Partial Beta-tubulin Gene Research Articles

Endophytic fungi isolated from Pelargonium sidoides DC: Antimicrobial interaction and isolation of a bioactive compound

The aim of this study was to investigate the antimicrobial activity of endophytes isolated from the root of Pelargonium sidoides DC., and to explore the antimicrobial interactions between endophytic fungi and the host plant. The hypothesis explored is that medicinal plants used as an anti-infective may not only rely on the properties of the medicinal plant alone but also on the residing endophytes within the botanical matrix. The study further aimed to isolate and identify secondary metabolites from the endophyte exhibiting promising antimicrobial activity. Fungal cultures were isolated from P. sidoides root material, and extracts were prepared and tested independently and in combination with the host plant to determine the interactions. Antimicrobial studies were undertaken using the minimum inhibitory concentration (MIC) assay against Gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 27853) bacteria. The bioactive compound was identified using High Performance Liquid Chromatography–Mass Spectrometry–Solid Phase Trapping–Nuclear Magnetic Resonance (HPLC–MS–SPT–NMR). The identification of the bioactive endophyte was undertaken by polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis of the internally transcribed spacer regions and intervening 5.8S nrRNA gene and partial beta-tubulin gene. The crude extract from the fungal isolate Penicillium skrjabinii exhibited antimicrobial activity against S. aureus and E. coli at 0.03 and 0.09 mg/mL, respectively. Bioassay-guided isolation was carried out and the major compound, dibutyl phthalate (which is known to be produced by micro-organisms) was isolated from the most active fraction. A number of endophytes displayed a synergistic interaction with the host plant. This is the first study reporting P. skrjabinii as an endophyte that produces dibutyl phthalate and further provides insight to endophytic interactions with the host plant (Pelargonium sidoides root).

Cutaneous Exophiala oligosperma Infection in a Patient with Bullous Pemphigoid with a Review of the Literature.

Phaeohyphomycosis is an infection caused by a heterogeneous group of melanized fungi. Human infections due to members of genus Exophiala are rare but may occur at any part of the body. We herein report a case of an 85-year-old male with a history of bullous pemphigoid who presented with a chronic, non-healing wound on his right dorsal hand for a month. Direct microscopy of a pus sample from the base of the ulcer revealed strands of pigmented, moniliform hyphae. The isolate was identified as E. oligosperma based on morphological characters and sequencing of the rDNA internal transcribed spacers (ITS) and partial beta-tubulin gene. The patient received a three-month course of oral itraconazole with no recurrence.

Aspergillus colonization in patients with chronic obstructive pulmonary disease.

Background and Purpose:Chronic obstructive pulmonary disease (COPD) has been recognized as a risk factor for invasive aspergillosis. Airway colonization by Aspergillus species is a common feature of chronic pulmonary diseases. Nowadays, the incidence of COPD has increased in critically ill patients. The aim of the present study was to isolate and identify Aspergillus colonies in the respiratory tract of COPD patients.Materials and Methods:This study was performed on 50 COPD patients, who were aged above 18 years, and were in intensive care units of three hospitals in Sari, Iran, for at least six days. All the samples obtained from sputum, bronchoalveolar lavage, and tracheal aspirates were cultured for fungi each week. According to the conventional techniques, Aspergillus isolates were initially based on growth and standard morphological characteristics. To confirm the identification of grown Aspergillus, the partial beta-tubulin gene was sequenced using specific primers. Results:A total of 50 patients, who met our inclusion criteria, were enrolled in the study during 2012-14. The results showed that 27 (54%) and 23 (46%) of the participants were male and female, respectively. The majority of the patients developed dyspnea followed by hemoptysis, chest pain, and high fever. Corticosteroids and broad-spectrum antibacterial agents were administered to 75% and 80% of the patients, respectively. Based on the conventional and molecular approaches, A. fumigatus (seven cases; 43.7%), A. flavus (five cases; 31.2%), A. niger (one case; 6.2%), A. terreus (one case; 6.2%), A. orezea (one case; 6.2%), and A. tubingensis (one case; 6.2%) were recovered.Conclusion:Recovery of Aspergillus species from the respiratory tract of COPD patients with pneumonia indicates two possibilities: either colonization or invasive aspergillosis.

담수에 자생하는 수생식물에서 분리된 내생균류의 지베렐린 생산과 동정

수생식물 샘플은 낙동강과 금호강이 만나는 달성습지에서 자라풀을 채집하였다. 자라풀의 뿌리에서 내생균류를 분리하고 형태가 다른 균주를 관찰하여 최종적으로 16개 균주를 선발하였다. 내생균류의 배양여과액은 식물생장촉진활성 검정을 위하여 난장이벼에 처리하여 스크리닝하였으며, HD1008 균주가 식물생장촉진활성이 가장 높은 것으로 확인되었다. HD1008 균주의 배양여과액을 HPLC와 GC/MS-SIM을 이용하여 분석하였고, HD1008 균주가 식물호르몬인 지베렐린 <TEX>$GA_1$</TEX> (1.2 ng/100 mL)과 <TEX>$GA_4$</TEX> (5 ng/100 mL) 를 생산하는 것을 정량분석을 통하여 확인하였다. 또한, HD1008균주의 beta-tubulin 유전자 염기서열을 이용하여 동정에 이용하였으며, 분자적인 방법과 형태적인 방법으로 관찰하였을 때, 지베렐린을 생산하는 새로운 P. trzebinskii로 동정되었다. Aquatic plant Hydrocharis dubia (Blume) Backer was collected from the Dalsung wetland in Daegu. Sixteen endophytic fungi with different colony morphologies were isolated from the roots of aquatic plants. Waito-c rice (WR) seedlings were treated with fungal culture filtrates (FCF) for screening plant growth-promoting activity. In the results, HD1008 strain isolated from aquatic plant showed highest plant growth-promoting activity. The FCF of HD1008 strain was analyzed using gas chromatography mass spectrometry (GC/MS) with selected ion monitoring (SIM). Analysis of the FCF of HD1008 strain found that it contained gibberellins (GA) (<TEX>$GA_1$</TEX>, 1.2 ng/100 mL; <TEX>$GA_4$</TEX>, 5 ng/100 mL). Phylogenetic tree of HD1008 strain was constructed by partial internal transcribed spacer (ITS) region and partial beta-tubulin gene sequences. Therefore, we describe HD1008 strain as a new gibberellin-producing Penicillium trzebinskii based on morphological and molecular characteristics.

Aspergillus clavatus Y2H0002 as a New Endophytic Fungal Strain Producing Gibberellins Isolated from Nymphoides peltata in Fresh Water

Eighteen endophytic fungi with different colony morphologies were isolated from the roots of Nymphoides peltata growing in the Dalsung wetland. The fungal culture filtrates of the endophytic fungi were treated to Waito-c rice seedling to evaluate their plant growth-promoting activities. Culture filtrate of Y2H0002 fungal strain promoted the growth of the Waito-c rice seedlings. This strain was identified on the basis of sequences of the partial internal transcribed spacer region and the partial betatubulin gene. Upon chromatographic analysis of the culture filtrate of Y2H0002 strain, the gibberellins (GAs: GA1, GA3, and GA4) were detected and quantified. Molecular and morphological studies identified the Y2H0002 strain as belonging to Aspergillus clavatus. These results indicated that A. clavatus improves the growth of plants and produces various GAs, and may participate in the growth of plants under diverse environmental conditions.

Identification and species status of the mango biotype of Colletotrichum gloeosporioides in Ghana

Research work was carried out to identify and ascertain the species status of the mango biotype of Colletotrichum gloeosporioides infecting mangoes in Ghana. Forty five isolates of Colletotrichum species were collected from 12 districts in Ghana while five each were obtained from mango fruits from Florida, Mexico and Puerto Rico. The entire internal transcribed spacer region, partial beta-tubulin gene and partial glyceraldehyde-3-phosphate dehydrogenase gene of isolates were sequenced and used in phylogenetic studies. The results of the sequence analysis of the first ribosomal transcribed spacer (ITS 1) region showed that 35 % of the isolates from Ghana and all the five isolates from Mexico were the mango biotype of C. gloeosporioides, while the others were not. Phylogenetic studies showed that the mango biotype of the pathogen was Colletotrichum asianum but not C. gloeosporioides as previously thought. However, the other isolates that were not the mango biotype were identified as Colletotrichum siamense and Colletotrichum species which had probably cross-infected mango from other fruit crops in the field.

First Report of Togninia minima Perithecia on Esca- and Petri-Diseased Grapevines in South Africa

Esca and petri diseases are important grapevine trunk diseases in South Africa and most other grape-producing countries. The causal pathogens are Phaeomoniella chlamydospora and several species of Phaeoacremonium. In total, 25 species of Phaeoacremonium have been isolated from grapevines of which seven species have been linked to Togninia teleomorphs obtained through in vitro mating studies (3). Of these species, only perithecia of T. minima, T. fraxinopennsylvanica, and T. viticola have been found on grapevines in California (1,2,4). T. minima is heterothallic, and although both mating types are present in South African vineyards, perithecia have never been observed (3). In the current study, grapevine cordons and trunks were collected from vineyards and rootstock mother vines within Western Cape Province for examination in the laboratory under a dissecting microscope. The grapevines displayed general decline symptoms, including reduced vegetative growth, dead or dying shoots and cordons, as well as internal vascular streaking and/or a red/black/brown margin next to decayed wood typically associated with esca and petri disease. Rootstock mother vines were apparently healthy, although many old, cracked pruning wounds were visible. Togninia-like perithecia with distinctive long necks were found along the wood crevices, often on old pruning wounds. The perithecia were removed and placed on microscope slides with sterile water. Structures were measured and slides were washed with 500 μl of sterile water onto potato dextrose agar amended with chloramphenicol (250 mg/liter). Ascospores were allowed to germinate overnight to obtain single ascospore colonies. Perithecia were found on cultivars Muscat d' Alexandrie and Pinotage (Vitis vinifera) at Stellenbosch in May 2011 and on Ramsey (V. champinii) rootstock mother vines at Slanghoek in June 2012. Perithecia were globose to subglobose, black, and often embedded in the wood tissue but also present on the surface of the wood. The length of the necks was 250 to 300 × 47.5 to 55 μm. The asci were hyaline and ranged from 16 to 25 × 3.5 to 5 μm. Ascospores were hyaline, ellipsoid, and ranged from 5 to 6 × 1.5 to 2 μm. These measurements were similar to those reported by Mostert et al. (3) and Rooney et al. (4). Colony growth was typical of T. minima. DNA was extracted from the colonies and the partial betatubulin gene was amplified and sequenced using the primers T1 and Bt2b. Sequences were deposited into GenBank (JX962864 to 67). Based on a megablast search of the NCBI's GenBank nucleotide database, 100% similarity was found with other T. minima sequences (JQ691670.1, HQ605018.1, HQ605014.1; identities = 647/647 [100%], gaps = 0/647 [0%]). To our knowledge, this is the first report on the occurrence of T. minima perithecia on grapevines in Western Cape Province of South Africa. The removal of dead spurs and cordons will be instrumental in lowering the inoculum originating from perithecia, especially in rootstock mother blocks where no control strategies are applied for petri disease or esca. Spore trapping studies are currently in progress to study spore release patterns in order to determine whether pruning wounds are at risk during traditional pruning periods.

Isolation and Identification of Fungi from a Meju Contaminated with Aflatoxins

A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial beta-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

Cyphellophora and its relatives in Phialophora: biodiversity and possible role in human infection

Cyphellophora is a genus of black yeast-like fungi characterised by having simple phialides with multiseptate, curved conidia. Judging from SSU and LSU data, Cyphellophora was found to be located in a well-supported clade within the Chaetothyriales comprising a number of species occurring on human skin and nail. Cyphellophora is phylogenetically close to Phialophora europaea, P. reptans and P. oxyspora, though morphologically these species produce single-celled phialoconidia rather than multiseptate ones. Pseudomicrodochium suttonii and P. fusarioides have dark colonies and phylogenetically fit in with Cyphellophora; the type species of Pseudomicrodochium, P. aciculare, has similar, septate conidia but has a hyaline thallus. In the present study, multilocus phylogenetic analyses were combined with morphology and physiology. Sequences of the internal transcribed spacer region, the DNA dependent RNA polymerase II largest subunit and the partial beta tubulin gene were analysed for a set of 30 strains. Two novel species, Cyphellophora pauciseptata and Phialophora ambigua were discovered. Cyphellophora eucalypti was reduced to synonymy of C. guyanensis. The role of the studied fungi between colonization and infection of human skin was discussed. Putative virulence factors for these black yeast-like fungi were hypothesized to be the ability to assimilate monoaromatic hydrocarbons, to produce melanin pigments, and to tolerate the temperature of epidermal human skin.

Babesia microti-like infections are prevalent in North American foxes

Babesia microti-like organisms have recently been identified as a cause of hemolytic anemia and azotemia in European dogs. A genetically and morphologically similar B. microti-like parasite has been identified in two foxes from North America. In order to assess the prevalence of this parasite in North American wild canids we screened blood samples from coyotes ( Canis latrans) and red foxes ( Vulpes vulpes) from eastern Canada and red foxes and gray foxes ( Urocyon cinereoargenteus) from North Carolina, USA for the presence B. microti-like DNA by polymerase chain reaction. Thirty-nine percent (50/127) of the red fox samples, 26% (8/31) of the gray fox samples and none (0/12) from the coyote samples tested positive for the presence of B. microti-like DNA. Partial 18S ribosomal ribonucleic acid and beta-tubulin genes from the North American B. microti-like parasites of foxes were sequenced and samples from six domestic dogs from Spain that were infected with a B. microti-like parasite were analyzed for comparison. Partial 18S ribosomal ribonucleic acid and beta-tubulin gene sequences from the North American B. microti-like parasites of foxes were nearly identical to those previously reported from foxes as well as those from domestic dogs from Spain characterized in this study. Interestingly, partial beta-tubulin gene sequences characterized from the B. microti-like parasites of domestic dogs from Spain in this study were different from those previously reported from a Spanish domestic dog sample which is believed to be a pseudogene. The ability of the North American B. microti-like parasite to infect and induce disease in domestic dogs remains unknown. Further studies investigating the pathogenic potential of the North American B. microti-like parasite in domestic dogs are indicated.

Eupenicillium saturniforme, a New Species Discovered from Northeast China

A new Eupenicillium species, E. saturniforme was isolated from soil in Jilin Province, northeast China. Morphologically, it resembled E. shearii and E. tropicum, but is distinguished from them by slowly maturing cleistothecia, lenticular ascospores with nearly smooth-walled convex surfaces, strictly velutinous colony texture with abundant conidiogenesis, robust biverticillate penicilli, apically vesiculate metulae and rough-walled stipes and conidia. The partial beta-tubulin gene sequence of the new species (EU644080) showed relationship with Penicillium glabrum in the BLAST search in GenBank. Further analyses of partial calmodulin and ribosomal DNA internally transcribed spacer 1-5.8S-internally transcribed spacer 2 (rDNA ITS1-5.8S-ITS2) sequence data confirmed that E. saturniforme fell in the clade with P. glabrum, P. lividum, P. purpurascens, P. spinulosum and P. thomii of Subgenus Aspergilloides. However, E. saturniforme is a distinctive species lacking close relatives among described species of penicillia.

In vitroactivity of nine antifungal agents against clinical isolates ofAspergillus calidoustus

This study analyzes Aspergillus isolates from the Spanish National Centre for Microbiology collection, which were identified morphologically as members of Aspergillus section Usti. Strains were identified through the analysis of the Internal Transcribed Spacer regions and partial beta tubulin gene sequences. One Aspergillus pseudodeflectus isolate and eight Aspergillus calidoustus isolates were detected in this panel of clinical strains. Terbinafine and the echinocandins micafungin and anidulafungin, were the drugs most active against these species.

Direct molecular evidence supports long‐spored microsclerotial isolates of Verticillium from crucifers being interspecific hybrids

There is no published evidence directly supporting the suggestion that long‐spored isolates of Verticillium dahliae are interspecific hybrids. Retention in the nuclear genome of long‐spored (amphihaploid) isolates of two sequence types where non‐hybrid short‐spored (haploid) isolates carry only single sequence types would provide such evidence. PCR amplicons for a partial β‐tubulin gene and a 5S rRNA‐associated sequence were cloned and sequenced to provide multiple sequences for individual isolates. For the 5S rRNA sequence, two sequence types were found for long‐spored isolates, but only one for the haploid isolates. For the β‐tubulin gene, two or three sequence types were found in long‐spored isolates, but only one in most haploid isolates. One haploid isolate gave two sequence types, one like that from the other haploid isolates and the second like the short‐sequence type from some long‐spored isolates. These results support the long‐spored isolates being interspecific hybrids between a ‘parental’ species similar to V. dahliae (haploid) and an unidentified second species. The presence of a sequence type in some long‐spored isolates similar to that in one haploid isolate suggests a third ‘parental’ species may have been involved, possibly via a short‐spored isolate that was itself hybrid.

Cylindrocarpon pauciseptatum sp. nov., with notes on Cylindrocarpon species with wide, predominantly 3-septate macroconidia

A Cylindrocarpon species with up to 10 μm wide, straight and predominantly 3-septate macroconidia, subglobose to ovoidal microconidia and chlamydospores, is described as Cyl. pauciseptatum. It is most similar to Cyl. austrodestructans but no chlamydospores and microconidia are formed in the latter. Similar macroconidia also occur in Cyl. theobromicola, which forms oval to ellipsoidal microconidia at least sparsely and has slightly curved macroconidia, and Cyl. destructans var. crassum, which forms abundant 1-celled microconidia. DNA sequence data of the internal transcribed spacer regions 1 and 2 plus the 5.8S rDNA and the partial beta-tubulin gene were used for phylogenetic inferences. Cylindrocarpon pauciseptatum and Cyl. macrodidymum are monophyletic and are closely related to other species of Cylindrocarpon sensu stricto including members of the Cyl. destructans (teleomorph, Neonectria radicicola) species complex, which accommodates Cyl. liriodendri (teleomorph, Neon. liriodendri), Cyl. destructans var. crassum and Cyl. austrodestructans (teleomorph, Neonectria austroradicicola comb. nov.). Cylindrocarpon theobromicola is distantly related to species of Cylindrocarpon sensu stricto or Neonectria sensu stricto. It clustered among cylindrocarpon-like species with curved macroconidia, of which some belong to the Neon. mammoidea group. Relatively voluminous cells in sporodochial conidiophores of Cyl. theobromicola resembled those described for Campylocarpon, which is closely related to members of the Neon. mammoidea group including Cyl. theobromicola. Cylindrocarpon pauciseptatum has been isolated from roots of Vitis spp. in South-eastern Europe (Slovenia) as well as New Zealand, where it also occurs on roots of Erica melanthera.

New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati

Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology, extrolite production and partial β-tubulin, calmodulin, and actin gene sequences. The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences. Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology, short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface. Aspergillus turcosus can be distinguished by velvety, gray turquoise colonies and short, loosely columnar conidial heads. The four new taxa also have unique extrolite profiles, which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis. A. turcosus produced kotanins. N.denticulata sp. nov., N. assulata sp. nov., N. galapagensis sp. nov., and A. turcosus sp. nov. are described and illustrated.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-007-9183-1) contains supplementary material, which is available to authorized users.

Genetic Variation Among Penicillium crustosum Isolates from Arctic and other Ecological Niches

Penicillium crustosum is an important and panglobal contaminant of lipid- and protein-rich foods and feeds. Although it is infrequent in extremely cold environments, we isolated a high number of P. crustosum strains from Arctic coastal, but particularly, subglacial environments in Svalbard, Norway. P. crustosum is extremely consistent in its phenotypic properties, including morphology, physiology, and secondary metabolite production. However, some Arctic isolates differed from other Arctic and non-Arctic strains in their weak growth on creatine and in the production of the secondary metabolite andrastin A. In this study, we characterized genetic variability of P. crustosum strains originating from different Arctic and non-Arctic environments using amplified fragment length polymorphism (AFLP) and, in addition, M13 minisatellite fingerprinting and partial beta-tubulin gene sequencing. Most of the Arctic strains (85%) showed a relatively low variability and polymorphism level. They produced nine different AFLP genotypes grouped into two clusters in accordance with glacier origin and creatine utilization. The rest of the Arctic isolates and isolates from various non-Arctic environments displayed a much greater degree of genetic variability. It seems that in stressful glacial environment low microbial genetic variation is represented by only a few adapted genotypes that were not recovered from nonpolar environments.

Penicillium svalbardense, a new species from Arctic glacial ice

During investigations of mycobiota in the coastal Arctic polythermal glaciers, different species of the ubiquitous genus Penicillium were isolated from the extreme subglacial environment. A group of Penicillium strains was obtained that did not belong to any known Penicillium species. This species was isolated in high numbers from the Kongsvegen subglacial ice and was not detected in the surrounding environment. A detailed analysis of secondary metabolite profiles, physiological and morphological characteristics, and partial beta-tubulin gene sequences showed that the proposed new species Penicillium svalbardense is closely related but not identical to Penicillium piscarium and Penicillium simplicissimum. It differs in the production of secondary metabolites and in the morphological features of conidia and penicilli, and it is therefore described as a new species.

Ophiostomatoid fungi associated with the northern spruce engraver, Ips perturbatus, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial beta-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.

Neonectria liriodendri sp. nov., the main causal agent of black foot disease of grapevines

Black foot disease is a serious disease of grapevine crops in most areas where vines are grown. Mainly two species of Cylindrocarpon, C. destructans and C. macrodidymum, are associated with this disease. Recent studies have revealed a tremendous molecular variation within the former but only slight molecular variation within the latter, indicating that C. destructans presents a complex of several species The present study elucidates the taxonomic status of C. destructans-like isolates associated with black foot disease of grapevines. Grapevine isolates were studied morphologically, subjected to DNA analyses of their ITS and partial beta-tubulin genes, and were mated in all combinations in vitro. Cylindrocarpon destructans strains isolated from grapevines in Europe and South Africa appeared morphologically and genetically identical, and had identical ITS and partial beta-tubulin gene sequences. Phylogenetic analyses placed these strains in a clade closely related but clearly distinct from other clades with C. destructans-like anamorphs obtained from various herbaceous or woody hosts. Only the ex-type strain of Cylindrocarpon liriodendri had identical sequences to strains isolated from grapevines, and could also not be distinguished by morphological characters. The grapevine isolates are therefore reidentified here as Cylindrocarpon liriodendri. Cylindrocarpn liriodendri formed perithecia in heterothallic conditions and the holomorph of this species is described as Neonectria liriodendri sp. nov. Neonectria liriodendri is genetically distinct from the ex-type strain of Neonectria radicicola, which originated from Cyclamen in Sweden. Both ex-type strains also differ from at least two other clades comprising additional C. destructans-like strains. Many of these strains originated from Panax sp., which is the host of the type of C. destructans. Our phylogenetic analyses indicate that C. destructans is not the anamorph of N. radicicola and that N. liriodendri, N. radicicola and several C. destructans-like taxa may have evolved independently within the same phylogenetic species complex.

Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and -tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.