Molecular phylogenetic analyses reveal three new Ceratocystis species and provide evidence for geographic differentiation of the genus in Africa

The emergence of wattle wilt disease on non-native Acacia mearnsii trees in Africa, caused by the indigenous fungus Ceratocystis albifundus, has highlighted a need to better understand the diversity, ecology and distribution of Ceratocystis species in natural African environments. In this study we applied phylogenetic inference to identify and characterize isolates of Ceratocystis collected in a natural savanna ecosystem in South Africa. Three new species were recognized and are described as C. cryptoformis sp. nov. in the C. moniliformis complex, as well as C. thulamelensis sp. nov. and C. zambeziensis sp. nov., both residing in the C. fimbriata complex. Incorporating the new species into global phylogenies of Ceratocystis provided insights into the patterns of evolution and biogeography of this group of fungi. Notably, the African continent was identified as an important centre of diversification of Ceratocystis spp., from which several lineages of these fungi were shown to have radiated.     

A new wilt and die-back disease of Acacia mangium associated with Ceratocystis manginecans and C. acaciivora sp. nov. in Indonesia

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.     

Constant association of ophiostomatoid fungi with the bark beetle <Emphasis Type="Italic">Ips subelongatus</Emphasis> invading Japanese larch logs

Ceratocystiopsis, Ceratocystis, Grosmannia, and Ophiostoma species were isolated from Ips subelongatus and beetle-infested Japanese larch logs collected at several areas in central and northern Honshu Island, Japan, to determine constant associates of I. subelongatus. Ceratocystiopsis minuta, two species of Ceratocystis, three species of Grosmannia, and four species of Ophiostoma were isolated. Of the fungi isolated in the present study, G. laricis, O. brunneociliatum, and O. piceae were constant associates of the beetles. Ceratocystis fujiensis, Ceratocystiopsis minuta, and Ophiostoma sp. F were occasionally isolated with high frequencies of occurrence but were not consistent associates. Ceratocystis fujiensis was most often isolated as the leading fungal invasion in the sapwood of Japanese larch logs invaded by I. subelongatus, confirming that the fungus acts as a primary invader of sapwood in beetle-attacked logs. Contribution No. 221, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba     

Genetic variation in the wattle wilt pathogenCeratocystis albofundus

Ceratocystis albofundus is an important wilt pathogen on exoticAcacia mearnsii trees in South Africa. It is known only from this country and has also been reported from nativeProtea spp., but it is not clear if the pathogen is native or introduced to South Africa. This study was conduced to determine the nuclear and mitochondrial gene diversity in a population ofC. albofundus and to compare this diversity with that of otherCeratocystis species. Isolates were collected from a number of geographic regions in South Africa. Total genomic, DNA was extracted from each isolate, digested withPstl and probed with the radioactively labelled oligonucleotide marker (CAT)₅ to determine nulear DNA diversity. For the determination of mitochondrial DNA diversity, the RFLPs ofHaeIII digests were scored directly without probing. Nei’s gene diversity (H) was determined and a distance matrix was developed for each set of markers and analyzed using UPGMA. TheC. albofundus population was found to have a high level of both nuclear and mitochondrial gene diversity when compared with published data of populations of otherCeratocystis spp. This further supports the hypothesis thatC. albofundus is native to South Africa.     

Ceratocystis species, including two new species associated with nitidulid beetles, on eucalypts in Australia

The genus Ceratocystis includes important fungal pathogens of trees, including Eucalyptus spp. Ironically, very little is known regarding the diversity or biology of Ceratocystis species on Eucalyptus species in Australia, where most of these trees are native. The aim of this study was to survey for Ceratocystis spp., and their possible insect associates, on eucalypts in Australia and thus to establish a foundation of knowledge regarding these fungi on the continent. Collections were made in three states of Australia from wounds on trees, as well as from nitidulid beetles associated with these wounds. Ceratocystis spp. were identified based on morphology and multigene sequence comparisons. Of the 54 isolates obtained, two previously unknown species of Ceratocystis were found and these are described here as Ceratocystis corymbiicola sp. nov. and Ceratocystis tyalla sp. nov. Furthermore, the distribution of Ceratocystis pirilliformis is expanded to include Eucalyptus spp. in Tasmania.     

<Emphasis Type="Italic">Ceratocystis ficicola</Emphasis> sp. nov., a causal fungus of fig canker in Japan

The causal fungus of Ceratocystis canker of fig in Japan is described as Ceratocystis ficicola sp. nov. This species is characterized by galeated ascospores and is similar to Ceratocystis fimbriata sensu lato. However, the perithecia of the new species are much larger than those of C. fimbriata. The fungus grows more rapidly than C. fimbriata sensu stricto at 25°C but more slowly at 30°C. Molecular analysis of the nucleotide sequences of rDNA ITS regions showed that C. ficicola is phylogenetically placed in the clade of C. fimbriata s. l. but is easily distinguishable from other species of C. fimbriata s. l.     

Ophiostomatoid fungi associated withIps cembrae in Japan and their pathogenicity of Japanese larch

Six ophiostomatoid fungi, i.e.,Ceratocystiopsis minuta, Ceratocystis laricicola, Ophiostoma brunneo-ciliatum, O. laricis, O. piceae andOphiostoma sp., were isolated from the galleries ofIps cembrae on Japanese Iarch (Larix kaempferi) logs in central Honshu, Japan. Japanese larch trees approximately 30 yr old were inoculated with all six fungi.Ceratocystis laricicola produced the largest lesions on the inner bark around the inoculation point and the largest dry zone in the sapwood. Furthermore,C. laricicola was the only fungus associated withI. cembrae that had the ability to kill Japanese larch, death occurring in 30-yr-old trees within 3.5 mo after inoculation.     

Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew

To investigate the phylogenetic relationships among the powdery mildew fungi of some economically important tropical trees belonging to Oidium subgenus Pseudoidium, we conducted molecular phylogenetic analyses using 30 DNA sequences of the rDNA internal transcribed spacer (ITS) regions and 26 sequences of the domains D1 and D2 of the 28S rDNA obtained from the powdery mildews on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp. The results indicate that the powdery mildew fungi isolated from these tropical trees are closely related to one another. These powdery mildews are also closely related to E. alphitoides (including Erysiphe sp. on Quercus phillyraeoides). Because of the obligate biotrophic nature of the powdery mildew fungi, the relationship between powdery mildews and their host plants is conservative. However, the present study suggests that a particular powdery mildew species has expanded its host ranges on a wide range of the tropical trees. This article also suggests that a powdery mildew fungus distributed in temperate regions of the Northern Hemisphere expanded its host ranges onto tropical plants and may be a good example of how geographical and host range expansion has occurred in the Erysiphales.     

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.     

Tyrosol and tryptophol produced by <Emphasis Type="Italic">Ceratocystis adiposa</Emphasis>

Extracellular tyrosol and tryptophol were produced and isolated from the phytopathogen fungus Ceratocystis adiposa cultivated in a rich broth. These compounds have not been previously reported in this Ceratocystis species, as its identification was established through spectroscopic methods (¹H NMR, ¹³C NMR and MS). Productivity of tyrosol and tryptophol was 65.3 and 44 μg l⁻¹ day⁻¹, respectively, which were values higher than those reported to date. Tyrosol is a compound of pharmaceutical interest showing antioxidating activity, a property used in the treatment of atherosclerosis. Tryptophol is reported to have antibiotic and phytotoxic activity.     

The oldest stylophoran echinoderm: a new <Emphasis Type="Italic">Ceratocystis</Emphasis> from the Middle Cambrian of Germany

A new stylophoran (Ceratocystis prosthiakida sp. nov.) from the early Middle Cambrian of the Franconian Forest (north-east Bavaria, Germany) is described with the aid of computed tomography, a powerful means of visualizing fossil anatomy. Investigation in this manner enabled previously inaccessible, buried parts of the fossil to be imaged and studied. In addition, a procedure for digitally correcting plate articulations was implemented. Ceratocystis prosthiakida sp. nov. possesses a thin anterior right spine which clearly differentiates it from other species of Ceratocystis. Similar to other Ceratocystis species, C. prosthiakida sp. nov. lived in storm-dominated shallow marine conditions. The species is from the Galgenberg Formation, which has an early Agdzian age in the unified West Gondwanan chronostratigraphy. As a result, it represents the oldest stylophoran known to date.     

New proteins orthologous to cerato-platanin in various <Emphasis Type="Italic">Ceratocystis</Emphasis> species and the purification and characterization of cerato-populin from <Emphasis Type="Italic">Ceratocystis populicola</Emphasis>

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A new species of Melampsora on Populus yunnanensis from China

Melampsora nujiangensis, a rust fungus found on Populus yunnanensis in China, is described as a new species. Light and scanning electron microscopy with herbarium specimens of the rust fungus show that the shape of its urediniospores differs from that of other known species of Melampsora, and its urediniospore walls are thinner than the other species. Furthermore, in phylogenetic trees based on the DNA sequences (28S and ITS) the rust fungus is phylogenetically separated by high bootstrap values. These results indicate that the fungus is an isolated species among the genus Melampsora. Contribution no. 202 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

First report of Albizia lebbeck dieback caused by Scytalidium dimidiatum in Oman

Over 200 Albizia lebbeck trees at Sultan Qaboos University campus wilted and died. The symptoms were dieback of large branches due to infection by Scytalidium dimidiatum. The fungus has also infected Ficus benghalensis, F. carica, F. retusa, Thespesia populnea, Delonix regia and Peltophorum petrocarpum. This is the first report of the fungus in Oman and on A. lebbeck, T. populnea, D. regia and P. petrocarpum. The strain of this fungus has not been found to cause human disease in Oman. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identity of a powdery mildew fungus occurring on Paeonia and its relationship with Erysiphe hypophylla on oak

A powdery mildew fungus found on Paeonia lutea at the Botanical Garden of Geneva (Switzerland) was identified as Erysiphe hypophylla based on morphological observations. The occurrence of E. hypophylla on Paeonia seemed curious, because host plants of this species have been restricted to a few Quercus species of the family Fagaceae. In this study, we determined the rDNA sequences of the powdery mildew specimens on Paeonia and E. hypophylla on Quercus to confirm the identity of the Paeonia fungus. The three sequences from the specimens on P. lutea were identical to one another in both ITS and 28S rDNA regions and also to the sequences of E. hypophylla on Q. robur, which supports the identification that the fungus on P. lutea is E. hypophylla. However, these sequences were also identical to the sequences of E. alphitoides on Quercus spp. and Oidium mangiferae on mango. This result suggests a possibility that E. hypophylla is conspecific to E. alphitoides. Further study is required to clarify whether E. hypophylla is a synonym of E. alphitoides or a distinct species.     

Fungal Infection of Mantis Shrimp (<Emphasis Type="Italic">Oratosquilla oratoria</Emphasis>) Caused by Two Anamorphic Fungi Found in Japan

Two fungal pathogens of the mantis shrimp (Oratosquilla oratoria) in Yamaguchi and Aichi Prefectures, Japan are described as the new species Plectosporium oratosquillae and Acremonium sp. (a member of the Emericellopsis marine clade). Both fungi infect the gills of the mantis shrimp, which become brown or black due to melanization. The former species is characterized by its slow growth on artificial seawater yeast extract peptone glucose (PYGS) agar, pale yellow to pale orange or grayish yellow colonies, short cylindrical solitary phialides with a wavy tip, and one-celled ellipsoidal conidia. Although lacking the two-celled conidia demonstrated by the type species Plectosporium tabacinum, the taxonomic placement of the new species was confirmed by DNA sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rDNA and ITS2). Acremonium sp., the other causal pathogen, differs from P. oratosquillae by its fast growth on PYGS agar, pale orange to salmon-colored colonies, long, slender conidiophores consisting of solitary phialides with tips lacking an undulate outline, and typically cylindrical conidia. Analysis of ITS and β-tubulin gene sequences placed this fungus within the phylogenetically distinct Emericellopsis (anam. Acremonium) marine clade. Various physiological characteristics of both pathogens were also investigated. This is the first report of a fungal infection found on the mantis shrimp in Japan.     

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.