Thomas C. Harrington

A fungal symbiont of the redbay ambrosia beetle causes a lethal wilt in redbay and other lauraceae in the Southeastern United States

Extensive mortality of redbay has been observed in the coastal plain counties of Georgia and southeastern South Carolina since 2003 and northeastern Florida since 2005. We show that the redbay mortality is due to a vascular wilt disease caused by an undescribed Raffaelea sp. that is a fungal symbiont of Xyleborus glabratus, an exotic ambrosia beetle. Trees affected by the disease exhibit wilt symptoms that include a black discoloration of the sapwood. Redbay trees and containerized seedlings died within 5 to 12 weeks after inoculation with the Raffaelea sp. When redbay seedlings were challenged with X. glabratus, the beetles tunneled into 96% of the plants, 70% died, and the Raffaelea sp. was recovered from 91%. X. glabratus and the Raffaelea sp. have also been associated with mortality of sassafras, and the Raffaelea sp. has been isolated from wilted pondberry and pondspice. Additional inoculation studies have shown that the Raffaelea sp. is pathogenic to sassafras, spicebush, and avocado, but not to red maple. Female adults of X. glabratus have paired mycangia near the mandibles, and the Raffaelea sp. is routinely isolated from the heads of beetles. The fungus is apparently introduced into healthy redbay during beetle attacks on stems and branches. The wilt currently affecting redbay and sassafras represents a major threat to other members of the Lauraceae indigenous to the Americas, including avocado in commercial production.

A PCR-based identification method for species of Armillaria

A portion of the Intergenic Spacer (IGS) of the ribosomal RNA operon of 74 isolates of 11 Ar? millaria species from Europe and North America was amplified using the polymerase chain reaction. Am- plifications were made from scrapes of living mycelium without DNA extraction. Alu I digests ofthe amplified product were electrophoresed in agarose and stained with ethidium bromide. With few exceptions, each tax? on had a unique combination of restriction fragments. Most taxa had a single Alu I pattern, but two restric? tion patterns were seen among isolates of A. borealis,

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 ...

New combinations in Raffaelea, Ambrosiella, and Hyalorhinocladiella, and four new species from the redbay ambrosia beetle, Xyleborus glabratus.

— Female adults of the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae), from the southeastern USA were individually macerated and serially diluted onto culture media for isolation of fungal symbionts. Six Raffaelea species were recovered: R. lauricola, R. arxii, and four new species: R. subalba, R. ellipticospora, R. fusca and R. subfusca. Phylogenetic analyses of LSU rDNA sequences placed these mycangial inhabitants and other species of Raffaelea, as well as some species of Ambrosiella associated with ambrosia beetles, into a monophyletic, asexual clade within Ophiostoma. New combinations in Raffaelea are made for some Ambrosiella species and Dryadomyces amasae. Ambrosia beetle symbionts with Ceratocystis affinities, including A. trypodendri comb. nov., are retained in Ambrosiella, but Ambrosiella species associated with bark beetles are transferred to the anamorph genus Hyalorhinocladiella as H. ips, H. macrospora, and H. tingens.

Defining Species in the Fungi

The species concept is central to biology and has received considerable debate, yet a universal definition of a species has not found widespread agreement. Much of the discussion has centered on animals and plants, while mycologists and their organisms have generally been peripheral to the debate. A recent symposium dedicated to species concepts in clonal organisms (Mishler and Budd, 1990) did not include the fungi, though they probably have more apomictic taxa than any other major group of organisms (Blackwell, 1993). Inclusion of the fungal kingdom, with its broad array of reproductive strategies, may complicate the debate, but new insights could be gained by using fungal models. This chapter attempts to bring fungi into the debate and aims to arrive at a workable definition for fungal species.

Phylogenetic and taxonomic evaluation of Chalara, Chalaropsis, and Thielaviopsis anamorphs associated with Ceratocystis

Parsimony analysis of sequences of the internal transcribed spacer region of the nuclear rDNA and partial sequences of the large subunit (LSU) place four anamorphic Chalara species as a monophyletic grouping within the teleomorph genus Ceratocystis. Chalara ovoidea, Ch. thielavioides, Ch. populi, and Ch. elegans (synanamorph: Thielaviopsis basicola) form aleurioconidia typical of the anamorph genus Thielaviopsis, to which the species are transferred. Three of these species (T. ovoidea, T. thielavioides, and T. populi) are morphologically similar to each other but are shown to be distinct by rDNA sequences. The anamorphic genera Chalaropsis and Hughesiella are considered synonyms of Thielaviopsis. Thielaviopsis punctulata, which forms aleurioconidia singly, is shown to be the anamorph of Ce. radicicola. The respective anamorphs for Ce. coerulescens, Ce. fagacearum, and Ce. eucalypti, which lack aleurioconidia, are also transferred to the amended genus Thielaviopsis as T. ungeri, T. quercina, and T. eucalypti. Although Ch. australis and Ch. neocaledoniae do not form aleurioconidia, they are placed in Thielaviopsis based on their endoconidial state and clear affinities to Ceratocystis eucalypti. Three apparently asexual Ambrosiella species belong in the Ce. moniliformis clade based on LSU rDNA sequences, but the cultures available are not suitable for detailed morphological study, and these species are not transferred to Thielaviopsis.

Phylogeny of the Ophiostoma stenoceras-Sporothrix schenckii complex

Ophiostoma stenoceras is a well-known sapwood-colonizing fungus occurring on some coniferous and hardwood hosts in the Northern Hemisphere. In the Southern Hemisphere, the fungus has been reported only from New Zealand. The human pathogen, Sporothrix schenckii, has been suggested to be the anamorph of O. stenoceras. The aim of this study was to gain a better understanding of the phylogenetic relationship between these two species. The study also provided the opportunity to confirm the identity of some Sporothrix and O. stenoceras-like isolates recently collected from wood and soil around the world. For this purpose, the DNA sequence of internal transcribed spacer (ITS) regions of the ribosomal RNA operon was determined. Isolates of O. nigrocarpum, O. albidum, O. abietinum, O. narcissi and O. ponderosae, all morphologically similar to O. stenoceras, were included in the study. From phylogenetic analyses of the sequence data, four main clades were observed. These represented O. stenoceras, O. nigrocarpum and two separate groups containing isolates of S. schenckii. Our results confirm earlier suggestions that S. schenckii should be classified within the teleomorph genus Ophiostoma but support studies separating O. stenoceras and S. schenckii. Ophiostoma albidum and O. ponderosae should be considered synonyms of O. stenoceras. The status of O. narcissi and O. abietinum needs further clarification. The two groups within S. schenckii might represent two species, but this needs to be confirmed. This study represents the first reports of O. stenoceras from Colombia, Kenya, Uruguay and South Africa.

Genetic Variability and Host Specialization in the Latin American Clade of Ceratocystis fimbriata

ABSTRACT The Ceratocystis fimbriata complex includes many undescribed species that cause wilt and canker diseases of many economically important plants. Phylogenetic analyses of DNA sequences have delineated three geographic clades within Ceratocystis fimbriata. This study examined host specialization in the Latin American clade, in which a number of lineages were identified using sequences of the internal transcribed spacer (ITS) region of the rDNA. Three host-associated lineages were identified from cacao (Theobroma cacao), sweet potato (Ipomoea batatas), and sycamore (Platanus spp.), respectively. Isolates from these three lineages showed strong host specialization in reciprocal inoculation experiments on these three hosts. Six cacao isolates from Ecuador, Trinidad, and Columbia differed genetically from other cacao isolates and were not pathogenic to cacao in inoculation tests. Further evidence of host specialization within the Latin American clade of Ceratocystis fimbriata was demonstrated in inoculation experiments in growth chambers using sweet potato, sycamore, Colocasia esculenta, coffee (Coffea arabica), and mango (Mangifera indica) plants; inoculation experiments in Brazil using Brazilian isolates from cacao, Eucalyptus spp., mango, and Gmelina arborea; and inoculation experiments in Costa Rica using Costa Rican isolates from cacao, coffee, and Xantho-soma sp. Hosts native to the Americas appeared to be colonized by only select pathogen genotypes, whereas nonnative hosts were colonized by several genotypes. We hypothesize that local populations of Ceratocystis fimbriata have specialized to different hosts; some of these populations are nascent species, and some host-specialized genotypes have been moved to new areas by humans.

The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers.

Dead and dying oak (Quercus) and numerous other woody ornamental trees and shrubs showing signs and symptoms of Armillaria root rot were identified in the Company Gardens, Cape Town, South Africa, which were established in the mid‐1600s by the Dutch East Indies Trading Company. Nineteen isolates from dying trees or from mushrooms were collected and analysed to identify and characterize the Armillaria sp. responsible for the disease. The AluI digestion of the amplified product of the first intergenic spacer region (IGS‐1) of the rRNA operon of 19 isolates from the Company Gardens was identical to that of some of the European isolates of A. mellea s. s. The IGS‐1 region and the internal transcribed spacers (ITS) were sequenced for some of the Cape Town isolates. Phylogenetic analyses placed the Cape Town isolates in the European clade of A. mellea, which is distinct from the Asian and North American clades of this species. Identification based on sexual compatibility was conducted using A. mellea tester strains in diploid–haploid pairings, which showed some compatibility between the Cape Town isolates and testers from Europe. Somatic compatibility tests (diploid–diploid pairings) and DNA fingerprinting with multilocus, microsatellite probes indicated that the Cape Town isolates were genetically identical and may have resulted from vegetative (clonal) spread from a single focus in the centre of the original Company Gardens (c. 1652). The colonized area is at least 345 m in diameter. Assuming a linear spread rate underground of 0.3 m/year to 1.6 m/year, the genet (clone) was estimated to be between 108 and 575 years old. These data suggest that A. mellea was introduced into Cape Town from Europe, perhaps on potted plants, such as grapes or citrus, planted in the Company Gardens more than 300 years ago.

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.