimed to analyse the genetic and pathogenic diversity of R. solani in Salta, NWA, where 97 isolates were recovered from commercial bean cultivars and wild beans showing WB symptoms in a major bean production area. The isolates were characterized on the basis of specific primers, rDNA‐ITS sequences and morphological characteristics. All the isolates were identified as R. solani AG 2‐2WB, and they exhibited considerable intragroup variation. The phylogenetic tree generated with the ITS sequences confirmed the isolates identification. Aggressiveness of the isolates towards bean seedlings was assessed in the greenhouse. A great variability in virulence was observed among the isolates analysed. On the basis of the disease reaction on foliar tissues, the isolates were grouped into three virulence categories as follows: weakly virulent (30%), moderately virulent (38%) and highly virulent (32%). However, no correlation between virulence and geographical origin was detected. The information generated in this study provides initial data on the population variability of the WB pathogen in north‐western Argentina and represents a valuable contribution to regional breeding programmes aimed to obtain cultivars with durable resistance.     

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.     

黄颡鱼卵水霉病病原的分离鉴定及其无性繁殖特性

【目的】对黄颡鱼卵水霉病病原进行分离鉴定,并对其无性繁殖特性进行研究。【方法】采用传统方法从患水霉病的黄颡鱼卵上进行丝状真菌的分离,然后通过人工感染实验证实分离菌株的致病性,通过形态学观察和ITS rDNA序列分析对致病菌株进行鉴定,并进一步通过单因子法研究其无性繁殖特性。【结果】从患水霉病的黄颡鱼卵上分离了4株丝状真菌,经人工感染试验证实其中一株丝状真菌HP对黄颡鱼卵具有致病性,并进一步研究了其形态与无性繁殖特性,开展了ITS rDNA序列分析。实验结果表明,菌株HP菌丝为透明管状结构,中间无横隔,分枝较少;游动孢子囊多数呈棒状,游动孢子发育成熟后从孢子囊中释放出来,并迅速游离;能够产生第二孢孢子;新孢子囊以内层出的方式产生;藏卵器呈球形,与雄器同枝或异枝。菌株HP的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类,同源性高达99%,与多子水霉菌株Arg4S(GenBank登录号GQ119935)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株HP为多子水霉(Saprolegnia ferax)。此外,菌株HP在5°C-35°C、pH 4-10范围内均能产生游动孢子,产生游动孢子的最适温度和pH分别为20°C和7,而且5-25 mg/L福尔马林和0.25 1.25 mg/L二硫氰基甲烷对菌株HP产生游动孢子具有明显的抑制作用。【结论】分离鉴定了黄颡鱼卵水霉病病原,并确定了其无性繁殖特性,可以作为该病防治用药的依据。     

Characterization of a newly discovered Beauveria bassiana isolate to Franklimiella occidentalis Perganda, a non-native invasive species in China

In this study, a new virulent Beauveria bassiana isolate (B. bassiana-CYT5) had been identified as a new member of the species B. bassiana. The B. bassiana-CYT5 isolate was compared with four other virulent B. bassiana isolates and found to be highly infectious and virulent against the Franklimiella occidentalis Perganda. The B. bassiana-CYT5 could approximately 93.08% mortality of F. occidentalis 6 days post inoculation in the concentration of 1×10(8) conidia/mL. The phylogenetic tree based on ITS and partial sequence of elongation factor 1-alpha (EF1-alpha) indicated that B. bassiana-CYT5 isolate was in a cluster of B. bassiana. Furthermore, B. bassiana-CYT5 isolate demonstrated high heat tolerance (60-100% relative germination) between 1-h and 2-h exposure at 37 °C, 38 °C, 39 °C and 40 °C, respectively. So our results suggested that B. bassiana-CYT5 isolate could be a new efficient biocontrol agent against F. occidentalis.     

产D-阿拉伯醇菌株的筛选、鉴定及其产D-阿拉伯醇条件的优化

摘要：【目的】产D-阿拉伯醇的耐高渗酵母的筛选、鉴定和产D-阿拉伯醇条件的优化。【方法】通过电镜、Biolog GN、(G+C)含量和26S rDNA D1/D2区序列分析法对所获得的菌株进行了描述。通过红外光谱、核磁共振氢谱和碳谱、质谱以及旋光度实验鉴定纯化产物的结构。通过单因素实验优化产D-阿拉伯醇的发酵条件。【结果】本文筛选得到一株产D-阿拉伯醇的新型菌株,经鉴定属于假丝酵母属并命名为Candida sp. H2。200 mL摇瓶发酵生产D-阿拉伯醇的单因素优化实验表明,最适发酵条件为：葡萄糖250     

Candida dubliniensis: an appraisal of its clinical significance as a bloodstream pathogen

A nine-year prospective study (2002-2010) on the prevalence of Candida dubliniensis among Candida bloodstream isolates is presented. The germ tube positive isolates were provisionally identified as C. dubliniensis by presence of fringed and rough colonies on sunflower seed agar. Subsequently, their identity was confirmed by Vitek2 Yeast identification system and/or by amplification and sequencing of the ITS region of rDNA. In all, 368 isolates were identified as C. dubliniensis; 67.1% came from respiratory specimens, 11.7% from oral swabs, 9.2% from urine, 3.8% from blood, 2.7% from vaginal swabs and 5.4% from other sources. All C. dubliniensis isolates tested by Etest were susceptible to voriconazole and amphotericin B. Resistance to fluconazole (≥8 μg/ml) was observed in 2.5% of C. dubliniensis isolates, 7 of which occurred between 2008-2010. Of note was the diagnosis of C. dubliniensis candidemia in 14 patients, 11 of them occurring between 2008-2010. None of the bloodstream isolate was resistant to fluconazole, while a solitary isolate showed increased MIC to 5-flucytosine (>32 μg/ml) and belonged to genotype 4. A review of literature since 1999 revealed 28 additional cases of C. dubliniensis candidemia, and 167 isolates identified from blood cultures since 1982. In conclusion, this study highlights a greater role of C. dubliniensis in bloodstream infections than hitherto recognized.     

产喜树碱内生真菌的筛选及鉴定

从喜树Camptotheca acuminate树皮和果实中分离得到27株内生真菌,发酵后经HPLC检测,筛选出一株菌丝产喜树碱的菌,产量达774μg/L。对其ITS序列进行系统发育分析,结合其培养特征和显微特征,鉴定为拟茎点霉属(Phomopsis sp.)。这是首次报道分离自喜树的该属真菌发酵产喜树碱。     

Partial purification and characterization of the organic solvent-tolerant lipase produced by Pseudomonas fluorescens RB02-3 isolated from milk

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50 °C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn2+, Co2+, Cu2+, Ni2+ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd2+, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

高效降解纤维素低温真菌的筛选、鉴定及发酵优化

【背景】纤维素的生物转化已经成为能源、环境和化工领域的研究热点,但可降解纤维素的低温真菌鲜有报道。【目的】从西藏高海拔的植物根际土壤中筛选具有高效降解纤维素能力的低温真菌,优化其产酶条件,为其工业化应用奠定基础。【方法】利用稀释平板涂布法、刚果红定性及酶活定量分析进行低温降解菌的筛选;根据菌株形态学特征及ITSrDNA序列分析对其进行鉴定;利用单因素实验和响应面优化法优化其产酶条件。【结果】分离筛选到一株高效产纤维素酶的低温真菌NLS-2;鉴定菌株NLS-2为青霉菌属;在低温15°C下,其产纤维素酶的最佳培养条件为稻草粉2.5%,酵母粉0.5%,KH2PO40.5%,发酵时间7d,pH6.5,摇床转速170r/min。【结论】青霉菌NLS-2可在低温条件下生长并具有较强的纤维素酶生产能力,具有良好的应用前景。     

Description of two new species of Glypthelmins Stafford, 1905 (Digenea: Macroderoididae) in Rana spp. from Mexico, based on morphology and mtDNA and rDNA sequences

Glypthelmins Stafford, 1905 includes 29 putative species commonly found in the intestine and liver of anurans from all over the world but mainly in the Americas. Partial sequences of the cytochrome c oxidase subunit 1 ( cox 1), ribosomal internal transcribed spacer region 2 (ITS2) and the large subunit 28S rDNA gene were obtained and analysed using pairwise distance matrices and parsimony methods in order to characterise the interrelationships between 14 isolates of four nominal species of Glypthelmins recognised on morphological grounds. The highest intra-specific sequence divergence occurred in the cox 1 (18.53%) sequence, followed by that of the ITS2 (5.44%) and 28S (4.63%). Genetic variability was detected between the three isolates originally identified as G. facioi Brenes et al., 1959 from two localities in Mexico and one locality in Costa Rica. Sequence divergence exhibited among these isolates ranged from 10.70 to 11.22%, from 0.48 to 0.97% and from 1.33 to 1.88% for cox 1, ITS2 and 28S, respectively. Phylogenetic analysis combining all three data-sets generated a single most parsimonious tree. The three isolates of G. facioi form a clade, with an isolate collected from frogs in Veracruz State as the sister group to an isolate from Tabasco State + G. facioi from Costa Rica. The information derived from pairwise distance of independent data-sets plus the phylogenetic information indicate that each of the two isolates from Mexico, identified a priori as G. facioi, represent separate species. A re-examination of specimens was carried out and a re-evaluation made of the morphological characters to find reliable differences that had been overlooked. As a consequence, G. brownorumae n. sp. from Tabasco and G. tuxtlasensis n. sp. from Veracruz are described based on molecular and morphological differences.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

Identification of Thai isolates assigned to the genus Gluconobacter based on 16S-23S rDNA ITS restriction analysis

Forty-four Thai isolates phenotypically assigned to the genus Gluconobacter were examined for 16S-23S rDNA ITS restriction analysis by MboII and SduI (=Bsp1286I) digestions. The Thai isolates tested were divided into seven groups: Group I for fourteen isolates, Group IX for one isolate, Group X for two isolates, Group V-2 for four isolates, Group XI for three isolates, Group IV for one isolate, and Group III for nineteen isolates. There were no isolates of either Group II or Group V-1 that were identified as G. cerinus. The isolates of Group III, Group IV, and Group XI were subjected to an additional 16S-23S rDNA ITS restriction analysis by AvaII, TaqI, BsoBI, and BstNI digestions. The isolates of Group III were divided into three groups and two subgroups: Group III-2 for five isolates, Group III-6 for two isolates, and Group III-4, which was divided into two subgroups, Subgroup III-4a for four isolates and Subgroup III-4b for eight isolates. The fourteen isolates of Group I were identified as G. oxydans, and the two isolates of Group X were temporarily identified as G. oxydans. The five isolates of Group III-2 and the one isolate of Group IV were identified as G. frateurii. The remaining twenty-two isolates of Group V-2, Group III-4, Group III-6, Group IX, and Group XI were not identified but are candidates for several new species.     

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

彭泽鲫卵源致病性水霉的鉴定及其生物学特性

从患病的彭泽鲫卵上分离3株丝状真菌,经人工感染试验证实其中1株丝状真菌JL1对彭泽鲫卵具有致病性,并进一步研究了其形态与生长特性,开展了ITS rDNA序列分析。实验结果表明,菌株JL1菌丝为透明管状结构,中间无横隔,分枝较少;游动孢子囊多数呈棒状,游动孢子呈多排排列,发育成熟后从孢子囊中释放出来,并迅速游离;藏卵器呈球形,与雄器同枝或异枝。菌株JL1的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类,同源性高达99%,与Saprolegnia sp.H(登录号:EF460351)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株JL1为水霉菌(Saprolegnia sp.)。此外,菌株JL1在5°C-30°C、pH 4-11范围内均能生长,最适生长温度和pH范围分别为25°C-30°C和6-9。同时菌株JL1对NaCl敏感,质量分数为2%的NaCl即可抑制其生长,可以作为该病防治的依据。