Cytochemical labeling for fungal and host components in plant tissues inoculated with fungal wilt pathogens.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

Fusarium foetens, a new species pathogenic to begonia elatior hybrids (Begonia x hiemalis) and the sister taxon of the Fusarium oxysporum species complex.

A new disease recently was discovered in begonia elatior hybrid (Begonia × hiemalis) nurseries in The Netherlands. Diseased plants showed a combination of basal rot, vein yellowing and wilting and the base of collapsing plants was covered by unusually large masses of Fusarium macroconidia. A species of Fusarium was isolated consistently from the discolored veins of leaves and stems. It differed morphologically from F. begoniae, a known agent of begonia flower, leaf and stem blight. The Fusarium species resembled members of the F. oxysporum species complex in producing short monophialides on the aerial mycelium and abundant chlamydospores. Other phenotypic characters such as polyphialides formed occasionally in at least some strains, relatively long monophialides intermingled with the short monophialides formed on the aerial mycelium, distinct sporodochial conidiomata, and distinct pungent colony odor distinguished it from the F. oxysporum species complex. Phylogenetic analyses of partial sequences of the mitochondrial small subunit of the ribosomal DNA (mtSSU rDNA), nuclear translation elongation factor 1α (EF-1α) and ß-tubulin gene exons and introns indicate that the Fusarium species represents a sister group of the F. oxysporum species complex. Begonia × hiemalis cultivars Bazan, Bellona and Netja Dark proved to be highly susceptible to the new species. Inoculated plants developed tracheomycosis within 4 wk, and most died within 8 wk. The new taxon was not pathogenic to Euphorbia pulcherrima, Impatiens walleriana and Saintpaulia ionantha that commonly are grown in nurseries along with B. × hiemalis. Inoculated plants of Cyclamen persicum did not develop the disease but had discolored vessels from which the inoculated fungus was isolated. Given that the newly discovered begonia pathogen is distinct in pathogenicity, morphology and phylogeny from other fusaria, it is described here as a new species, Fusarium foetens.

Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis).

ABSTRACT The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

First Report of Phytophthora nicotianae on Limonium in Europe.

Limonium (statice or sea-lavendar, family Plumbaginaceae) is grown in the Netherlands as a perennial (Limonium sinense) or annual (Limonium sinuatum) crop. Plants have tufted leaves and numerous clustered flowers of different colors and are used for flower arrangements. In August 2000, we received diseased plants of L. sinense cv. Diamond and L. sinuatum. Disease symptoms consisted of leaf wilting followed by plant collapse. The base of the leaves showed progressive necrotic areas that later turned dark brown to black. The cortex of the stem and roots was water-soaked and dark brown to black. Longitudinal sections of stems and roots of diseased plants displayed discoloration of tissues. Rotted root tissue was brown with a characteristic black margin. Rotted vascular tissues and other stem parts were also dark brown. Pith parenchyma turned gray-brown and had a firm, wet rot. In plants with advanced disease symptoms, a cavity in the stem parenchyma was observed. Isolations were made from sections of symptomatic leaves, stems and roots of both Limonium species on cherry and water agar (WA), followed by incubation at 20°C. Phytophthora sp. was isolated consistently from the base of leaves, stems, and roots of diseased plants and identification of isolates was based on morphological characteristics and by isozyme analysis (3). Observations of colony morphology and growth at 35°C were made on V8 agar. Mating type was determined in dual cultures with mating type A2 (P. nicotianae, P 1923 [4]) and A1 (P. nicotianae, PD98/8/10402). Sporangial features were observed from liquid cultures of the isolates (autoclaved soil-extract or sterile distilled water). All isolates formed colonies consisting of loose, fluffy aerial mycelia. Sporangia and chlamydospores were present in all fungal isolates and all isolates were able to grow at 35°C. Few sporangia were produced on solid media (WA and V8 juice agar), but were abundant in liquid cultures. Sporangia were borne singly or in simple sympodial sporangiophores (3 to 4 sporangia), and were ovoid/spherical, obturbinate with rounded base and had prominent papillae (some had two papillae). Sporangia measured 40 to 64 × 24 to 56 µm, (average 50.4 × 38.4 µm) and had an average length:breath ratio of 1.3:1. Chlamydospores were terminal and intercalary and measured 18 to 44 µm (average 31.6 µm). Hyphal swellings with hyphal outgrowths were present. Isolates of the fungus were heterothallic and produced oogonia and oospores rapidly and abundantly on V8 agar at 22°C only with the A1 mating type of P. nicotianae. We concluded that all isolates from Limonium had the A2 compatibility type. Antheridia were amphigynous. Oogonia were spherical and ranged from 20 to 30 µm, (average 27.5 µm). Oospores ranged from 18 to 27 µm, (average 23.1 µm). The observed characteristics are similar to those described for P. nicotianae. Isozyme analysis, using the dimeric enzymes malic enzyme (EC 1.1.1.40) and malate dehydrogenase (EC 1.1.1.37), revealed the presence of the Mdhp100 allele and the Mdh-2100 allele. Both alleles are characteristic for P. nicotianae (3). Based on morphological features and isozyme genotyping, isolates of Phytophthora from diseased Limonium plants could be assigned to P. nicotianae van Breda de haan (1). A report from Florida associated Phytophthora sp. with root rot of Limonium plants (2) but did not identify the species. According to the multi-decade records at the Netherlands Plant Protection Service (unpublished data) Phytophthora has never been observed on Limonium before. This is the first report of P. nicotianae associated with root rot and basal rot of Limonium plants in Europe. References: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN. (2) D. F. Farr et al. 1989. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN. (3) W. A. Man in 't Veld et al. Phytopathology 88:922-929, 1998. (4) P. Oudemans and M. D. Coffey. Mycol. Res. 95:1025-1046, 1991.

Molecular Relationships of Fungi Within the Fusarium redolens-F. hostae Clade.

ABSTRACT The evolutionary relationships of fungi in the Fusarium redolens-F. hostae clade were investigated by constructing nuclear and mitochondrial gene genealogies for 37 isolates representing the known genetic and pathogenic diversity of this lineage, together with 15 isolates from putative sister groups that include the Gibberella fujikuroi and F. oxysporum species complexes and related species. Included in the analyses were 29 isolates of F. redolens from Asparagus, Convallaria, Dianthus, Fritillaria, Hebe, Helleborus, Hordeum, Linum, Pisum, Pseudotsuga, and Zea spp., and from soil. Isolates of F. hostae analyzed included two reference isolates from Hosta spp. and six isolates from Hyacinthus spp. that originally were classified as F. oxysporum f. sp. hyacinthi. DNA sequences from a portion of the nuclear translation elongation factor 1alpha (EF-1alpha) gene and the mitochondrial small subunit (mtSSU) ribosomal RNA (rRNA) were analyzed individually and as a combined data set based on results of the nonparametric Wilcoxon signed ranks Templeton combinability test. Maximum parsimony analysis of the combined data set identified the F. redolens-F. hostae clade as a sister group to a phylogenetically diverse clade in which the G. fujikuroi species complex formed the most basal lineage. Also included in this latter clade were two unnamed Fusarium spp. that are morphologically similar to F. oxysporum and putative sister taxa comprising the F. oxysporum complex and a F. nisikadoi-F. miscanthi clade. Phylogenetic diversity in F. redolens was small; all isolates were represented by only three EF-1alpha and two mtSSU rDNA haplotypes. Both the isolates of F. redolens f. sp. asparagi and those of F. redolens f. sp. dianthi were nearly evenly distributed in the combined molecular phylogeny between the two major subclades within F. redolens.

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum.

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae x P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.

Gene Genealogies and AFLP Analyses in the Fusarium oxysporum Complex Identify Monophyletic and Nonmonophyletic Formae Speciales Causing Wilt and Rot Disease.

ABSTRACT The monophyletic origin of host-specific taxa in the plant-pathogenic Fusarium oxysporum complex was tested by constructing nuclear and mitochondrial gene genealogies and amplified fragment length polymorphism (AFLP)-based phylogenies for 89 strains representing the known genetic and pathogenic diversity in 8 formae speciales associated with wilt diseases and root and bulb rot. We included strains from clonal lineages of F. oxysporum f. spp. asparagi, dianthi, gladioli, lilii, lini, opuntiarum, spinaciae, and tulipae. Putatively nonpathogenic strains from carnation and lily were included and a reference strain from each of the three main clades identified previously in the F. oxysporum complex; sequences from related species were used as outgroups. DNA sequences from the nuclear translation elongation factor 1alpha and the mitochondrial small subunit (mtSSU) ribosomal RNA genes were combined for phylogenetic analysis. Strains in vegetative compatibility groups (VCGs) shared identical sequences and AFLP profiles, supporting the monophyly of the two single-VCG formae speciales, lilii and tulipae. Identical genotypes were also found for the three VCGs in F. oxysporum f. sp. spinaciae. In contrast, multiple evolutionary origins were apparent for F. oxysporum f. spp. asparagi, dianthi, gladioli, lini, and opuntiarum, although different VCGs within each of these formae speciales often clustered close together or shared identical EF-1alpha and mtSSU rDNA haplotypes. Kishino-Hasegawa analyses of constraints forcing the monophyly of these formae speciales supported the exclusive origin of F. oxysporum f. sp. opuntiarum but not the monophyly of F. oxysporum f. spp. asparagi, dianthi, gladioli, and lini. Most of the putatively nonpathogenic strains from carnation and lily, representing unique VCGs, were unrelated to F. oxysporum f. spp. dianthi and lilii, respectively. Putatively nonpathogenic or rot-inducing strains did not form exclusive groups within the molecular phylogeny. Parsimony analyses of AFLP fingerprint data supported the gene genealogy-based phylogram; however, AFLP-based phylogenies were considerably more homoplasious than the gene genealogies. The predictive value of the forma specialis naming system within the F. oxysporum complex is questioned.