Fusarium oligoseptatum sp. nov., a mycosymbiont of the ambrosia beetle Euwallacea validus in the Eastern U.S. and typification of F. ambrosium.

Fusarium oligoseptatum sp. nov. was isolated from the invasive Asian ambrosia beetle Euwallacea validis (Coleoptera, Scolytinae, Xyleborini) and from the galleries that females had constructed in dying Ailanthus altissima (tree-of-heaven) symptomatic for Verticillium wilt in south-central Pennsylvania, USA. This ambrosia fungus was cultivated by Euwallacea validis as the primary source of nutrition together with a second symbiont, Raffaelea subfusca . Female beetles transport their fungal symbionts within and from their natal galleries in paired pre-oral mycangia. Fusarium oligoseptatum was distinguished phenotypically from the 11 other known members of the Ambrosia Fusarium Clade (AFC) by uniquely producing mostly 1-2 septate clavate sporodochial conidia that were swollen apically. Phylogenetic analysis of multilocus DNA sequence data resolved F. oligoseptatum as a genealogically exclusive species-level lineage but evolutionary relationships with other members of the AFC were unresolved. Published studies have shown that F. oligoseptatum can be identified via phylogenetic analysis of multilocus DNA sequence data or a PCR multiplex assay employing species-specific oligonucleotide primers. In addition, to provide nomenclatural stability, an epitype was prepared from an authentic strain of F. ambrosium that was originally isolated from a gallery constructed in Chinese tea (Camellia sinensis ) by E. fornicatus in India, together with its lectotypification based on a published illustration.

Fusarium oxysporum f. sp. palmarum, a Novel Forma Specialis Causing a Lethal Disease of Syagrus romanzoffiana and Washingtonia robusta in Florida.

A new disease of Syagrus romanzoffiana (queen palm) and Washingtonia robusta (Mexican fan palm) has spread across the southern half of Florida during the past 5 years. The initial foliar symptom is a one-sided chlorosis or necrosis of older leaf blades, with a distinct reddish-brown stripe along the petiole and rachis and an associated discoloration of internal tissue. Within 2 to 3 months after onset of symptoms, the entire canopy becomes desiccated and necrotic but the leaves do not droop or hang down around the trunk. Based on pathogenicity and morphological and molecular characterization, the etiological agent has been identified as a new forma specialis of Fusarium oxysporum, designated f. sp. palmarum. Sequence analysis of a portion of the translation elongation factor 1-α gene (EF-1α) separated 27 representative isolates into two EF-1α groups, which differed by two transition mutations. Members of both EF-1α groups are pathogenic on both species of palm. A phylogenetic analysis inferred from partial EF-1α sequences from a genetically diverse set of F. oxysporum isolates, including three other formae speciales pathogenic on palm (i.e., f. sp. albedinis, f. sp. canariensis, and f. sp. elaeidis), suggested that f. sp. palmarum and f. sp. albedinis may be more closely related to one another than either is to the two other palm pathogens.

Multilocus phylogenetic diversity of Fusarium avenaceum pathogenic on lisianthus.

Fusarium avenaceum is a globally distributed fungus commonly isolated from soil and a wide range of plants. Severe outbreaks of crown and stem rot of the flowering ornamental, lisianthus (Eustoma grandiflorum), have been attributed to F. avenaceum. We sequenced portions of the translation elongation factor 1-alpha (tef) and beta-tubulin (benA) protein coding genes as well as partial intergenic spacer (IGS) regions of the nuclear ribosomal genes in 37 Fusarium isolates obtained from lisianthus and other host plants. Isolates that were previously identified morphologically as F. acuminatum were included as an outgroup. Phylogenetic analyses of tef, benA, and IGS sequences showed that F. avenaceum isolates were an exclusive group with strong bootstrap support and no significant incongruence among gene genealogies. Isolates from lisianthus were scattered within this clade and did not form distinct groups based on host species or locality. Pathogenicity tests of F. avenaceum isolates obtained from several other hosts showed an ability to cause disease on lisianthus, suggesting that F. avenaceum may be pathogenic on lisianthus regardless of its phylogenetic origin. These findings have management implications and suggest that any host that supports F. avenaceum may serve as a source of inoculum for lisianthus growers.

First Report of a Novel Fusarium Species Causing Yellowing Decline of Sugar Beet in Minnesota.

In the United States, yellows disease of sugar beet (Beta vulgaris), which causes wilt, early death, and yield reduction, is caused primarily by Fusarium oxysporum f. sp. betae (3,4), but F. graminearum (2) has also been implicated. During the past 3 years, a similar disease causing yellowing and severe decline appeared in some sugar beet fields of central and southwest Minnesota planted with cultivars resistant to yellows. The disease has become a concern to the local sugar beet industry, which produces 56% of sugar beets in the United States. From 2005 to 2007, isolations were made from sugar beets collected in commercial fields and from a Fusarium screening nursery showing symptoms of yellowing, interveinal chlorosis, scorching, stunting, vascular discoloration of the taproot, and early death of plants. Of 96 Fusarium isolates recovered and used in root-dip inoculation trials in the greenhouse, 58 were pathogenic to sugar beets. On the basis of morphology, 12 were identified as F. oxysporum, 6 as F. graminearum, and 40 as a novel Fusarium species. The remaining 38 isolates were nonpathogenic. All three pathogenic Fusarium species were isolated from taproots, but only the novel Fusarium was isolated from petioles. In culture, the novel Fusarium exhibited a bright orange color on the underside of potato dextrose agar medium and produced micro- and macroconidia sparsely. Hyphal tip isolates of all novel Fusarium isolates were pathogenic, causing typical yellowing symptoms and plant death to the Fusarium yellows susceptible sugar beet cv. VDH46177 in replicated greenhouse trials. Isolates were successfully reisolated from the symptomatic plants, fulfilling Koch's postulates. Restriction fragment length polymorphism (RFLP) endonuclease digestion patterns (Alu1, Fnu4HI, HaeIII, and HhaI) of the internal transcribed spacer (ITS) region of 40 pathogenic novel isolates showed a distinct pattern compared with known Fusarium species. Thin layer chromatography analysis of 13 novel isolates detected the type A trichothecenes neosolaniol and 4,15-diacetoxyscirpenol. Partial sequences of the translation elongation factor 1-α (TEF) from 12 single-spored novel Fusarium isolates were generated. BLAST analysis of the TEF sequence against the FUSARIUM-ID (1) and GenBank databases did not match any known Fusarium species. On the basis of pathogenicity, morphology, RFLP patterns, mycotoxin production, and TEF sequence analysis it appears that this is a new species of Fusarium, but additional multilocus phylogenetic analyses are warranted. The natural occurrence of this novel Fusarium pathogen in sugar beet may have implications in breeding for resistance to Fusarium yellows, since yellow decline has been observed in purportedly Fusarium-tolerant cultivars in the Minnesota and North Dakota production regions. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) L. E. Hanson. Plant Dis. 90:686, 2006. (3). L. E. Hanson. Plant Dis. 90:1554, 2006. (4) C. E. Windels et al. Plant Dis. 89:341, 2005.

Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae.

We investigated the sexual reproductive mode of the two most important etiological agents of soybean sudden death syndrome, Fusarium tucumaniae and Fusarium virguliforme. F. tucumaniae sexual crosses often were highly fertile, making it possible to assign mating type and assess female fertility in 24 South American isolates. These crosses produced red perithecia and oblong-elliptical ascospores, as is typical for sexual members of the F. solani species complex. Genotyping of progeny from three F. tucumaniae crosses confirmed that sexual recombination had occurred. In contrast, pairings among 17 U.S. F. virguliforme isolates never produced perithecia. Inter-species crosses between F. tucumaniae and F. virguliforme, in which infertile perithecia were induced only in one of the two F. tucumaniae mating types, suggest that all U.S. F. virguliforme isolates are of a single mating type. We conclude that the F. tucumaniae life cycle in S. America includes a sexual reproductive mode, and thus this species has greater potential for rapid evolution than the F. virguliforme population in the U.S., which may be exclusively asexual.

The current status of species recognition and identification in Aspergillus.

The species recognition and identification of aspergilli and their teleomorphs is discussed. A historical overview of the taxonomic concepts starting with the monograph of Raper & Fennell (1965) is given. A list of taxa described since 2000 is provided. Physiological characters, particularly growth rates and the production of extrolites, often show differences that reflect phylogenetic species boundaries and greater emphasis should be placed on extrolite profiles and growth characteristics in species descriptions. Multilocus sequence-based phylogenetic analyses have emerged as the primary tool for inferring phylogenetic species boundaries and relationships within subgenera and sections. A four locus DNA sequence study covering all major lineages in Aspergillus using genealogical concordance theory resulted in a species recognition system that agrees in part with phenotypic studies and reveals the presence of many undescribed species not resolved by phenotype. The use of as much data from as many sources as possible in making taxonomic decisions is advocated. For species identification, DNA barcoding uses a short genetic marker in an organism"s DNA to quickly and easily identify it to a particular species. Partial cytochrome oxidase subunit 1 sequences, which are used for barcoding animal species, were found to have limited value for species identification among black aspergilli. The various possibilities are discussed and at present partial beta-tubulin or calmodulin are the most promising loci for Aspergillus identification. For characterising Aspergillus species one application would be to produce a multilocus phylogeny, with the goal of having a firm understanding of the evolutionary relationships among species across the entire genus. DNA chip technologies are discussed as possibilities for an accurate multilocus barcoding tool for the genus Aspergillus.

First Report of Fusarium Wilt Caused by Fusarium oxysporum on Roselle in the United States.

Roselle, Hibiscus sabdariffa var. sabdariffa, is an annual that is grown primarily for its inflated calyx, which is used for drinks and jellies. It is native from India to Malaysia, but was taken at an early date to Africa and is now widely grown in the tropics and subtropics (2). In late 2005, dying plants were noted by a producer in South Florida. Plants wilted, became chlorotic, and developed generally unthrifty, sparse canopies. Internally, conspicuous vascular discoloration was evident in these plants from the roots into the canopy. After 5 days on one-half-strength potato dextrose agar (PDA), salmon-colored fungal colonies grew almost exclusively from surface-disinfested 5 mm2 pieces of vascular tissue. On banana leaf agar, single-spored strains produced the following microscopic characters of Fusarium oxysporum: copious microconidia on monophialides, infrequent falcate macroconidia, and terminal and intercalary chlamydospores. Partial, elongation factor 1-α (EF1-α) sequences were generated for two of the strains, O-2424 and O-2425, and compared with previously reported sequences for the gene (3). Maximum parsimony analysis of sequences showed that both strains fell in a large, previously described clade of the F. oxysporum complex (FOC) that contained strains from agricultural hosts, as well as human clinical specimens (2; clade 3 in Fig. 4); many of the strains in this clade have identical EF1-α sequences. Strains of F. oxysporum recovered from wilted roselle in Egypt, O-647 and O-648 in the Fusarium Research Center collection, were distantly related to the Florida strains. We are not aware of other strains of F. oxysporum from roselle in other international culture collections. Roselle seedlings were inoculated with O-2424 and O-2425 by placing a mycelial plug (5 mm2, PDA) over a small incision 5 cm above the soil line and then covering the site with Parafilm. Parafilm was removed after 1 week, and plants were incubated under ambient temperatures (20 to 32°C) in full sun for an additional 5 weeks (experiment 1) or 7 weeks (experiment 2). Compared with mock-inoculated (wound + Parafilm) control plants, both O-2424 and O-2425 caused significant (P < 0.05) vascular disease (linear extension of discolored xylem above and below wound site) and wilting (subjective 1 to 5 scale); both isolates were recovered from affected plants. F. oxysporum-induced wilt of roselle has been reported in Nigeria (1) and Malaysia (4) where the subspecific epithet f. sp. rosellae was used for the pathogen. We are not aware of reports of this disease elsewhere. To our knowledge, this is the first report of F. oxysporum-induced wilt of roselle in the United States. Research to determine whether the closely related strains in clade 3 of the FOC are generalist plant pathogens (i.e., not formae speciales) is warranted. References: (1) N. A. Amusa et al. Plant Pathol. J. 4:122, 2005. (2) J. Morton. Pages 81-286 in: Fruits of Warm Climates. Creative Resource Systems, Inc., Winterville, NC, 1987. (3) K. O'Donnell et al. J. Clin. Microbiol. 42:5109, 2004. (4) K. H. Ooi and B. Salleh. Biotropia 12:31, 1999.

Fusarium species of the Gibberella fujikuroi complex and fumonisin contamination of pearl millet and corn in Georgia, USA.

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and O'Donnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB(1) and FB(2) were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 microg/g.

First Report of a Wilt Disease of Hiemalis Begonias Caused by Fusarium foetens in the United States.

During 2003, 10% of the Hiemalis begonias (Begonia × hiemalis Fotsch) developed wilt symptoms in a commercial greenhouse in Connecticut. Foliage turned a dull green, and stems developed a dark watersoaked discoloration near the soil line and had vascular discoloration. Stems, petioles, and leaves collapsed and became covered with sporodochia of a Fusarium spp. Single conidia were isolated from sporodochia and cultured on carnation leaf agar (CLA) and potato dextrose agar for 10 days. Isolates resembled Fusarium oxysporum, but the profuse sporulation with minimal aerial mycelium and the rare occurrence of polyphialides was consistent with the description of F. foetens (2). A comparison of a partial sequence of the 1-α elongation factor gene showed a 100% match with F. foetens. Inocula from five isolates were grown on CLA, washed from the plate, and adjusted to 106 conidia per ml. Suspension (50 µl) was injected into stems of healthy 6-week-old Hiemalis begonias cv. Barkos (one plant per isolate). Controls received distilled water. After 4 weeks, all inoculated plants turned dark and collapsed, and the same fungus was reisolated from these plants. Control stems remained healthy. An isolate (O-2348) has been deposited at the Fusarium Research Center at Pennsylvania State University, University Park. F. foetens has recently been described in association with a new disease of Hiemalis begonias in Europe (1). References: (1) R. Schrage, Phytomedizinischen Gesellschaft 33:68, 2003. (2) H.-J. Schroers et al. Mycologia 96:393, 2004.