Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Candida khmerensis sp. nov., a novel cation-tolerant yeast isolated from dry salted shrimp and sewage in Cambodia

Two cation-tolerant yeasts with powdered colonies, K28-3-2T and K26-1-4, were isolated from dry salted shrimp and sewage, respectively, in Siem Reap province, Cambodia. The D1/D2 sequences of the 26S rDNA data showed that the two isolates were conspecific and related to the Pichia burtonii and Candida fennica. Two isolates were examined by a polyphasic taxonomic approach, including molecular phylogenetic analysis, morphological, physiological and biochemical tests, DNA hybridization and MSP-PCR fingerprinting, in comparison with P. burtonii and C. fennica. The two isolates were found to grow by multilateral budding with true and pseudo-mycelium, to not produce ascospores, and to contain ubiquinone Q-8 similar to that of P. burtonii and C. fennica. The two isolates were not differentiated from the two closest species, P. burtonii and C. fennica, by the phenotypic character examined, except for the cation (Li+)-tolerance. From DNA-DNA reassociation studies, however, the two isolates showed low similarities to the closest two species. Based on D1/D2 sequences of 26S rDNA and DNA-DNA reassociation data, they were shown to be a new distinct species from P. burtonii and C. fennica. Therefore, a novel species is proposed, Candida khmerensis sp. nov., represented by strain K28-3-2T (=JCM 13262(T)=CBS 9784T). The novel species, Candida khmerensis sp. nov. can be clearly distinguished from P. burtonii and C. fennica by either the 26S rDNA D1/D2 or ITS region with 5.8S rDNA sequencing, or by the MSP-PCR fingerprinting pattern.     

KN-62 enhances Chlamydia pneumoniae-induced p44/p42 mitogen-activated protein kinase activation in murine fibroblasts and attenuates in vitro infection

The fungus Spilocaea oleagina causes peacock leaf spot in olive. Virtually nothing is known about S. oleagina despite the loss of crop yield caused by this fungus. In order to get insight, an in vitro culture of the fungus has been established and its identity confirmed by amplified fragment length polymorphism analysis. Using this in vitro culture, we have cloned and analysed the DNA sequences of the 18S and 28S ribosomal RNA genes (rDNA) as well as the internal transcribed spacers (ITS) and 5.8S rDNA region of S. oleagina. Sequence analysis and comparison to other fungi determined that this fungus belongs to the Dothideomycetes class. We have also determined that S. oleagina is an anamorphic phase of a yet unidentified Venturia species based on phylogenetic analysis using the 28S rDNA and ITS sequences.     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species

The polymerase chain reaction was used to amplify a rDNA fragment containing the internal transcribed spacers (ITS1-5.8S-ITS2) and the D1/D2 variable domains of the 28S rDNA from 10 species of the genus Botryosphaeria (Fungi, Ascomycota). Restriction analysis of the amplicons with frequent-cutting endonucleases (amplified ribosomal DNA restriction analysis) allowed the definition of 12 rDNA haplotypes. Each of the rDNA haplotypes could be unambiguously assigned to a single Botryosphaeria species, thus allowing clear identification of all the species tested. Intraspecific polymorphism was very low and detected only in Botryosphaeria parva and Botryosphaeria dothidea. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations. The method described in this paper provides a simple and rapid procedure for the differentiation and identification of Botryosphaeria isolates at the species level.     

A new species of Leptobacillim,L. symbioticum,isolated from mites and sori of soybean rust

A verticillium-like fungus forming a whitish colony and mainly solitary phialides that produced fusiform to cylindrical conidia in chains was isolated from uredinia of Phakopsora pachyrhizi, a causal agent of soybean rust. In addition, two similar looking isolates were obtained from Prostigmata mites. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA and LSU rDNA D1/D2 region sequences revealed that the three isolates were the same species and assigned to the genus Leptobacillium. These isolates represent a new species, morphologically and phylogenetically distinguished from the type species, L. leptobactrum, and we propose L. symbioticum sp. nov. In addition, Simplicillium chinense and S. coffeanum are assigned to the genus Leptobacillium.     

Morphological and Molecular Characterization of Fusarium solani Isolates

Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF-1α) distinguished the ' F. solani complex' in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment-length polymorphism (AFLP) and mini-microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum , were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well-supported clade including F. solani f. sp . pisi , nine into a clade including only isolates of F. solani f. sp . radicicola and four into a clade including F. solani f. sp . cucurbitae , but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.     

Rhodotorula lamellibrachii sp. nov., a new yeast species from a tubeworm collected at the deep-sea floor in Sagami Bay and its phylogenetic analysis

A new species of the genus Rhodotorula was isolated from a tubeworm (Lamellibrachia sp.) collected at a depth of 1156 m in Sagami Bay, Japan. Strain SY-89 had physiological properties quite similar to R. aurantiaca. Two phylogenetic trees, one based on internal transcribed spacer (ITS) regions and 5.8S rDNA sequences and the other based on the D1/D2 region of the large subunit (26S) rDNA sequences, united strain SY-89 to the type strain of Sakaguchia dacryoides through a considerable evolutionary distance. Strain SY-89 was differentiated from S. dacryoides by the G+C content of the nuclear DNA and differences in the ability to utilize specific carbon and nitrogen compounds. The low complementarity of strain SY-89 DNA to that of the type strain of S. dacryoides confirmed that this strain was genetically unrelated to previously known species. The tubeworm isolates are described as R. lamellibrachii sp. nov. The type strain of R. lamellibrachii is strain SY-89 (= JCM 10907). R. lamellibrachii formed a cluster with Erythrobasidium hasegawianum, R. lactosa, S. dacryoides and Sporobolomyces elongatus on the ITS and 5.8S rDNA phylogenetic tree. These five species shared a signature sequence in 26S rDNA, although this relationship was not supported by phylogeny based on the D1/D2 region of 26S rDNA.     

Stereumin A-E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024

Five cadinane sesquiterpenoids, named stereumin A (1), B (2), C (3), D (4) and E (5) were isolated from the CHCl(3) extract of the culture broth of the fungal strain CCTCC AF 207024. Based on the sequences at the internal transcribed spacer (ITS) region and partial 28S rDNA, this fungus was identified as a Stereum sp. The structures of the five compounds were elucidated using spectroscopic data from 1D, 2D NMR and HRESIMS experiments, and the structures of 1 and 2 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1-5 showed nematicidal activities against the nematode Panagrellus redivivus at 400 mg l(-1). Among these five compounds, compounds 3 and 4 killed 84.4% and 94.9% of P. redivivus, respectively in 48 h.     

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.     

金耳与其近似种的rDNA-ITS序列分析

对金耳(Tremella aurantialba)的担子果、酵母状分生孢子培养物和菌丝培养物的核糖体DNA内部转录间隔区(ITS)序列进行PCR扩增和测序。结果表明金耳担子果的ITS区PCR产物均为碱基数不同的两条带,片段长度和序列与酵母状分生孢子培养物、菌丝培养物一致。通过对ITS1和ITS2联合进行系统发育分析表明金耳酵母状分生孢子培养物归属于银耳属的金耳,参与组成担子果的寄主菌丝为毛韧革菌(Stereum hirsutum)。结合GenBank中登录的金黄银耳、脑状银耳、橙黄银耳等近似种构建了的系统发育树,结果支持形态学证据,表明金耳是一个独立种。     

Erysiphe corylopsidis sp. nov., a new powdery mildew fungus found on Corylopsis spicata and C. pauciflora

A powdery mildew fungus occurring on leaves of Corylopsis pauciflora and C. spicata in Japan is described as a new species, Erysiphe corylopsidis. This species is characterized by fewer than 15 appendages on a chasmothecium, primary branches of the appendages occasionally elongated, and a relatively small number (2–5) of ascospores per ascus. Molecular phylogenetic analyses based on rDNA ITS and 28S rDNA sequences indicate that this fungus forms an independent lineage in the genus Erysiphe.     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析*

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

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.     

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.     

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 110244^T (= CBS 14272^T?=?InaCCY703^T?=?AK 01).