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Seven new Entomocorticium species (Peniophoraceae) are described based on morphology and phylogenetic analyses. Along with the type species (E. dendroctoni), Entomocorticium comprises eight species of nutritional symbionts of pine bark beetles in North America. Entomocorticium cobbii is the mycangial associate of the southern pine beetle, Dendroctonus frontalis, and E. parmeteri is the mycangial associate of the western pine beetle, D. brevicomis. Entomocorticium whitneyi, E. portiae, E. kirisitsii, E. oberwinkleri and the previously described E. dendroctoni have been isolated from galleries of D. ponderosae and D. jeffreyi in western North America. Entomocorticium sullivanii forms an ambrosia-like layer of basidia and basidiospores in the pupal chambers of Ips avulsus in the southeastern USA. Entomocorticium is phylogenetically placed within Peniophora, a corticioid genus of wood decay fungi with wind-dispersed basidiospores. At least four species of Entomocorticium produce basidiospores on basidia with reduced sterigmata that apparently do not forcibly discharge basidiospores. Another basidiomycete, Gloeocystidium ipidophilum, was described from Ips typographus galleries in Europe, but it is phylogenetically and taxonomically placed in another genus of wood decay fungi as Cylindrobasidium ipidophilum (Physalacriaceae). The free-living wood-decay fungus Phlebiopsis gigantea (Phanerochaetaceae) has been occasionally associated with bark beetles but is unrelated to C. ipidophilum or Entomocorticium.

Extensive mortality of Metrosideros polymorpha (`ōhi`a) trees has been associated with Ceratocystis spp. on Hawai`i Island and was named rapid `ōhi`a death (ROD). Both C. lukuohia and C. huliohia have been associated with ROD, although C. lukuohia appears to be the more important pathogen. Crown observations and dissections of forest trees either wound-inoculated with, or naturally infected by, C. lukuohia were conducted to confirm pathogenicity and document patterns of host colonization. In pathogenicity trials, one of three and two of three trees inoculated with the fungus in February and August, respectively, exhibited crown wilt symptoms at 92 and 69 days after inoculation. Extensive, radial, black staining of the sapwood was found in main stems, while no crown wilt or xylem staining was found in control trees. Xylem staining, necrotic phloem, and fungus presence was noted in six trees inoculated in May to June and harvested 37 to 42 days later, and these observations were compared with those in two naturally infected trees felled in early August. Contiguous xylem staining was found in the main stems and into crowns of all diseased trees, while discontinuous streaks of xylem staining extended into the main forks and side branches. Necrotic phloem associated with xylem staining occurred on the lower stems of inoculated trees. Aside from the necrotic phloem and radial staining of the sapwood, symptom development in `ōhi`a infected with C. lukuohia is similar to other systemic wilt diseases on hardwood trees. We propose Ceratocystis wilt of `ōhi`a as the official name of the disease.

Two recently introduced fungal plant pathogens (Ceratocystis lukuohia and Ceratocystis huliohia) are responsible for Rapid ‘ōhi‘a Death (ROD) in Hawai‘i. Despite being sexually incompatible, the two pathogens often co-occur in diseased ‘ōhi‘a sapwood, where genetic interaction is possible. We sequenced and annotated 33 mitochondrial genomes of the two pathogens and related species, and investigated 35 total Ceratocystis mitogenomes. Ten mtDNA regions [one group I intron, seven group II introns, and two autonomous homing endonuclease (HE) genes] were heterogeneously present in C. lukuohia mitogenomes, which were otherwise identical. Molecular surveys with specific primers showed that the 10 regions had uneven geographic distribution amongst populations of C. lukuohia. Conversely, identical orthologs of each region were present in every studied isolate of C. huliohia regardless of geographical origin. Close relatives of C. lukuohia lacked or, rarely, had few and dissimilar orthologs of the 10 regions, whereas most relatives of C. huliohia had identical or nearly identical orthologs. Each region included or worked in tandem with HE genes or reverse transcriptase/maturases that could facilitate interspecific horizontal transfers from intron-minus to intron-plus alleles. These results suggest that the 10 regions originated in C. huliohia and are actively moving to populations of C. lukuohia, perhaps through transient cytoplasmic contact of hyphal tips (anastomosis) in the wound surface of ‘ōhi‘a trees. Such contact would allow for the transfer of mitochondria followed by mitochondrial fusion or cytoplasmic exchange of intron intermediaries, which suggests that further genomic interaction may also exist between the two pathogens.

Ambrosia beetles farm fungal cultivars (ambrosia fungi) and carry propagules of the fungal mutualists in storage organs called mycangia, which occur in various body parts and vary greatly in size and complexity. The evolution of ambrosia fungi is closely tied to the evolution and development of the mycangia that carry them. The understudied ambrosia beetle tribe Xyloterini included lineages with uncharacterized ambrosia fungi and mycangia, which presented an opportunity to test whether developments of different mycangium types in a single ambrosia beetle lineage correspond with concomitant diversity in their fungal mutualists. We collected representatives of all three Xyloterini genera (Trypodendron, Indocryphalus, and Xyloterinus politus) and characterized their ambrosia fungi in pure culture and by DNA sequencing. The prothoracic mycangia of seven Trypodendron species all yielded Phialophoropsis (Microascales) ambrosia fungi, including three new species, although these relationships were not all species specific. Indocryphalus mycangia are characterized for the first time in the Asian I. pubipennis. They comprise triangular prothoracic cavities substantially smaller than those of Trypodendron and unexpectedly carry an undescribed species of Toshionella (Microascales), which are otherwise ambrosia fungi of Asian Scolytoplatypus (Scolytoplatypodini). Xyloterinus politus has two different mycangia, each with a different ambrosia fungus: Raffaelea cf. canadensis RNC5 (Ophiostomatales) in oral mycangia of both sexes and Kaarikia abrahamsonii (Sordariomycetes, genus incertae sedis with affinity for Distoseptisporaceae), a new genus and species unrelated to other known ambrosia fungi, in shallow prothoracic mycangia of females. In addition to their highly adapted mycangial mutualists, Trypodendron and X. politus harbor a surprising diversity of facultative symbionts in their galleries, including Raffaelea. A diversity of ambrosia fungi and mycangia suggest multiple ancestral cultivar captures or switches in the history of tribe Xyloterini, each associated with unique adaptations in mycangium anatomy. This further supports the theory that developments of novel mycangium types are critical events in the evolution of ambrosia beetles and their coadapted fungal mutualists.

The North American clade (NAC) of Ceratocystis includes pathogenic species that infect a wide range of woody hosts. Previous phylogenetic analyses have suggested that this clade includes cryptic species and a paraphyletic C. variospora. In this study, we used morphological data and phylogenetic analyses to characterize NAC taxa, including Ceratocystis isolates causing a serious disease of almond trees in California. Phylogenetic analyses based on six gene regions supported two new species of Ceratocystis. Ceratocystis destructans is introduced as the species causing severe damage to almond trees in California, and it has also been isolated from wounds on Populus and Quercus in Iowa. It is morphologically similar to C. tiliae, a pathogen on Tilia and the most recently characterized species in the NAC. Ceratocystis betulina collected from Betula platyphylla in Japan is also newly described and is the sister taxon to C. variospora. Our six-locus phylogenetic analyses and morphological characterization resolved several cryptic species in the NAC.

Ambrosia beetles farm specialised fungi in sapwood tunnels and use pocket-like organs called mycangia to carry propagules of the fungal cultivars. Ambrosia fungi selectively grow in mycangia, which is central to the symbiosis, but the history of coevolution between fungal cultivars and mycangia is poorly understood. The fungal family Ceratocystidaceae previously included three ambrosial genera (Ambrosiella, Meredithiella, and Phialophoropsis), each farmed by one of three distantly related tribes of ambrosia beetles with unique and relatively large mycangium types. Studies on the phylogenetic relationships and evolutionary histories of these three genera were expanded with the previously unstudied ambrosia fungi associated with a fourth mycangium type, that of the tribe Scolytoplatypodini. Using ITS rDNA barcoding and a concatenated dataset of six loci (28S rDNA, 18S rDNA, tef1-α, tub, mcm7, and rpl1), a comprehensive phylogeny of the family Ceratocystidaceae was developed, including Inodoromyces interjectus gen. & sp. nov., a non-ambrosial species that is closely related to the family. Three minor morphological variants of the pronotal disk mycangium of the Scolytoplatypodini were associated with ambrosia fungi in three respective clades of Ceratocystidaceae: Wolfgangiella gen. nov., Toshionella gen. nov., and Ambrosiella remansi sp. nov. Closely-related species that are not symbionts of ambrosia beetles are accommodated by Catunica adiposa gen. & comb. nov. and Solaloca norvegica gen. & comb. nov. The divergent morphology of the ambrosial genera and their phylogenetic placement among non-ambrosial genera suggest three domestication events in the Ceratocystidaceae. Estimated divergence dates for the ambrosia fungi and mycangia suggest that Scolytoplatypodini mycangia may have been the first to acquire Ceratocystidaceae symbionts and other ambrosial fungal genera emerged shortly after the evolution of new mycangium types. There is no evidence of reversion to a non-ambrosial lifestyle in the mycangial symbionts.

The native ‘ōhi’a lehua (Metrosideros polymorpha) has cultural, biological and ecological significance to Hawai’i, but it is seriously threatened by a disease commonly referred to as rapid ‘ōhi’a death (ROD). Preliminary investigations showed that a Ceratocystis species similar to C. fimbriata s.lat. was the cause of the disease. In this study, we used a combination of the phylogenetic, morphological and biological species concepts, as well as pathogenicity tests and microsatellite analyses, to characterise isolates collected from diseased ‘ōhi’a trees across Hawai’i Island. Two distinct lineages, representing new species of Ceratocystis, were evident based on multigene phylogenetic analyses. These are described here as C. lukuohia and C. huliohia. Ceratocystis lukuohia forms part of the Latin American clade (LAC) and was most closely associated with isolates from Syngonium and Xanthosoma from the Caribbean and elsewhere, including Hawai’i, and C. platani, which is native to eastern USA. Ceratocystis huliohia resides in the Asian-Australian clade (AAC) and is most closely related to C. uchidae, C. changhui and C. cercfabiensis, which are thought to be native to Asia. Morphology and interfertility tests support the delineation of these two new species and pathogenicity tests show that both species are aggressive pathogens on seedlings of M. polymorpha. Characterisation of isolates using microsatellite markers suggest that both species are clonal and likely represent recently-introduced strains. Intensive research is underway to develop rapid screening protocols for early detection of the pathogens and management strategies in an attempt to prevent the spread of the pathogens to the other islands of Hawai’i, which are currently disease free.

Meredithiella norrisii (Microascales, Ceratocystidaceae) is an ambrosia fungus carried in mycangia of the North American ambrosia beetle, Corthylus punctatissimus. Reports on the identity of the fungal symbionts of other species of Corthylus have been inconsistent. This study tested the hypothesis that Meredithiella spp. are the primary symbionts of Corthylus spp. Cultures and/or internal transcribed spacer (ITS) rDNA barcode sequences of Meredithiella spp. were obtained consistently from beetles and galleries of nine Corthylus spp. The ITS sequences of three putative species of Meredithiella were associated with C. consimilis and C. flagellifer in Mexico and C. calamarius in Costa Rica. The symbiont of C. columbianus in the USA was identified as M. norrisii. Two new Meredithiella spp. are described: M. fracta from C. papulans in Florida and Honduras, and M. guianensis associated with C. crassus and two unidentified Corthylus spp. in French Guyana. The Meredithiella spp. propagate in the mycangia of adult females by thallic-arthric growth, and the ambrosia growth in larval cradles comprises bead-like hyphal swellings or conidiophores, with or without terminal aleurioconidia. Bayesian phylogenetic analysis of a combined 18S and 28S nuc rDNA and translation elongation factor 1-α (TEF1-α) data set demonstrated that Meredithiella is a distinct monophyletic clade within the Ceratocystidaceae, but its phylogenetic placement with regard to the other ambrosial genera in the family remains ambiguous. The mycangia of C. punctatissimus and C. papulans are also compared using light microscopy and micro-computed tomography (micro-CT) imaging, revealing that they differ in both size and shape, but these differences may not correlate with different lineages of Meredithiella.

Ceratocystis fimbriata Ellis & Halsted recently was recorded causing seed and seedling blight on Carapa guianensis Aubl. (andiroba), a tree species native to the Amazon Rainforest and prized for its valuable timber and medicinal seed oil. C. fimbriata more commonly causes wilt type diseases in woody hosts, especially on non-native host trees. However, on andiroba the disease occurs on seedlings and seeds, affecting the species regeneration. We studied 73 isolates of C. fimbriata on andiroba from three regions of the Amazon Basin to see if they represented natural or introduced populations. Analysis of ITS rDNA sequences and phylogenetic analysis of mating type genes revealed new haplotypes of C. fimbriata from the Latin American Clade that were closely related to other Brazilian populations of the fungus. In mating experiments, andiroba isolates were inter-fertile with tester strains of C. fimbriata from Brazil and elsewhere, confirming that they belong to a single biological species. Using microsatellite markers, 14 genotypes and populations with intermediate levels of genetic variability were found, suggesting that the fungus is indigenous to the Amazon Basin. Inoculation tests indicated that the andiroba isolates are host-specialized on andiroba, supporting the proposition of the special form C. fimbriata f. sp. carapa. Made available in DSpace on 2019-03-20T00:43:38Z (GMT). No. of bitstreams: 1 26788.pdf: 3260751 bytes, checksum: e216b037faec9a95d9551042e8f53907 (MD5) Previous issue date: 2019

An examination of the isolates of Ceratocystis fimbriata from corms of Araceae ( Colocasia esculenta , taro, and Xanthosoma sagittifolium ) from Hawaii and Fiji found that they represent a new species, Ceratocystis uchidae . Phylogenetic analyses showed that the new species is in the Asian-Australian clade of the C. fimbriata complex, and it is most closely related to C. polychroma from Indonesia and C. cercfabiensis from China. Ceratocystis uchidae differs from these species in its shorter perithecial necks, longer ostiolar hyphae, and reduced growth at 32 °C compared to 30 °C. An unnamed species on Eucalyptus sp. from Yunnan, China was similar to C. uchidae , but C. uchidae forms shorter perithecial necks, and the two species were not interfertile in mating tests. Crossing of mating testers demonstrated that C. uchidae and C. polychroma also were not interfertile. Isolates of C. cercfabiensis showed substantial variation in mycelial morphology, with frequent sectoring. Perithecial necks of C. cercfabiensis varied greatly in length, and perithecia with the longest necks failed to exude ascospores. Also, tester strains of C. cercfabiensis were partially interfertile with tester strains of C. uchidae . Ceratocystis cercfabiensis appears to be an aberrant species and may be a hybrid between two other species of Ceratocystis .

We report the association of Cristulariella moricola (Hino) Redhead with a zonate leaf spot on Eucalyptus benthamii (Myrtaceae). We observed the disease outbreak across a series of experimental E. benthamii plantings in Barnwell County, South Carolina, USA in October 2015. The disease caused nearly complete foliar necrosis of most trees in one experimental planting, but disease incidence remained at low levels ( C. moricola in other tree species.

Genera of ambrosia beetles in the tribe Xyleborini with large, mesonotal mycangia host unique fungal symbionts in the genus Ambrosiella. The symbiont of a recent invasive to the USA from Asia, Anisandrus maiche Stark, had not been previously characterized. We found the mycangium anatomy of An. maiche collected in Ohio to be similar to that of Anisandrus dispar and consistently isolated a novel fungus, Ambrosiella cleistominuta sp. nov., from An. maiche mycangia and galleries. The fungus was distinguished from other named Ambrosiella by morphological characters and DNA sequences (ITS rDNA and tef-1α). The mycangial symbionts of ambrosia beetles had been assumed to be strictly asexual, but A. cleistominuta produces cleistothecious ascomata with ascospores in beetle galleries and in culture. In contrast to ascomata of other Ceratocystidaceae, the relatively small ascomata of A. cleistominuta are neckless and without ostioles. The ascospores are relatively large, and single-ascospore colonies produced ascomata and ascospores in culture, showing that A. cleistominuta is homothallic.

Laurel wilt is a vascular wilt disease caused by Raffaelea lauricola, a mycangial symbiont of an ambrosia beetle, Xyleborus glabratus. The fungus and vector are native to Asia but were apparently introduced to the Savannah, GA, area 15 or more years ago. Laurel wilt has caused widespread mortality on redbay (Persea borbonia) and other members of the Lauraceae in the southeastern United States, and the pathogen and vector have spread as far as Texas. Although believed to be a single introduction, there has been no extensive study on genetic variation of R. lauricola populations that would suggest a genetic bottleneck in the United States. Ten isolates of R. lauricola from Japan, 55 from Taiwan, and 125 from the United States were analyzed with microsatellite and 28S rDNA markers, and with primers developed for two mating-type genes. The new primers identified isolates as either MAT1 or MAT2 mating types in roughly equal proportions in Taiwan and Japan, where there was also high genetic diversity within populations based on all the markers, suggesting that these populations may have cryptic sex. Aside from a local population near Savannah and a single isolate in Alabama that had unique microsatellite alleles, the U.S. population was genetically uniform and included only the MAT2 mating type, supporting the single introduction hypothesis. This study suggests the importance of preventing a second introduction of R. lauricola to the United States, which could introduce the opposite mating type and allow for genetic recombination.

Ceratocystis fimbriata is a native, soilborne pathogen in South America that causes a lethal wilt disease on a broad range of economically important plants. Ceratocystis wilt on kiwifruit (Actinidia spp.) was first recognized in 2010 in the state of Rio Grande do Sul, Brazil. The genetic variation among kiwifruit isolates was analized to determine if a single introduced strain of the pathogen was responsible for the epidemic or if there was substantial genetic variation in the population, suggesting that the fungus was soilborne and indigenous to the region. We used 14 microsatellite (simple sequence repeat, SSR) markers to identify 18 genotypes of C. fimbriata among 76 isolates from eight kiwifruit farms. The 18 genotypes clustered into three groups based on UPGMA analysis of the microsatellite alleles. The largest group comprised 60 isolates of 11 closely-related microsatellite genotypes obtained from seven of the eight farms. These genotypes appeared to have originated from a single farm that had supplied cuttings for grafting to the other farms. The population of the pathogen from the farm that supplied the cuttings had the highest level of genotypic diversity and relatively high gene diversity, suggesting that this source population represented an indigenous, soilborne population. Phylogenetic analyses of the DNA sequences of the mating type locus (including portions of MAT1-1-2 and MAT1-2-1) placed the isolates into three groups, corresponding to the three microsatellite groups. Most of the isolates, including all the tested isolates from the farm that supplied the cuttings, had mating type gene sequences that were distinct from other Brazilian populations of C. fimbriata. A second group comprised isolates from one farm that had mating type gene sequences typical of Mata Atlântica (Rio de Janeiro and Sao Paulo) populations of C. fimbriata on Colocasia esculenta and Mangifera indica. Three farms purchased kiwifruit plants or rootstocks from commercial nurseries in Brazil as well as scions from the source farm, and some of the isolates from these farms were genetically similar to Eucalyptus isolates of C. fimbriata from Bahia and Minas Gerais, Brazil. The kiwifruit epidemic in Rio Grande do Sul is the southern-most report of C. fimbriata in Brazil, and the primary pathogen population on kiwifruit appears to be indigenous and originated from a single farm that distributed the pathogen in grafting material. In addition, commercial nursery stock was also implicated as sources of C. fimbriata genotypes. The disease is a major limiting factor for kiwifruit production in southern Brazil, and the results suggest that clean planting stock will be important to successful production.

Ocasio-Morales, R. G., Tsopelas, P., and Harrington, T. C. 2007. Origin of Ceratocystis platani on native Platanus orientalis in Greece and its impact on natural forests. Plant Dis. 91:901-904. Canker stain of plane tree recently was reported in a small area of southwestern Greece on natural populations of the important riparian species, oriental plane tree, Platanus orientalis. The fungus Ceratocystis platani (= C. fimbriata f. platani) was successfully isolated from infected, stained wood of 15 dead or dying trees on the Peloponnese Peninsula. Genetic analyses of these 15 isolates from Greece, using nuclear and mitochondrial DNA fingerprints, showed the fungus to be identical to the genotype reported from Italy, France, and Switzerland. A polymerase chain reaction-based microsatellite analysis of eight polymorphic loci discovered a new microsatellite allele in one of the isolates from Greece, but this may be due to a mutation after introduction of a single strain. Earlier studies indicated that the most common European genotype had been introduced from eastern North America to Italy during World War II. The recent introduction to Greece appears to have originated from Italy, France, or Switzerland, rather than from eastern North America, where the fungus is native. The pathogen is having a dramatic impact on the natural population of P. orientalis in southwestern Greece, and containment measures should be imposed before it spreads throughout the natural range of this ecologically and historically important host.

Ceratocystis fagacearum (T.W. Bretz) J. Hunt is considered to be the most destructive vascular pathogen of oaks (Fagaceae: Quercus spp.) in the eastern, north-central, and south-central United States. (1,2,4). All red oak species (subgenus Quercus: section Lobatae) are highly susceptible to C. fagacearum, and infected trees typically die within 3 months of first symptom expression. However, members of the white oak group (subgenus Quercus: section Quercus) are moderately to highly resistant to C. fagacearum and rarely die from oak wilt (1,3). In early August of 2008, we received branch samples taken from wilting red oaks (Q. rubra L.) growing in a residential neighborhood in Scotia, NY (Schenectady County). The endoconidial state of the oak wilt fungus, Thielaviopsis quercina (B.W. Henry) A.E. Paulin, T.C. Harr. & McNew, was consistently isolated from the xylem in the branches. The cultures were identified based on hyphal and conidial morphology on acidified potato dextrose agar (aPDA) as well as sequences of the internal transcribed spacer (ITS) (GenBank Accession No. FJ347031) and large subunit (LSU) (GenBank Accession No. FJ347030) regions of nuclear ribosomal DNA (rDNA). The nucleotide identities for the ITS and LSU sequences were a precise match, 99 and 100%, to rDNA sequences (GenBank Accession Nos. AF043598 and AF222483, respectively) of other isolates of C. fagacearum. According to the homeowners at the site, 12 red oaks have died during the last 3 years, and each tree died within one growing season after oak wilt-like symptoms were noted. In a brief survey of nearby properties in late August of 2008, we found 12 additional trees that were either expressing crown symptoms of oak wilt (e.g., premature leaf casting, bronzing of leaf margins, and water-soaked leaves) or were standing dead and within close proximity (5 to 10 m) to symptomatic trees. Branch samples from four of the symptomatic trees revealed limited (spotted) or no vascular discoloration; however, C. fagacearum was isolated from each suspect tree on aPDA. Remnants of gray mycelial mats and associated pressure cushions were observed beneath the bark of one standing dead oak. The sweet fruit-like odor characteristic of the oak wilt fungus was immediately evident once the bark overlying the mats was removed. Prior to this discovery, the Susquehanna River in north-central Pennsylvania was considered to be the northeastern limit for oak wilt occurrence in the United States (2,4). To our knowledge, this is the first report of the fungus from New York and expands the known range of C. fagacearum to the northeast by at least 300 km, supporting the hypothesis that the range of this fungus continues to expand via animal vectors and/or human activities (2). An isolate of C. fagacearum from New York has been deposited at the Centraalbureau voor Schimmelcultures (CBS 123913). References: (1) D. N. Appel. Ann. Rev. Phytopathol. 33:103, 1995. (2) J. Juzwik et al. Ann. Rev. Phytopathol. 46:13, 2008. (3) W. L. MacDonald et al. European oaks-susceptible to oak wilt? Page 131 in: Shade Tree Wilt Diseases. C. L. Ash, ed. The American Phytopathological Society, St. Paul, MN, 2001. (4) USDA Forest Service. Oak Wilt Distribution. Northeast Area, State and Private Forestry, St. Paul, MN. Online publication, 2005.

A single Japanese walnut (Juglans ailantifolia Carrière, an ornamental, deciduous tree) with symptoms of witches'-brooms and branch dieback, consistent with those associated with the walnut witches'-broom (WWB) phytoplasma (1), was observed near Ames, IA. No other Japanese walnut trees were present in the planting and the numerous black walnut (Juglans nigra L.) trees were asymptomatic. Leaf samples were collected in September 2009 from witches'-brooms as well as from two asymptomatic branches from the Japanese walnut tree and from three branches each from two nearby (10 m and 100 m away, respectively) black walnut trees. The presence of phytoplasma was tested using DNA extracted (sodium dodecyl sulfate and potassium acetate methods) from the midvein of individual leaves and PCR with universal phytoplasma primers P1 and P7, which amplify from the beginning of the 16S rDNA to the beginning of the 23S rDNA gene (4). Each of the five symptomatic leaves yielded a PCR product, but the two asymptomatic leaves from the sole Japanese walnut tree did not. One of the three asymptomatic leaves from a black walnut tree (100 m away) was also positive. In a subsequent round of PCR, with the nested primers R16F2 and R16R2 (4), three additional asymptomatic leaves from the two black walnut trees were positive. The P1/P7 or R16F2/R16R2 products from each of the three trees were directly sequenced or cloned into a TA vector and sequenced using vector primers. The BLAST searches (v. 2.2.2.4) of these sequences most closely matched the sequences of the WWB phytoplasma and other members of the 16SrIII group (peach X-disease). The closest matches for the full P1/P7 sequence from the Japanese walnut (GenBank Accession No. HQ221553, 1,814 bp) were with those of phytoplasmas associated with WWB from two black walnut trees in Georgia (AF190227, 1,812 of 1,815 bp matching; and AF190226, 1,808 of 1,815 bp matching), spiraea stunt (AF190228, 1,808 of 1,814 bp), and western X (AF533231, 1,807 of 1,814 bp). The iPhyClassifier restriction fragment length polymorphism similarity coefficient was 0.99 for L33733 (Canadian peach X phytoplasma) and 0.98 for AF190226. Sequence HQ221553 differed by 10 bp from the sequence from the asymptomatic black walnut tree that was 100 m away (HQ221554, 1,815 bp), which matched closest to one of the black walnut samples from Georgia (AF190227, 1,807 of 1,816 bp). A 1,029-bp fragment from the second black walnut tree (10 m away) differed by 1 or 2 bp from the Georgia WWB accessions. To our knowledge, this is the first report of WWB symptoms in Iowa and the first identification of the WWB phytoplasma outside of Georgia (1). The disease, however, is more widely known (Illinois, Indiana, and Ohio) and may cause serious reduction in nut production (1–3). It can be lethal to Juglans spp., especially to exotic species such as Japanese walnut (2,3). The native black walnut is thought to be relatively resistant to tolerant of WWB (2,3) and may only show growth decline with no symptoms, except for broom production from cut surfaces (3). Care should be taken in moving planting stock of black walnut (4) because asymptomatic trees may harbor the phytoplasma. References: (1) J. Chen et al. Plant Dis. 76:1116, 1992. (2) C. E. Seliskar. For. Sci. 22:144, 1976. (3) W. A. Sinclair et al. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY, 1987. (4) L. Ward. Juglans (Walnut). Post-Entry Quarantine Testing Manual. Biosecurity New Zealand, Ministry of Agriculture and Forestry, Auckland, 2008.

Laurel wilt, caused by Raffaelea lauricola, has been responsible for extensive losses of redbay (Persea borbonia) in South Carolina and Georgia since 2003. Symptoms of the disease have been noted in other species of the Lauraceae such as the federally endangered pondberry (Lindera melissifolia) and the threatened pondspice (Litsea aestivalis). Pondberry and pondspice seedlings were inoculated with R. lauricola from redbay, and both species proved highly susceptible to laurel wilt. Field assessments found substantial mortality of pondberry and pondspice, but in many cases the losses were not attributable to laurel wilt. R. lauricola was isolated from only 4 of 29 symptomatic pondberry plants at one site, but the fungus was not recovered from three plants at another site. R. lauricola was isolated from one of two symptomatic pondspice plants at one site, and from five of 11 plants at another site, but not from any plant at a third site. Insect bore holes, similar to those produced by Xyleborus glabratus (the vector of laurel wilt), were found in some pondberry and pondspice stems, but adults were not found. Damage caused by Xylosandrus compactus was found in pondberry stems, but this ambrosia beetle does not appear to be a vector of R. lauricola. Xyleborinus saxeseni adults were found in a dying pondspice with laurel wilt, and R. lauricola was recovered from two of three adults. Isolates of R. lauricola from pondberry, pondspice, and X. saxeseni had rDNA sequences that were identical to previously characterized isolates, and inoculation tests confirmed that they were pathogenic to redbay. Because pondberry and pondspice tend to be shrubby plants with small stem diameters, these species may not be frequently attacked by X. glabratus unless in close proximity to larger diameter redbay.

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite ‘E’ (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite ‘E’. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5′-AGTGCGGTGGTATCGACAAG) and EFCF6 (3′-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Laurel wilt, caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, is responsible for extensive mortality of native redbays (Persea borbonia and P. palustris) in the coastal plains of the southeastern United States (1). The wilt also affects the more widespread sassafras, Sassafras albidum, particularly in areas where diseased redbays are common and populations of X. glabratus are high. Because sassafras stems were thought to lack chemicals that are attractive to the beetle, and sassafras tends to be widely scattered in forests, it was believed that the advance of the laurel wilt epidemic front might slow once it reached the edge of the natural range of redbay, which is restricted to the coastal plains of the Gulf and Atlantic Coasts (2). In July and August of 2011, wilt-like symptoms (i.e., wilted and dead leaves, and streaks of black discoloration in the xylem) were observed on 1 to 10 sassafras trees (15 to 23 cm diameter; 6 to 9 m height) at each of three locations, which were approximately 6 km from one another in Marengo Co., Alabama. Samples of the discolored wood from five trees were plated on malt agar amended with cycloheximide and streptomycin (CSMA), and a fungus morphologically identical to R. lauricola was isolated from each tree (1). For confirmation, a portion of the large subunit (28S) of the rDNA region of three of the isolates was sequenced (3); in each case, the sequence matched exactly that of other isolates of R. lauricola (EU123077) from the United States. Symptomatic trees were found at all three sites when revisited in April 2012, and approximately 20 sassafras trees in various stages of wilt were observed at one location, where only one diseased tree had been noted in 2011. Bolts were cut from the main stem of a symptomatic tree, and eggs, larvae, and adults of X. glabratus were commonly found in tunnels, and R. lauricola was isolated from the discolored xylem. Three container-grown sassafras saplings (mean height 193 cm, mean diameter 2.1 cm at groundline) were inoculated as previously described (1) with conidia (~600,000) from an isolate of R. lauricola. Three additional sassafras saplings were inoculated with sterile, deionized water, and all plants were placed in a growth chamber at 25°C with a 15-h photoperiod. Inoculated plants began to exhibit wilt symptoms within 14 days, and at 30 days all inoculated plants were dead and xylem discoloration was observed. Control plants appeared healthy and did not exhibit xylem discoloration. Pieces of sapwood from 15 cm above the inoculation points were plated on CSMA, and R. lauricola was recovered from all wilted plants but not from control plants. This is the first record of laurel wilt in Alabama and is significant because the disease appears to be spreading on sassafras in an area where redbays have not been recorded (see http://www.floraofalabama.org ). The nearest previously documented case of laurel wilt is on redbay and sassafras in Jackson Co., Mississippi (4), approximately 160 km to the south. The exact source of the introduction of X. glabratus and R. lauricola into Marengo Co. is not known. The vector may have been transported into the area with storms, moved with infested firewood, or shipped with infested timber by companies that supply mills in the area. References: (1) S. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. Hanula et al. Econ. Ent. 101:1276, 2008. (3) T. Harrington et al. Mycotaxon 111:337, 2010. (4) J. Riggins et al. Plant Dis. 95:1479, 2011.

Genotypes of the Latin American wilt pathogen Ceratocystis fimbriata have been moved around the world in vegetatively propagated material of various crop plants, including Ipomoea batatas (sweet potato), Colocasia esculenta (taro), and Eucalyptus spp. When compared to a worldwide collection of isolates of C. fimbriata, isolates from taro, Punica granatum (pomegranate), and Eriobotrya japonica (loquat) from Yunnan Province, China were found to have sequences of internal transcribed spacer (ITS) rDNA and mating type genes that were identical to isolates from Eucalyptus in Brazil. Analyses of 35 isolates with 14 microsatellite markers revealed that the Yunnan population was nearly uniform, consisting of only 19 alleles and seven closely related genotypes, suggesting that the population is not natural and is the result of an introduction. As in comparisons of sequences of ITS rDNA and mating type genes, the microsatellite alleles of the Yunnan isolates were most similar to those of Eucalyptus isolates from Minas Gerais and Bahia, Brazil, where C. fimbriata is native, soilborne, and commonly infects cuttings of Eucalyptus spp. used for rooting in nurseries. Thus, the Yunnan population, which is causing severe losses on pomegranate, may have been indirectly derived from introductions of C. fimbriata in contaminated Eucalyptus cuttings from Brazil.

Chinese isolates of Ceratocystis fimbriata from sweet potato (Ipomoea batatas) and pomegranate (Punica granatum) were genetically compared with a worldwide collection of isolates from a variety of hosts. Isolates from black-rotted storage roots of sweet potato in China, Japan, Australasia, and the United States had identical internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequences and only minor variation in microsatellite alleles. Sequences of their mating type genes were most similar to those of isolates from various hosts in Ecuador, a center of diversity for sweet potato. Isolates from Colocasia esculenta (taro) and pomegranate from Yunnan and Sichuan had only one ITS rDNA sequence (haplotype ITS5). This haplotype, sequences of mating type genes, and microsatellite alleles linked these isolates to isolates from Eucalyptus stumps in South China and diseased Eucalyptus trees in Brazil, supporting the hypothesis that the pomegranate population originated from Brazil via cuttings of Eucalyptus. Isolates from sweet potato and pomegranate in China were interfertile with tester strains of C. fimbriata, confirming that the causes of the two epidemics in China belong to a single biological species. However, other isolates from Eucalyptus stumps were intersterile with the tester strains and had ITS rDNA sequences typical of the Asian species, C. cercfabiensis.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml−1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml−1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml−1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.

Ceratocystis fimbriata causes wilt and death of plants in several important crops and native vegetation in Brazil. Ceratocystis wilt was recently found to be causing substantial mortality in kiwifruit (Actinidia deliciosa and A. chinensis) in the Farroupilha region of Rio Grande do Sul. The aim of this study was to describe the symptomatology of the new disease and characterise isolates of C. fimbriata obtained from kiwifruit plants. Affected plants showed substantial discoloration of the woody xylem and wilt-type symptoms of the foliage, with eventual death of the whole plant. Phylogenetic analysis of ITS rDNA of 39 isolates from kiwifruit identified five haplotypes, which were placed in the Latin American clade of C. fimbriata. The ITS haplotypes were morphologically similar to isolates of C. fimbriata from other hosts in Brazil. Eight isolates of four ITS haplotypes varied somewhat in aggressiveness to three kiwifruit cultivars (Monty, Hayward and Elmwood), but all isolates were pathogenic and ...

The genus Ambrosiella accommodates species of Ceratocystidaceae (Microascales) that are obligate, mutualistic symbionts of ambrosia beetles, but the genus appears to be polyphyletic and more diverse than previously recognized. In addition to Ambrosiella xylebori, Ambrosiella hartigii, Ambrosiella beaveri, and Ambrosiella roeperi, three new species of Ambrosiella are described from the ambrosia beetle tribe Xyleborini: Ambrosiella nakashimae sp. nov. from Xylosandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella grosmanniae sp. nov. from Xylosandrus germanus. The genus Meredithiella gen. nov. is created for symbionts of the tribe Corthylini, based on Meredithiella norrisii sp. nov. from Corthylus punctatissimus. The genus Phialophoropsis is resurrected to accommodate associates of the Xyloterini, including Phialophoropsis trypodendri from Trypodendron scabricollis and Phialophoropsis ferruginea comb. nov. from Trypodendron lineatum. Each of the ten named species was distinguished by ITS rDNA barcoding and morphology, and the ITS rDNA sequences of four other putative species were obtained with Ceratocystidaceae-specific primers and template DNA extracted from beetles or galleries. These results support the hypothesis that each ambrosia beetle species with large, complex mycangia carries its own fungal symbiont. Conidiophore morphology and phylogenetic analyses using 18S (SSU) rDNA and TEF1α DNA sequences suggest that these three fungal genera within the Ceratocystidaceae independently adapted to symbiosis with the three respective beetle tribes. In turn, the beetle genera with large, complex mycangia appear to have evolved from other genera in their respective tribes that have smaller, less selective mycangia and are associated with Raffaelea spp. (Ophiostomatales).

Ceratocystis wilt is among the most important diseases on mango (Mangifera indica) in Brazil, Oman, and Pakistan. The causal agent was originally identified in Brazil as Ceratocystis fimbriata, which is considered by some as a complex of many cryptic species, and four new species on mango trees were distinguished from C. fimbriata based on variation in internal transcribed spacer sequences. In the present study, phylogenetic analyses using DNA sequences of mating type genes, TEF-1α, and β-tubulin failed to identify lineages corresponding to the four new species names. Further, mating experiments found that the mango isolates representing the new species were interfertile with each other and a tester strain from sweet potato (Ipomoea batatas), on which the name C. fimbriata is based, and there was little morphological variation among the mango isolates. Microsatellite markers found substantial differentiation among mango isolates at the regional and population levels, but certain microsatellite genotypes were commonly found in multiple populations, suggesting that these genotypes had been disseminated in infected nursery stock. The most common microsatellite genotypes corresponded to the four recently named species (C. manginecans, C. acaciivora, C. mangicola, and C. mangivora), which are considered synonyms of C. fimbriata. This study points to the potential problems of naming new species based on introduced genotypes of a pathogen, the value of an understanding of natural variation within and among populations, and the importance of phenotype in delimiting species.

Species in the North American clade (NAC) of the Ceratocystis fimbriata complex are mostly weak pathogens that infect native tree hosts through fresh wounds. Isolations from discolored tissue of wounded Tilia americana (basswood) in Iowa and Nebraska yielded a Ceratocystis species that was similar to but distinct from isolates of C. variospora from other hosts. Sequences of 28S rDNA showed that isolates from basswood did not differ from C. variospora, but there were minor differences in ITS rDNA sequences. The DNA sequences of a portion of the Cerato-platanin gene and TEF1α showed the basswood fungus to be a unique lineage. Cross inoculations in two experiments showed that the basswood isolates and C. variospora isolates from Quercus spp. were most aggressive to their respective hosts. Isolates from basswood grew slower and were less pigmented than C. variospora isolates from Quercus spp. The basswood fungus thus is distinguished from C. variospora based on phylogenetic analyses and phenotype and is herein described as C. tiliae sp. nov.

Morphological comparisons and phylogenetic analyses of Tubakia species from leaves of Quercus spp. in Iowa and other areas of eastern USA revealed three novel species: Tubakia hallii, Tubakia macnabbii, and Tubakia tiffanyae. These species, as well as Tubakia dryina and Tubakia iowensis, are common leaf endophytes and pathogens on Quercus and Castanea in eastern USA, as is Tubakia americana comb. nov, originally described from Quercus in New Jersey as Actinopelte americana. New combinations of species on leaves of other hosts in the eastern USA include Tubakia gloeosporioides, Tubakia liquidambaris, and Tubakia nyssae. Asian species of Tubakia are phylogenetically compared, and the new combination Tubakia supraseptata is made to accommodate a Japanese endophyte known only by its sexual state. The earlier description of Dicarpella dryina as the sexual state of T. dryina is questioned. The new combination Tubakia stellata is made to accommodate an unusual species from Brazil.

Summary Laurel wilt, caused by Raffaelea lauricola, is responsible for extensive mortality of redbay and other American members of the Lauraceae in the southeastern United States. Raffaelea lauricola is a mycangial symbiont of the redbay ambrosia beetle (Xyleborus glabratus), and the beetle and fungus were accidentally introduced from Asia. Branch dieback of camphortree (Cinnamomum camphora), an Asian member of the Lauraceae, has been occasionally observed in areas where laurel wilt has decimated redbay populations, and R. lauricola was isolated from such camphortrees. However, the role of X. glabratus and R. lauricola in this branch dieback remains unclear. Examination of camphortrees on Jekyll Island, Georgia showed that healthy-appearing trees and those with branch dieback had been attacked by X. glabratus, but the trees with branch dieback had four times as many beetle attacks. Raffaelea lauricola was routinely isolated from discoloured xylem near beetle tunnels in healthy trees and those with dieback. Single-point inoculations with R. lauricola on stems of mature, healthy camphortree trees failed to induce wilt-like symptoms or branch dieback, although areas of discoloration were scattered throughout the xylem, and R. lauricola was reisolated irregularly at various heights in some inoculated trees. In growth chamber experiments, single-point inoculations with R. lauricola resulted in systemic colonization but no wilt symptoms or branch dieback in camphortree saplings. In contrast, inoculations at multiple points along the stem (simulating multiple attacks by the vector) caused branch dieback and wilt-like symptoms, including a brownish, diffuse discoloration of the xylem. Camphortree appears to be more resistant than American species of Lauraceae to the vascular wilt caused by R. lauricola. The fungus does colonize camphortrees systemically, however, and can apparently cause branch dieback. This suggests that the fungus may provide brood material for X. glabratus in Asia as it does in the southeastern United States.

Isolations from the granulate ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae: Scolytinae: Xyleborini), collected in Georgia, South Carolina, Missouri and Ohio, yielded an undescribed species of Ambrosiella in thousands of colony-forming units (CFU) per individual female. Partial sequences of ITS and 28S rDNA regions distinguished this species from other Ambrosiella spp., which are asexual symbionts of ambrosia beetles and closely related to Ceratocystis spp. Ambrosiella roeperi sp. nov. produces sporodochia of branching conidiophores with disarticulating swollen cells, and the branches are terminated by thick-walled aleurioconidia, similar to the conidiophores and aleurioconidia of A. xylebori, which is the mycangial symbiont of a related ambrosia beetle, X. compactus. Microscopic examinations found homogeneous masses of arthrospore-like cells growing in the mycangium of X. crassiusculus, without evidence of other microbial growth. Using fungal-specific primers, only the ITS rDNA region of A. roeperi was amplified and sequenced from DNA extractions of mycangial contents, suggesting that it is the primary or only mycangial symbiont of this beetle in USA.

Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) and sassafras trees that died from laurel wilt were discovered in a 3-parish area of northern Louisiana, USA, in Sep 2014. Redbay, a species that has been severely affected by the disease in the coastal plains forests of the southeastern USA, is not found in this area of northern Louisiana, suggesting that sassafras is attractive to X. glabratus and an adequate reproductive host for the beetle. This is the first report of X. glabratus and laurel wilt in Louisiana and the first report of the beetle and disease west of the Mississippi River. Sumario En septiembre del 2014, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) y arboles de sasafras que murieron a causa de la marchitez del laurel fueron descubiertos en un area de 3 municipios del norte de Louisiana, EE.UU. El aguacatillo ( Persea borbonia ), una especie que se ha visto gravemente afectada por la enfermedad en los bosques de la llanura costero del sudeste de EE.UU., no se encuentra en esta zona del norte de Louisiana, lo que sugiere que el sasafras es atractivo para X. glabratus y un hospedero reproductivo adecuado para el escarabajo. Este es el primer informe de X. glabratus y la marchitez del laurel en Louisiana y el primer informe del escarabajo y la enfermedad al oeste del rio Mississippi. View this article in BioOne

Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi.

Summary Ceratocystis wilt, caused by Ceratocystis fimbriata, has become the most important disease in eucalyptus (Eucalyptus spp. and hybrids) plantations in Brazil. To further our understanding of the epidemiology of this disease, we surveyed eucalyptus plantations in the states of Minas Gerais and Bahia that were known to have Ceratocystis wilt or were thought to have been planted with infected rooted cuttings. There was generally higher disease incidence in the Minas Gerais plantations, which were on former Cerrado forest sites and likely had soilborne inoculum prior to planting eucalyptus. In such plantations, disease incidence was not evident before 20 months after planting but slowly increased up to 50% at 74 months. The symptomatic and killed trees were aggregated, perhaps from uneven distribution of inoculum in the soil. Also, the progression of cumulative disease incidence best fit a monomolecular model, which is typical of soilborne diseases (fixed level of initial inoculum with little or no secondary inoculum during the crop rotation). However, plots where some trees had been harvested during the rotation showed very high levels of disease incidence in the sprouts that arose from stumps, suggesting secondary spread of the pathogen on harvesting tools or machinery. Most of the Bahia plantations were on pastureland prior to eucalyptus cultivation, and the pathogen was likely introduced with infected nursery stock. In such plots, symptoms were evident as soon as 7 months after planting, and most of the mortality occurred within 12 months. The diseased trees on former pastureland sites were sometimes aggregated within planting rows, suggesting that bunches of infected nursery stock were planted together within the rows. Care should be taken in planting disease-free planting material and spreading the pathogen on tools, but on sites with soilborne inoculum, use of resistant clones may be the only management option.

A newly recognized, late-season leaf disease of Quercus macrocarpa (bur oak) has become increasingly severe across Iowa and in neighboring states since the 1990s. Vein necrosis and leaf death may occur over the whole crown or only on the lower branches. Symptoms typically intensify year-to-year in individual trees, and there appears to be substantial variation in susceptibility. Distinctive conidiomata (pycnothyria with a shield of radiating, setae-like hyphae) of a Tubakia sp. are found along the necrotic leaf veins. The same species produces a second type of pycnothyrium with a crustose covering and smaller conidia on the petioles of killed leaves, which remain on the tree through the winter and provide the primary inoculum to infect newly emerging shoots and leaves in spring. Comparison of the Tubakia sp. on bur oak with T. dryina and other species of Tubakia led to the conclusion that the species on bur oak is new, distinct from T. dryina, which herein is defined more narrowly. Inoculation studies confirmed that Tubakia iowensis sp. nov. is the cause of bur oak blight. Bur oak blight appears to be particularly severe on Q. macrocarpa var. oliviformis, which is well adapted to the dry, upland sites where the disease is found most frequently. The recent climatic trend in Iowa to higher spring precipitation might have led to increased severity of the disease.

The laurel wilt pathogen Raffaelea lauricola was hypothesized to have been introduced to the southeastern USA in the mycangium of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia. To test this hypothesis adult X. glabratus were trapped in Taiwan and on Kyushu Island, Japan, in 2009, and dead beetles were sent to USA for isolation of fungal symbionts. Individual X. glabratus were macerated in glass tissue grinders, and the slurry was serially diluted and plated onto malt agar medium amended with cycloheximide, a medium semiselective for Ophiostoma species and their anamorphs, including members of Raffaelea. R. lauricola was isolated from 56 of 85 beetles in Taiwan and 10 of 16 beetles in Japan at up to an estimated 10 000 CFUs per beetle. The next most commonly isolated species was R. ellipticospora, which also has been recovered from X. glabratus trapped in the USA, as were two other fungi isolated from beetles in Taiwan, R. fusca and R. subfusca. Three unidentified Raffaelea spp. and three unidentified Ophiostoma spp. were isolated rarely from X. glabratus collected in Taiwan. Isolations from beetles similarly trapped in Georgia, USA, yielded R. lauricola and R. ellipticospora in numbers similar to those from beetles trapped in Taiwan and Japan. The results support the hypothesis that R. lauricola was introduced into the USA in mycangia of X. glabratus shipped to USA in solid wood packing material from Asia. However differences in the mycangial mycoflora of X. glabratus in Taiwan, Japan and USA suggest that the X. glabratus population established in USA originated in another part of Asia.

The redhaired pine bark beetle Hylurgus ligniperda (F.) is native to Europe but was discovered in Los Angeles, California, in 2003. This root- and stump-feeding beetle is a common vector of Ophios- tomatales, which are potential tree pathogens or causes of blue stain of conifer sapwood. In this study Ophiostomatales were isolated on a cycloheximide- amended medium from 118 adult H. ligniperda collected from infested logs of Pinus halepensis and P. pinea at two sites in California. In total eight species of Ophiostomatales were identified and seven species that occasionally were isolated were unidentified. The most frequently isolated species were Ophiostoma ips and Grosmannia galeiforme ,w hich were isolated respectively from 31% and 23% of the 118 beetles. The other species isolated included O. piceae (isolated from 9% of the beetles), O. querci (8% )a nd Leptographium tereforme sp. nov. (6%). Grosmannia huntii, L. serpens ,t hreeSporothrix species, O. floccosum, O. stenoceras, two unidentified Hyalorhino- cladiella sp. and a sterile fungus each were isolated from fewer then 5% of beetles. Most of the identified species already were known in USA and have been found in association with H. ligniperda in other countries. However the new species, L. tereforme, and G. galeiforme were recorded from USA for the first time, and this is the first report of L. serpens from western North America.

Harrington, T. C., and Fraedrich, S. W. 2010. Quantification of propagules of the laurel wilt fungus and other mycangial fungi from the redbay ambrosia beetle, Xyleborus glabratus. Phytopathology 100:11181123. The laurel wilt pathogen, Raffaelea lauricola, is a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia and was believed to have brought R. lauricola with it to the southeastern United States. Individual X. glabratus beetles from six populations in South Carolina and Georgia were individually macerated in glass tissue grinders and serially diluted to quantify the CFU of fungal symbionts. Six species of Raffaelea were isolated, with up to four species from an individual adult beetle. The Raffaelea spp. were apparently within the protected, paired, mandibular mycangia because they were as numerous in heads as in whole beetles, and surface-sterilized heads or whole bodies yielded as many or more CFU as did nonsterilized heads or whole beetles. R. lauricola was isolated from 40 of the 41 beetles sampled, and it was isolated in the highest numbers, up to 30,000 CFU/beetle. Depending on the population sampled, R. subalba or R. ellipticospora was the next most frequently isolated species. R. arxii, R. fusca, and R. subfusca were only occasionally isolated. The laurel wilt pathogen apparently grows in a yeast phase within the mycangia in competition with other Raffaelea spp.

Mating studies showed that isolates of the insect-associated wilt pathogen Ceratocystis fimbriata from Eucalyptus spp., mango, fig, inhame (Colocasia esculenta), Gmelina arborea and sweet potato were interfertile, and progeny from those crosses showed normal segregation for microsatellite markers. Genetic diversity was compared among 13 populations of C. fimbriata collected from six states in Brazil using 15 highly polymorphic microsatellite markers. The gene diversity values of most eucalyptus and mango populations from Minas Gerais, Bahia, Rio de Janeiro and Sao Paulo states were similar to putatively native populations of Ceratocystis platani and C. cacaofunesta, two other species in the C. fimbriata complex that are homothallic. Index of association values indicated substantial asexual reproduction or selfing in populations on mango and eucalyptus. Most of these eucalyptus and mango populations were not highly differentiated from each other, and these populations and genotypes appeared to be more closely related to each other than to other populations by UPGMA analyses. By contrast, the G. arborea population from Paraand the fig and inhame populations from Sao Paulo had relatively low lev- els of diversity and were highly differentiated from each other and all other studied populations, suggesting that they were from different origins and had gone through genetic bottlenecks. One of the eucalyptus populations in Bahia consisted of a single genotype and may have been introduced to the site in infected cuttings from another Bahia location. Similarly, a mango population from Mato Grosso do Sul consisted of as ingle genotype, which was identical to one of the genotypes found on mango in Sao Paulo. Aside from introductions by humans, mating studies and genetic analyses suggest that limited dispersal distance and a high degree of selfing or asexual reproduction lead to local populations of C. fimbriata that have limited diversity but are highly differentiated from other populations.

Worrall, J. J., Harrington, T. C., Blodgett, J. T., Conklin, D. A., and Fairweather, M. L. 2010. Heterobasidion annosum and H. parviporum in the southern Rocky Mountains and adjoining states. Plant Dis. 94:115-118. Collections of Heterobasidion spp. from Nebraska, Colorado, Arizona, and New Mexico were identified based on the sequence of the internal transcribed spacer region of the ribosomal DNA. The North American variant of Heterobasidion annosum sensu stricto was found on Pinus ponderosa and Juniperus virginiana in central Nebraska, southern Colorado, central Arizona, and southern New Mexico. The North American variant of H. parviporum was found on Abies concolor and Picea engelmannii in southern Colorado and central New Mexico. The pathogens were not found in a survey of conifer forests in Wyoming and the Black Hills of South Dakota. Historical records of annosus root disease are reviewed by host group to gain more insight into the potential distributions of the respective pathogens. An apparent lack of overlap in host range suggests that substitution of tree species may be a useful management approach in some cases.

The oak wilt pathogen, Ceratocystis fagacearum, may be another example of a damaging, exotic species in forest ecosystems in the United States. Though C. fagacearum has received much research attention, the origin of the fungus is unknown. The pathogen may have been endemic at a low incidence until increased disturbances, changes in land use, and forest management created conditions favorable for disease epidemics. The host genus Quercus contains some relatively resistant species native to the United States, further supporting the hypothesis that the pathogen is native in origin. However, there are also many common, highly susceptible Quercus species—a characteristic typical of introduced pathogens. Most convincingly, studies have shown that the known populations of C. fagacearum have experienced a severe genetic bottleneck that can only be explained by a single introduction. The weight of evidence indicates that C. fagacearum is an introduced pathogen, with possible origins in Central or South America, or Mexico.

A restriction fragment length polymorphism (RFLP)-based technique was developed to identify members of the sooty blotch and flyspeck (SBFS) disease complex on apple because these fungi are difficult to identify using agar-plate isolation and morphological description. The method includes polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using a fungal-specific forward primer (ITS1-F) and an SBFS-specific reverse primer (Myc1-R), followed by digestion of the PCR product by the HaeIII restriction enzyme. When applied to previously identified isolates of 24 SBFS-causing species in nine genera, the PCR-RFLP assay produced 14 unique banding patterns. Different genera never shared the same RFLP pattern. To evaluate performance in vivo, the technique was applied to DNA extracted directly from SBFS colonies on apple fruit from three Iowa orchards. The primers amplified the rDNA of only SBFS fungi, with the exception of a Cladosporium sp.; however, its RFLP banding pattern was distinct from those of SBFS fungi. The majority (60%) of SBFS colonies in the in vivo trial were identified to genus by RFLP analysis. The PCR-RFLP assay greatly streamlined the identification process by minimizing the need for culturing, indicating its value as a tool for field studies of the SBFS complex.

Ceratocystis cacaofunesta (= Ceratocystis fimbriata) causes a lethal wilt disease of cacao (Theobroma cacao) in Latin America. Polymorphic microsatellite markers, (CAT)5 nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS‐rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.

Ceratocystis fimbriata causes diseases on a wide variety of plants in Brazil, including rubber tree (Hevea brasiliensis), on which it causes gray mold or moldy rot on tapping panels affecting latex yield. However, C. fimbriata isolated from rubber tree have not been critically studied. Phylogenetic analyses of sequences of ITS rDNA and a mating type gene placed rubber tree isolates from Acre and Bahia among Brazilian isolates of C. fimbriata from other hosts. In the analyses of 14 microsatellite loci, the rubber tree isolates from Bahia were identical to each other and had alleles similar to those of Brazilian isolates from mango and eucalyptus. The microsatellite alleles of the Acre rubber tree isolates were identical to each other but distinct from other Brazilian isolates. The rubber tree isolates were morphologically indistinguishable from each other and very similar to the isolates of C. fimbriata on Ipomoea batatas, on which the species was originally described. Based on inoculation experiments results, the Bahia and Acre rubber tree isolates do not appear to be host specialized, which is typical for Brazilian isolates of C. fimbriata sensu stricto.

Mango branch blight disease, caused by Ceratocystis fimbriata , is endemic to the municipality of Sao Fidelis in northern Rio de Janeiro State. In addition to mango, C. fimbriata was found associated with sugar apple trees (Annona squamosa) showing symptoms of branch blight in Sao Fidelis. Sugar apple and mango isolates from the same region had the same morphology and showed similar ITS-rDNA sequences. These sequences were also similar to other Brazilian isolates of C. fimbriata sensu stricto. Cross inoculation of such isolates obtained from diseased sugar apple and mango resulted in diseased symptoms on both plant species. This is the first record of A. squamosa as a host for C. fimbriata . Additional keywords: Host range, sugar apple, Mangifera indica, pathogenic variability. RESUMO Annona squamosa, nova hospedeira de Ceratocystis fimbriata . A seca-da-mangueira, causada pelo fungo Ceratocystis fimbriata , e uma doenca endemica no municipio de Sao Fidelis, no norte do Estado do Rio de Janeiro. Ceratocystis fimbriata foi encontrado associado a arvores de pinha (Annona squamosa) apresentando sintomas de seca dos ramos. Isolados obtidos de pinha e manga, originarios da mesma regiao, apresentaram a mesma morfologia e sequencias espacadoras ITS do rDNA similares. Estas sequencias foram tambem similares a de outros isolados brasileiros de C. fimbriata sensu stricto. Inoculacoes cruzadas dos isolados de pinha e manga resultaram no desenvolvimento de sintomas em ambas especies de plantas. Este e o primeiro relato de A. squamosa como hospedeira de C. fimbriata . Palavras-chave adicionais: Gama de hospedeiros, pinha, Mangifera indica, variabilidade patogenica.

Sooty blotch and flyspeck (SBFS) is a late-season disease of apple and pear fruit that cosmetically damages the cuticle, resulting in produce that is unacceptable to consumers. Previous studies reported that four species of fungi comprise the SBFS complex. We examined fungal morphology and the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA of 422 fungal isolates within the SBFS complex from nine orchards in four Midwestern states (USA) and compared them to previously identified species. We used LSU sequences to phylogenetically place the isolates at the order or genus level and then used ITS sequences to identify lineages that could be species. We used mycelial and conidial morphology on apple and in culture to delimit putative species. Thirty putative species found among the Midwest samples were shown to cause SBFS lesions on apple fruit in inoculation field trials. Among them Peltaster fructicola and Zygophiala jamaicensis have been associated previously with SBFS in North Carolina. The LSU analyses inferred that all 30 SBFS fungi from Midwestern orchards were Dothideomycetes; one putative species was within the Pleosporales, 27 were within Dothideales, and two putative species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella.

Ceratocystis fimbriata is a complex of many cryptic, host-specialized species that causes wilt and canker of woody species and rot diseases of storage roots and corms of many economically important plants worldwide. With the exception of the family Araceae, all confirmed hosts of C. fimbriata are dicotyledonous plants. We hypothesized that the isolates from members of the family Araceae would form a monophyletic lineage specialized to infect these unique hosts. Analyses of sequences of the internal transcribed spacer (ITS) region of nuclear rDNA indicate that isolates and herbarium specimens of C. fimbriata from the family Araceae represent three different groups: an Xanthosoma/Syngonium group on corms of Xanthosoma spp. from the Caribbean region and on ornamental S. podophyllum from greenhouses in Florida, Hawaii, Australia, and Brazil; an inhame group on corms of Colocasia esculenta in Brazil; and a distantly related taro group on Colocasia esculenta in Hawaii and China and on X. sagittifolium in Fiji. Inoculations of three species of Araceae (Caladium bicolor, S. podophyllum, and Colocasia esculenta) showed that isolates from all three groups are pathogenic to these three hosts. Brazilian isolates from Mangifera indica and Ficus carica were only weakly pathogenic to Caladium and Syngonium sp. and were not pathogenic to Colocasia sp. Syngonium plants appeared to be most susceptible to isolates of the Xanthosoma/Syngonium group, and Colocasia plants were least susceptible to isolates from Syngonium spp. Thus, it appears that adaptations to the family Araceae have evolved more than once in the C. fimbriata complex. It is hypothesized that the three groups of C. fimbriata on the family Araceae are native to the Caribbean, Brazil, and Asia, respectively, but they have been spread elsewhere by humans.