An expanded concept of Ceratocystis manginecans and five new species in the Latin American clade of Ceratocystis.

The genus Ceratocystis contains a number of emerging plant pathogens, mostly members of the Latin American Clade (LAC), in which there are several unresolved taxonomic controversies. Among the most important are Brazilian pathogens in the C. fimbriata complex, C. manginecans and C. eucalypticola. Representatives of C. manginecans and C. eucalypticola from India and China, respectively, were shown to be fully interfertile in laboratory matings, and hybrids between the putative species were identified on Punica in India. An Indian tester strain was sexually compatible with representatives of what has been considered C. fimbriata on numerous hosts across Brazil. In this revision of the LAC, the name C. fimbriata is restricted to the widely dispersed Ipomoea strain, and C. manginecans is recognized as a Brazilian species that is important on Mangifera, Eucalyptus, and many other crops. C. mangivora and C. mangicola are also considered synonyms of C. manginecans. Based on phylogenetics and mating studies, two other Brazilian species are recognized: C. atlantica, sp. nov., and C. alfenasii, sp. nov., each with wide host ranges. Three new Caribbean species are recognized based on phylogenetics and earlier inoculation studies: C. costaricensis, sp. nov., on Coffea, C. cubensis, sp. nov., on Spathodea, and C. xanthosomatis, sp. nov., on the vegetatively propagated aroids Xanthosoma and Syngonium. Some of the other Ceratocystis species were based primarily on unique internal transcribed spacer (ITS) rDNA sequences, but the unreliability of rDNA sequences was demonstrated when intraspecific crossing of isolates with differing ITS sequences generated single-ascospore progeny with intragenomic variation in ITS sequences and others with new ITS sequences. Species recognition in Ceratocystis should use phenotype, including intersterility tests, to help identify which lineages are species. Although some species remain under-studied, we recognize 16 species in the LAC, all believed to be native to Latin America, the Caribbean region, or eastern USA.

Corticioid basidiomycetes associated with bark beetles, including seven new Entomocorticium species from North America and Cylindrobasidium ipidophilum, comb. nov.

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.

Patterns of coevolution between ambrosia beetle mycangia and the Ceratocystidaceae, with five new fungal genera and seven new species.

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.

Taxonomic revision and multi-locus phylogeny of the North American clade of Ceratocystis.

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.

New Ceratocystis species associated with rapid death of Metrosideros polymorpha in Hawai'i.

The native 'ohi'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 'ohi'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 'ohi'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.

A re-evaluation of Tubakia, including three new species on Quercus and six new combinations.

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.

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

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 ß-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.

Phenology of Infection on Apple Fruit by Sooty Blotch and Flyspeck Species in Iowa Apple Orchards.

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 tiliae sp. nov., a wound pathogen on Tilia americana.

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.

Intraspecific and intragenomic variability of ITS rDNA sequences reveals taxonomic problems in Ceratocystis fimbriata sensu stricto.

Fourteen new species in the Latin American Clade (LAC) of the Ceratocystis fimbriata complex recently were distinguished from C. fimbriata sensu stricto largely based on variation in ITS rDNA sequences. Among the 116 isolates representing the LAC, there were 41 ITS haplotypes. Maximum parsimony (MP) analysis of ITS sequences produced poorly resolved trees. In contrast, analyses of mating-type genes (MAT1-1-2 and MAT1-2-1) resolved a single MP tree with branches of high bootstrap and posterior probability support. Four isolates showed intragenomic variation in ITS sequences. Cloning and sequencing of PCR products from a single haploid strain identified two or more ITS sequences differing at up to 16 base positions and representing two described species. Isolates from introduced populations that appeared to be clonal based on microsatellite markers varied at up to 14 bp in ITS sequence. Strains of seven Brazilian ITS haplotypes and an isolate from Ipomoea batatas (on which the species name C. fimbriata was based) were fully interfertile in sexual crosses. These analyses support three phylogenetic species that differ in pathogenicity: C. platani, C. cacaofunesta and C. colombiana. Five ITS species (C. manginecans, C. mangicola, C. mangivora, C. acaciivora, C. eucalypticola) appear to be ITS haplotypes that have been moved from or within Brazil on nursery stock. The taxonomic status of other species delineated primarily by ITS sequences (C. diversiconidia, C. papillata, C. neglecta, C. ecuadoriana, C. fimbriatomima, C. curvata) needs further study, but they are considered doubtful species.

First Report of Laurel Wilt, Caused by Raffaelea lauricola, on Sassafras (Sassafras albidum) in Alabama.

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.

Temporal patterns in appearance of sooty blotch and flyspeck fungi on apples.

Sooty blotch and flyspeck (SBFS) is a complex of about 80 fungal species that blemish the surface of apple fruit in humid regions worldwide. The dark colonies become visible in mid- to late summer, reducing the value of fresh fruit. Although many SBFS species can co-occur in the same orchard and even on the same apple, little is known about temporal patterns of these species, including the timing of colony appearance. To test the hypothesis that colonies of SBFS species appear on apples at characteristic times during the growing season, 50 apples were monitored weekly at three Iowa orchards in 2006 and six orchards in 2007 and 2008. However, a mean of 24.3 apples per orchard was assessed at harvest because of apple drop throughout the season. Colonies were marked with colored pens as they appeared. After harvest and after storage of apples at 2 °C for 3 months, SBFS colonies on each fruit were counted and classified by morphology, and a representative subset of colonies was excised from the fruit and preserved on dried peels for species identification using rDNA. Seventeen species were identified. Stomiopeltis spp. RS1 and RS2 appeared on apples 10 to 14 days before other SBFS taxa. Dissoconium aciculare was generally the last species to appear on apple fruit, and it continued to appear during postharvest storage. The most prevalent taxa in Iowa orchards were also the most abundant. Diversity of SBFS fungi in an orchard was positively correlated with cumulative hours of surface wetness hours due to rainfall or dew, which is believed to favor growth of SBFS fungi. Species-specific information about temporal patterns of appearance on apple fruit may lead to improved SBFS management strategies.

First Report of the Walnut Witches'-Broom Phytoplasma on Japanese and Black Walnut in Iowa.

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.

First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia.

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.

First Report of Laurel Wilt Caused by Raffaelea lauricola on Sassafras in Mississippi.

Laurel wilt is caused by the fungus Raffaelea lauricola T.C. Harrin., Aghayeva & Fraedrich and is lethal to redbay (Persea borbonia (L.) Spreng.), sassafras (Sassafras albidum (Nutt.) Nees), and other species in the Lauraceae (1). The fungus is carried by the redbay ambrosia beetle (Xyleborus glabratus Eichh.), which is native to Asia. After being discovered in Georgia in 2002 (1), X. glabratus and R. lauricola have spread rapidly, causing extensive redbay mortality in South Carolina, Georgia, Florida, and Mississippi (1,4). The disease has also been confirmed on sassafras in Florida, South Carolina (1), and Georgia. Questions remain as to whether laurel wilt will continue to spread on sassafras, which often occurs as scattered trees in the eastern United States. In June 2010, a homeowner reported that a sassafras tree north of Van Cleave, MS (30.668°N, 88.686°W) had begun wilting in late May. This landscape tree had three 10-m high stems (~20 cm in diameter at breast height). Dark staining in the xylem was observed around the entire circumference of all three stems and nearly all leaves were bronze colored and wilted. No ambrosia beetle tunnels were observed in the stems. No other symptomatic Lauraceae were encountered in the wooded area within 300 m. The nearest known location with laurel wilt on redbay was ~15 km away (4). A Lindgren funnel trap baited with manuka oil (2) was placed at the site in June and monitored biweekly until November, but no X. glabratus adults were captured. Chips from discolored xylem of the sassafras were surface sterilized, plated on cycloheximide-streptomycin malt agar, and R. lauricola was readily isolated (1). Identity of the fungus (isolate C2792 in collection of T. Harrington) was confirmed by using partial sequences of the 28S rDNA (3). The sassafras sequence was identical to that of all known sequences of R. lauricola in the United States, including GenBank No. EU123076 (the holotype isolate from redbay). To confirm pathogenicity, isolate C2792 was grown on malt extract agar and three redbay (average: 141 cm high and 12 mm in diameter at soil interface) and three sassafras (average: 170 cm high and 17 mm in diameter at soil interface) potted plants were wound inoculated with 0.2 ml of a spore suspension (4.9 × 106 conidia/ml) (1). Three control plants of each species were inoculated with sterile deionized water. After 8 weeks in a growth chamber at 26°C, all inoculated redbay and sassafras plants exhibited xylem discoloration above and below the inoculation point, two of the redbay and two of the sassafras had died, and the other plant of each species exhibited partial wilt (the main terminal or one or more branches). All control plants were asymptomatic. R. lauricola was reisolated from all inoculated symptomatic plants but not from controls. To our knowledge, this is the first report of laurel wilt on sassafras in Mississippi. Both redbay (4) and sassafras appear to be highly susceptible to the disease as it moves westward. Sassafras is less attractive than redbay to X. glabratus and it was thought that this might contribute to slowing the spread of laurel wilt once outside the range of redbay (2). Nonetheless, our observations confirm that sassafras can be infected where laurel wilt on redbay is not in the immediate vicinity. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. L. Hanula et al. J. Econ. Entomol. 101:1276, 2008. (3) T. C. Harrington et al. Mycotaxon 111:337, 2010. (4) J. J. Riggins et al. Plant Dis. 94:634, 2010.

Susceptibility to Laurel Wilt and Disease Incidence in Two Rare Plant Species, Pondberry and Pondspice.

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.

Quantification of propagules of the laurel wilt fungus and other mycangial fungi from the redbay ambrosia beetle, Xyleborus glabratus.

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.

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.

An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple.

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.

Association of Ophiostoma novo-ulmi with Scolytus schevyrewi (Scolytidae) in Colorado.

The smaller European elm bark beetle, Scolytus multistriatus, has been the primary vector of the Dutch elm disease fungus, Ophiostoma novo-ulmi, in elm trees in Colorado since 1948. An exotic from Asia, the banded elm bark beetle, Scolytus schevyrewi, was found in Siberian elm, Ulmus pumila, in Colorado in April of 2003; this was the first report of S. schevyrewi in North America. S. schevyrewi is now found throughout much of Colorado and in at least 21 other states. The similarities in breeding and feeding habits between S. schevyrewi and S. multistriatus have raised concerns about the ability of S. schevyrewi to serve as a vector for O. novo-ulmi. The objective of this preliminary study was to determine if O. novo-ulmi could be isolated from adult S. schevyrewi emerging from diseased elm trees. S. schevyrewi and S. multistriatus were allowed to infest diseased stem segments of American elm, Ulmus americana. The infested stem segments were caged and isolations were made from the adult brood that emerged. O. novo-ulmi was isolated from most of the adults of both beetle species, showing that S. schevyrewi could acquire the pathogen as effectively as S. multistriatus. Future studies are needed to determine if S. schevyrewi can effectively transmit the pathogen to healthy trees.