Mitochondrial Small Subunit rRNA Gene Research Articles

Initial study and phylogenetic analysis of hard ticks (Acari: Ixodidae) in Nantong, China along the route of avian migration.

The growing concern about migratory birds potentially spreading ticks due to global warming has become a significant issue. The city of Nantong in this study is situated along the East Asia-Australasian Flyway (EAAF), with numerous wetlands serving as roosting sites for migratory birds. We conducted an investigation of hard ticks and determined the phylogenetic characteristics of tick species in this city. We utilized three different genes for our study: the mitochondrial cytochrome oxidase subunit 1 (COX1) gene, the second internal transcribed spacer (ITS2), and the mitochondrial small subunit rRNA (12S rRNA) gene. The predominant tick species were Haemaphysalis flava (H. flava) and Haemaphysalis longicornis (H. longicornis). Additionally, specimens of Haemaphysalis campanulata (H. campanulata) and Rhipicephalus sanguineus (R. sanguineus) were collected. The H. flava specimens in this study showed a close genetic relationship with those from inland provinces of China, as well as South Korea and Japan. Furthermore, samples of H. longicornis exhibited a close genetic relationship with those from South Korea, Japan, Australia, and the USA, as well as specific provinces in China. Furthermore, R. sanguineus specimens captured in Nantong showed genetic similarities with specimens from Egypt, Nigeria, and Argentina.

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

BackgroundThe supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered.ResultsWe sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome.ConclusionsOur data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P.jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1 <CT<40.0 indicates the presence of a low load of the organism and needs further determination of either colonization or probable/possible Pneumocystis pneumonia. Overall, the new assay demonstrates excellent analytical and clinical performance and may be more sensitive than mtLSU PCR target for the detection of P. jirovecii.

Peltaster gemmifer: A new species in the sooty blotch and flyspeck species complex from the United States

ABSTRACTSooty blotch and flyspeck (SBFS) fungi infect the cuticle of fruit, including apple fruit, and produce pigmented colonies. A new member of this fungal complex in the genus Peltaster is described on the basis of molecular and morphological evidence. The SBFS complex is a diverse group of ectophytic fungi that reside primarily within the order Capnodiales. Sooty blotch and flyspeck isolates from apple orchards in the central United States were subjected to parsimony and Bayesian analyses based on the internal transcribed spacer regions of nuc rDNA, the partial translation elongation factor 1-α gene, and the partial mitochondrial small subunit rRNA gene. Phylogenetic analysis delineated a new species, Peltaster gemmifer, from P. cerophilus and P. fructicola. Peltaster gemmifer conidiophores bear primary conidia that produce secondary conidia either through budding or through microcyclic conidiation; these were not seen in cultures of P. cerophilus and P. fructicola. On cellulose membrane that was placed on water agar amended with apple juice, P. gemmifer produced brown to black pycnothyria in a superficial brownish mycelial mat, similar to the colonies produced on apple fruit. Findings from the present study add to the >80 named and putative SBFS species so far described worldwide.

Citeromyces cibodasensis sp. nov., a novel yeast species isolated from leaf litter in Indonesia

A novel yeast species was isolated from leaf litter of Macropanax dispermus obtained from the Cibodas Botanical Garden, West Java, Indonesia. The two strains of the species displayed typical characteristics of the genus Citeromyces. Phylogenetic analysis based on the gene sequences of the D1/D2 domains of large subunit (LSU) rDNA, internal transcribed spacer (ITS) including 5.8S rDNA, mitochondrial small-subunit rRNA gene (MtSm), and translation elongation factor-1α (EF-1α) showed that the novel strains were clearly separated from the other four existing species of the genus Citeromyces. Therefore, the two strains were proposed to represent a novel species within the genus Citeromyces, for which the name Citeromyces cibodasensis is proposed; the type strain is NBRC 110244T (= CBS 14272T = InaCCY703T = AK 01).

A study on the colonization of Pneumocystis jirovecii among outpatients during cancer chemotherapy and among healthy smokers.

Pneumocystis Jirovecii (PJ) is regarded as an agent of fungal infection and in cases of pneumocystis pneumonia (PCP) in immune-compromised patients including cancer patients. It is not clear what kinds of cancer, treatments, and environment need prophylaxis for PCP. In this study, we have analyzed the detectability of PJ DNA from sputum, and discussed prophylaxis and risk factors regarding PCP. A total of forty-nine materials (twenty-four from outpatients during cancer chemotherapies and twenty-five from healthy control subjects) was collected. Their PJ DNAs were amplified using nested PCR with specific primers of the PJ gene (the mitochondrial small subunit rRNA gene). PJ DNA was detectable in 46% of specimens (sputum) from cancer patients during chemotherapies, and incidences of not significantly different among types of cancer and chemotherapy regimens. Prophylactic use of Sulfamethoxazole/Trimetoprim (ST) reduced the detection of PJ DNA. Detection of PJ DNA is not high among healthy non-smokers (20%) and high among healthy smokers (47%). Prophylactic use of ST may be necessary for cancer patients during chemotherapies. Also, smoking may be associated with PJ colonization in the airway and air vesicles, and may increase the mortality rate for PCP. All patients undergoing cancer chemotherapies should cease smoking.

Peltaster cerophilus is a new species of the apple sooty blotch complex from Europe

Adopting the currently used concept for the genus Peltaster, the sooty blotch fungus Peltaster cerophilus is newly described from the cuticle of ripening or ripe apples. It forms a punctate phenotype consisting of superficially formed pycnothyria and a superficial mycelial mat consisting of a net of brown or brownish black hyphae. The pycnothyria are olivaceous brown to brown but have a spot in the center that is less strongly pigmented. Pycnothyria on the holotype of P. fructicola are homogeneously pigmented. On synthetic nutrient-poor agar, P. cerophilus is largely indistinguishable from P. fructicola. It forms delicate, spreading hyphae and intercalary conidiogenous cells with short, lateral, apically thick-walled conidiogenous necks forming blastic, unpigmented, one-celled conidia in basipetal succession. Conidia can swell and become one-septate. The species has microcyclical conidiation in proximate parts of colonies. DNA sequence analyses based on the ITS and the partial nuclear small and large subunit ribosomal RNA genes, the partial mitochondrial small subunit rRNA gene and the partial translation elongation factor 1-α gene support the distinction of the European P. cerophilus from P. fructicola, which is known from North America and Europe. The nuclear small ribosomal RNA subunit gene sequences of P. cerophilus contain two group I introns at locations known to accommodate introns in certain other, unrelated taxa. One of these, for which the code “SSU-1506 intron” was adopted, is 1459 base pairs long and located between the universal primer sites ITS5 and ITS1. Similar or positional-differing introns were encountered also in three currently undescribed Peltaster species. Representative strains of Peltaster fructicola did not accommodate introns in the nuclear small subunit ribosomal RNA gene.

Molecular survey of hard ticks in endemic areas of tick-borne diseases in China

Over the past several years, there was a substantial increase in the number of cases of known and novel tick-borne infections in humans in China. To better understand the ticks associated with these infections, we collected hard ticks from animals or around livestock shelters in 29 localities in 5 provinces (Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang) where cases of tick-borne illness were reported. We collected 2950 hard ticks representing 7 species of 4 genera (Dermacentor sinicus, Haemaphysalis flava, Haemaphysalis longicornis, Ixodes granulatus, Ixodes persulcatus, Rhipicephalus microplus, and Rhipicephalus sanguineus). These ticks were identified to species using morphological characters initially. We then sequenced the mitochondrial small subunit rRNA (12S rRNA) gene, cytochrome oxidase subunit 1 (COI) gene, and the second internal transcribed spacer (ITS2) gene of these ticks, and conducted phylogenetic analyses. Our analyses showed that the molecular and morphological data are consistent in the identification of the 7 tick species. Furthermore, all these 7 tick species from China were genetically closely related to the same species or related species found outside China. Rapid and accurate identification and long-term monitoring of these ticks will be of significance to the prevention and control of tick-borne diseases in China.

Kockiozyma gen. nov., for Zygozyma suomiensis: the phylogeny of the Lipomycetaceous yeasts

In phylogenetic trees based on 18S rRNA gene, 26S rRNA gene, mitochondrial small subunit rRNA gene and the EF-1α gene sequences, and the concatenated sequences of the latter four regions derived from the neighbor-joining method, the four species of the genus Zygozyma constituted four clusters, respectively, with low bootstrap values. In phylogenetic trees based on the concatenated sequences of 18S rRNA genes, 26S rRNA genes, mitochondrial small subunit rRNA genes and the EF-1α genes derived from the neighbor-joining method, the maximum parsimony method and the maximum likelihood method, Lipomyces species including Lipomyces starkeyi (the type species of the genus Lipomyces), Lipomyces tetrasporus and so on, Lipomyces lipofer (= Waltomyces lipofer), Lipomyces japonicus (= Smithiozyma japonica), Babjevia anomala (= Lipomyces anomalus) and Dipodascopsis uninucleata constituted their own respective clusters. The four species of the genus Zygozyma, viz., Zygozyma oligophaga (the type species of the genus Zygozyma), Zygozyma suomiensis, Zygozyma arxii (= Kawasakia arxii) and Zygozyma smithiae (= Limtongia smithiae) were separated from one another and phylogenetically independent from other Lipomycetaceous yeast species. From the data obtained, all four species of the genus Zygozyma were distinguished from one another at the generic level. The name of Kockiozyma was newly suggested for Zygozyma suomiensis, and Kockiozyma suomiensis was introduced as a new combination.

Low genetic diversity ofPneumocystis jiroveciiamong Cuban population based on two-locus mitochondrial typing

Genotypes of two different loci of the Pneumocystis jirovecii mitochondrial gene were studied in specimens from a total of 75 Pneumocystis pneumonia patients in Spain, France and Cuba. A new genotype of the mitochondrial small subunit rRNA gene of P. jirovecii (160A/196T) was identified, which was revealed to be the most common in these three countries, especially in Cuba where its proportion reached 93.8%. Our data imply that the new genotype might be circulating worldwide and also suggests that the distribution of P. jirovecii genotypes could be narrower in islands such as Cuba.

국내에서 분리된 Verticillium dahliae의 유전적 유연관계 분석

국내 Verticillium dahliae 균주를 공시하여 다른 Verticillium 종과의 유전적 차이와 다양성을 밝히고자 국내 두 지역으로 부터 분리한 V. dahliae를 포함한 7종의 Verticillium속 14균주를 대상으로 mitochondrial small subunit rRNA gene (rns) 영역 염기서열 분석과 random-amplified polymorphic DNA(RAPD)를 실시하였다. rns 영역 염기서열의 분석에서 국내 분리균인 5개의 V. dahliae균주는 Verticillium속 내 다른 종들과 달리 하나의 그룹을 형성하였고, 외국 균주들과도 동일한 그룹을 이루었다. rns 영역 염기서열 분석뿐만 아니라 RAPD 분석에서도 Verticillium속의 다른 종들과 구별되어 V. dahliae가 한 그룹을 형성하였으나 V. dahliae 균주간에 형성된 세 개의 소그룹을 통하여 동일한 국화과 작물로 부터 분리한 V. dahliae 간에 분리된 지역에 따라 유전적 다양성이 존재함을 확인하였다. 이러한 결과는 국내 V. dahliae의 유전적 다양성연구와 유연관계분석에 기초적인 자료로 사용될 것이다. To provide basic information for Verticillium spp., molecular methods were applied to analyze genetic characteristics within Verticillium spp. including Verticillium dahliae, isolated from diseased plants in two regions of Korea. Five Korean isolates of V. dahliae causing Verticillium wilt on chrysanthemum were analyzed, together with six other Verticillium spp., using mitochondrial small subunit rRNA gene (rns) sequence and random-amplified polymorphic DNA (RAPD). In a phylogenetic tree based on rns region sequences, Korean V. dahliae isolates formed a single clade with foreign isolates, whereas the other Verticillium spp. formed separate groups. In addition to rns sequence analysis, a dendrogram based on RAPD fragment patterns also showed clustering of all V. dahliae isolates into one group, separate from the six different Verticillium spp., and the V. dahliae isolates formed three subgroups which corresponded to the regions of origin, Kumi, Busan city and Canada. This indicates that high genetic variation exists between regions, although the fungus was isolated from the same host plant, chrysanthemum. These results provide the foundation for the study of genetic diversity and relationships among V. dahliae isolates in Korea.

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.

A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

A major obstacle in the culture-independent estimation of the abundance of bacteria associated with plants is contamination with plant organelles, which precludes the use of universal rRNA bacterial primers in quantitative PCR applications. We present here a PCR-based method that allows a priori determination of the degree of chloroplast and mitochondrial contamination in DNA samples from plant environments. It is based on differential digestibility of chloroplast, mitochondrial and bacterial small subunit rRNA gene amplicons with the restriction enzymes AfeI and BbvCI. Using this method, we demonstrated for field-grown lettuce plants that even a gentle washing protocol, designed to recover the microbial community and its metagenome from the leaf surface, resulted in substantial contamination with chloroplast DNA. This finding cautions against the use of universal primer pairs that do not exclude chloroplast DNA from amplification, because they risk overestimation of bacterial population sizes. In contrast, contamination with mitochondrial 18S rRNA was minor in the lettuce phyllosphere. These findings were confirmed by real-time PCR using primer sets specific for small subunit rRNA genes from bacteria, chloroplasts, and mitochondria. Based on these results, we propose two primer pairs (534f/783r and mito1345f/mito1430r) which between them offer an indirect means of faithfully estimating bacterial abundances on plants, by deduction of the mito1345f/mito1430r-based mitochondrial count from that obtained with 534f/783r, which amplifies both bacterial and mitochondrial DNA but excludes chloroplast. In this manner, we estimated the number of total bacteria on most leaves of field-grown lettuce to be between 10 5 and 10 6 g − 1 of leaf, which was 1–3 orders of magnitudes higher than the number of colony-forming units that were retrieved from the same leaf surfaces on agar plates.

Candida ogataesp. nov., an anamorphic member of theKuraishiaclade

Three methanol-assimilating yeast strains representing a hitherto undescribed species were isolated from rotten wood and freshwater samples collected in Hungary. Analysis of the D1/D2 large subunit rRNA gene sequences placed the strains in the Kuraishia clade; however, no ascospore formation was observed. These strains differ from Candida hungarica, the genetically most closely related recognized species, by four and five substitutions in D1/D2 and by >1% and 4% differences in the internal transcribed spacer and in the mitochondrial small subunit rRNA gene regions, respectively. Some phenotypic differences were also observed. Candida ogatae, a novel yeast species, is proposed to accommodate these isolates. The type culture is NCAIM Y.01845(T) (CBS 10924, NRRL Y-48474).

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.

Multigene phylogenetic analysis of the Lipomycetaceae and the proposed transfer ofZygozymaspecies toLipomycesandBabjevia anomalatoDipodascopsis

Phylogenetic relationships among species assigned to genera of the family Lipomycetaceae were determined from analysis of the nearly entire large, subunit rRNA gene, the small subunit rRNA gene, mitochondrial small subunit rRNA gene and the translation elongation factor-1alpha gene. Monophyly of the Lipomycetaceae was strongly supported, and currently described species appear genetically unique. The multigene analysis provided no support for maintaining the genera Kawasakia, Smithiozyma, Waltomyces or Zygozyma, and it is proposed that species in these genera be assigned to the genus Lipomyces. The monotypic genus Babjevia is a member of the Dipodascopsis clade and it is proposed to reassign Babjevia anomala to Dipodascopsis. The proposed changes will result in the Lipomycetaceae having two ascosporic genera, Lipomyces and Dipodascopsis, and the anamorphic genus Myxozyma.

Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and 14 new species combinations

Relationships among species assigned to the ascosporic yeast genera Sporopachydermia, Stephanoascus, Trichomonascus, Wickerhamiella and Zygoascus, and to the associated anamorphic genera Arxula, Blastobotrys, Sympodiomyces and Trigonopsis, were determined from phylogenetic analyses of gene sequences from the nearly complete large-subunit rRNA gene, the mitochondrial small-subunit rRNA gene, and cytochrome oxidase II. The genus Stephanoascus is polyphyletic, resulting in reassignment of two species to the older genus Trichomonascus and the third to Sugiyamaella gen. nov. (type species Sugiyamaella smithiae). The genera Sporopachydermia, Wickerhamiella and Zygoascus appear to be monophyletic. The species Pichia ofunaensis and P. tannicola are proposed for transfer to Zygoascus. Arxula, Blastobotrys and Sympodiomyces are members of the Trichomonascus clade, with the genus Blastobotrys having taxonomic priority for anamorphic states. Trigonopsis variabilis and three species of Candida represent a distinct clade. From the foregoing gene sequence analyses, the new ascosporic genus Sugiyamaella is proposed, as are 14 new species combinations and the new family Trichomonascaceae.

Identification and Distribution of Acanthamoeba Species Genotypes Associated with Nonkeratitis Infections

Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight- threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.

Multigene Phylogenetic Analysis of Pathogenic Candida Species in the Kazachstania ( Arxiozyma ) telluris Complex and Description of Their Ascosporic States as Kazachstania bovina sp. nov., K. heterogenica sp. nov., K. pintolopesii sp. nov., and K. slooffiae sp. nov

A yeast causing widespread infection of laboratory mice was identified from 26S rRNA gene sequences as Candida pintolopesii. To determine the relationship of C. pintolopesii with other members of the Kazachstania (Arxiozyma) telluris species complex, nucleotide sequences from domains 1 and 2 of the 26S rRNA gene, the mitochondrial small-subunit rRNA gene, and the RNA polymerase II gene were phylogenetically analyzed. That analysis resolved the 48 strains examined into five closely related species: K. telluris, Candida bovina, C. pintolopesii, Candida slooffiae, and a previously unknown species. One or more strains of each of the last four species formed an ascosporic state much like that of K. telluris. To place these ascosporogenous strains taxonomically, it is proposed that they be assigned to the teleomorphic genus Kazachstania as K. bovina (type strain NRRL Y-7283, CBS 9732, from the nasal passage of a pigeon), K. heterogenica (type strain NRRL Y-27499, CBS 2675, from rodent feces), K. pintolopesii (type strain NRRL Y-27500, CBS 2985, from the peritoneal fluid of a dead guinea pig), and K. slooffiae (type strain NRRL YB-4349, CBS 9733, from the cecum of a horse). On the basis of multigene sequence analyses, K. heterogenica appears to be a hybrid of K. pintolopesii and a presently unknown species. With the exception of K. bovina, the phylogenetically defined species show a moderate degree of host specificity.

Detection and Quantification of Fusarium solani f. sp. glycines in Soybean Roots with Real-Time Quantitative Polymerase Chain Reaction

Fusarium solani f. sp. glycines is the causal organism of soybean sudden death syndrome (SDS). This organism is difficult to detect and quantify because it is a slow-growing fungus with variable phenotypic characteristics. Reliable and fast procedures are important for detection of this soybean pathogen. Protocols were optimized for extraction of DNA from pure fungal cultures and fresh or dry roots. A new procedure to test polymerase chain reaction (PCR) inhibitors in DNA extracts was developed. Novel real-time quantitative PCR (QPCR) assays were developed for both absolute and relative quantification of F. solani f. sp. glycines. The fungus was quantified based on detection of the mitochondrial small-subunit rRNA gene, and the host plant based on detection of the cyclophilin gene of the host plant. DNA of F. solani f. sp. glycines was detected in soybean plants both with and without SDS foliar symptoms to contents as low as 9.0 × 10-5 ng in the absolute QPCR assays. This is the first report of relative QPCR using the comparative threshold cycle (Ct) method to quantify the DNA of a plant pathogen relative to its host DNA. The relative QPCR assay is reliable if care is taken to avoid reaction inhibition and it may be used to further elucidate the fungus-host interaction in the development of SDS or screen for resistance to the fungus.