J. Steimel

Phylogeny and taxonomy of the Ophiostoma piceae complex and the Dutch elm disease fungi

The Ophiostoma piceae complex forms a monophyletic group of insect-dispersed pyrenomycetes with synnemata (Pesotum) and micronematous (Sporothrix) synanamorphs. Other species of Ophiostoma outside ...

Genetic variation in eastern North American and putatively introduced populations of Ceratocystis fimbriata f. platani

The plant pathogenic fungus Ceratocystis fimbriata f. platani attacks Platanus species (London plane, oriental plane and American sycamore) and has killed tens of thousands of plantation trees and street trees in the eastern United States, southern Europe and Modesto, California. Nuclear and mitochondrial DNA fingerprints and alleles of eight polymorphic microsatellite markers of isolates of C. fimbriata from these regions delineated major differences in gene diversities. The 33 isolates from the eastern United States had a moderate degree of gene diversity, and unique genotypes were found at each of seven collection sites. Fingerprints of 27 isolates from 21 collection sites in southern Europe were identical with each other; microsatellite markers were monomorphic within the European population, except that three isolates differed at one locus each, due perhaps to recent mutations. The genetic variability of C. fimbriata f. platani in the eastern United States suggests that the fungus is indigenous to this region. The genetic homogeneity of the fungus in Europe suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction of a single genotype. This supports the hypothesis that the pathogen was introduced to Europe through Naples, Italy during World War II on infected crating material from the eastern United States. The Californian population may also have resulted from introduction of one or a few related genotypes because it, too, had a single nuclear and mitochondrial genotype and limited variation in microsatellite alleles.

Deletion of the MAT-2 mating-type gene during uni-directional mating-type switching in Ceratocystis.

Abstract Ceratocystis eucalypti is strictly heterothallic, with single ascospore strains representing one of two opposite mating types. Most other Ceratocystis species, including C. virescens, C. pinicola, and C. fimbriata, are homothallic. In the homothallic species, the MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. The current hypothesis is that self-fertility of MAT-2 strains is due to the deletion of the MAT-2 mating-type gene, resulting in the expression of the MAT-1 mating type. These mutant MAT-1 strains are able to cross with MAT-2 strains. Part of the MAT-2 mating-type gene in C. eucalypti, C. pinicola, and C. fimbriata was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA-binding motif. The expected approximately 300-bp PCR products were cloned and sequenced. Specific primers were designed that amplified 210-bp fragments only in MAT-2 isolates of C. eucalypti, C. virescens, C. pinicola, and C. fimbriata. These fragments were present in self-fertile field isolates and self-fertile progeny but were absent in the self-sterile (MAT-1) progeny from selfings of C. virescens, C. pinicola, and C. fimbriata, thus supporting the hypothesis that the MAT-2 mating-type gene is deleted during uni-directional mating-type switching. A Southern-blot analysis was performed to confirm the deletion of MAT-2 gene in self-sterile progeny. The DNA sequence data for the C. eucalypti MAT-2 mating-type gene was increased to 1371-bp using TAIL-PCR and uneven PCR, representing a portion of the complete MAT-2 gene DNA sequence.

Isozyme variation and species delimitation in the Ceratocystis coerulescens Complex

Nineteen electrophoretic phenotypes (unique combinations of electromorphs) were found among 98 isolates of Ceratocystis coerulescens and mor? phologically similar species using 10 isozymes. Analysis of the isozyme data and morphological comparisons suggested that there are five variants of C. coerulescens found on conifers: three are associated with blue-stain of Picea or Pinus, one (C. coerulescens f. douglasii) with blue-stain of Pseudotsuga, and one associated with the bark beetle Dendroctonus rufipennis on Picea. Ceratocystis polonica and C. laricicola, associated with bark beetle species in the genus Ips on Picea and Larix, respec? tively, had similar isozymes, are morphologically in- distinguishable from each other, and should probably be synonymized. Ceratocystis virescens, cause of stain of hardwoods and sapstreak disease of Acer saccharum, is distinct from the conifer species of Ceratocystis in iso? zyme electromorphs and anamorph morphology. Iso? zymes of C. virescens show some similarity to those of two Australian species, an undescribed species of Cer? atocystis from Eucalyptus and Chalara australis. The Chalara states of these three hardwood species and Chalara neocaledoniae are morphologically similar.

Comparison of isozymes, rDNA spacer regions and MAT-2 DNA sequences as phylogenetic characters in the analysis of the Ceratocystis coerulescens complex

The genus Ceratocystis sensu stricto in- cludes species that are insect-vectored, plant patho- gens, occurring mainly on angiosperm hosts. Species in the Ce. coerulescens complex form a morphologi- cally distinct group that are pathogens on gymno- sperms (Pinaceae) and angiosperms. In recent years, species in the Ce. coerulescens complex have been in- tensively studied based on their morphology and iso- zymes, as well as on their molecular characteristics. The aim of this study was to compare the phylogeny of these species inferred from the MAT-2 HMG box DNA sequences to the phylogenies inferred from partial rDNA sequences and isozyme data. Part of the MAT-2 HMG box of species in the Ce. coerulescens complex was amplified using PCR. The PCR frag- ments were sequenced and phylogenetically analysed using parsimony, with Ce. fimbriata as the outgroup taxon. Species in the Ce. coerulescens complex on gymnosperms formed a single clade, inferred to be a sister-taxon to the clade that included the species on angiosperm hosts, thus supporting the hypothesis that the adaptation to gymnosperms was a single evo- lutionary event in the history of Ceratocystis. The phy- logeny inferred from the MAT-2 HMG box sequenc- es was found to be similar to the phylogeny based on the internal transcribed spacer (ITS) regions of the

Species Delimitation and Host Specialization of Ceratocystis laricicola and C. polonica to Larch and Spruce

Ceratocystis laricicola and C. polonica are fungal symbionts of bark beetle species of the genus Ips that attack species of Larix and Picea, respectively, across Eurasia. Earlier studies found that these fungal species were morphologically identical, had similar isozymes patterns, and had identical internal transcribed spacer (ITS) sequences of the rDNA operon. We analyzed 27 isolates from Europe, southwestern Siberia (Russia) and Japan, representing the known geographic ranges of the two species. Phylogenetic analysis of the DNA sequences of a portion of the MAT-2 idiomorph showed these species to be distinct, with the Japanese isolates of C. laricicola having a sequence slightly different (5 bp) from those of the Russian and European isolates of C. laricicola. Sexual compatibility tests showed full interfertility among isolates of C. polonica from Europe, Russia and Japan, but isolates of C. polonica were not fully interfertile with isolates of C. laricicola. A Russian and a European isolate of C. laricicola mated with each other but not with the Japanese isolates of C. laricicola. Mature L. sibirica and P. obovata were inoculated with isolates of C. laricicola and C. polonica from Europe, Russia, and Japan, and measurement of lesions in the inner bark/cambium region demonstrated strong host specialization. The data suggest that the two fungal species are very closely related and are distinguished primarily by their physiological specialization to the hosts of their bark beetle vectors.

Genetic variation in the wattle wilt pathogen Ceratocystis 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.

Genetic relationships among Leptographium terebrantis and the mycangial fungi of three western Dendroctonus bark beetles

Morphology, mitochondrial DNA (mtDNA) restriction fragment polymorphisms (RFLPs) and nuclear DNA (nDNA) fingerprinting were used to clarify relationships among the morphologically similar Ophiostoma and Leptographium species associated with mycangia of three Dendroctonus bark beetles (Ophiostoma clavigerum associated with both D. ponderosae and D. jeffreyi, and L. pyrinum associated with D. adjunctus), as well as a closely related nonmycangial bark beetle associate (L. terebrantis). Most isolates of O. clavigerum form long (40–70 μm), septate conidia, while all isolates of L. terebrantis and L. pyrinum form conidia less than 17.0 μm in length. The conidia of L. pyrinum are pyriform, with truncate bases, while the conidia of the other species form only slightly truncate bases. Conidial masses of L. terebrantis are creamy yellow, while the conidial masses of the other species are white. Nuclear DNA fingerprints resulting from probing PstI restrictions with the oligonucleotide probe (CAC)5 and HaeIII and MspI restrictions of mtDNA, exhibited three major clusters. In the dendrogram developed from mtDNA RFLPs, the L. pyrinum isolates formed one cluster, while the majority of O. clavigerum isolates, including all D. jeffreyi isolates, formed another. A third cluster was composed of all L. terebrantis isolates, as well as several O. clavigerum isolates from D. ponderosae. The inclusion of some O. clavigerum isolates in the L. terebrantis cluster suggests that horizontal transfer of mtDNA has occurred among these fungi. The nDNA dendrogram also exhibited three clusters, and most isolates of L. pyrinum, L. terebrantis and O. clavigerum grouped separately; however, one isolate of O. clavigerum grouped with the L. terebrantis isolates, while one isolate of L. terebrantis grouped with O. clavigerum. No genetic markers were found that distinguished between O. clavigerum associated with D. ponderosae and O. clavigerum associated with D. jeffreyi. Ophiostoma clavigerum might be a recently diverged morphological variant of L. terebrantis, with special adaptations for grazing by young adults of D. jeffreyi and D. ponderosae. The anamorph of O. clavigerum, Graphiocladiella clavigerum, is transferred to Leptographium.