New and Interesting Fungi. 2.

One order, seven families, 28 new genera, 72 new species, 13 new combinations, four epitypes, and interesting new host and / or geographical records are introduced in this study. Pseudorobillardaceae is introduced for Pseudorobillarda (based on P. phragmitis). New genera include: Jeremyomyces (based on J. labinae) on twigs of Salix alba (Germany); Neodothidotthia (based on N. negundinicola) on Acer negundo (Ukraine); Neomedicopsis (based on N. prunicola) on fallen twigs of Prunus padus (Ukraine); Neophaeoappendicospora (based on N. leucaenae) on Leucaena leucocephala (France) (incl. Phaeoappendicosporaceae); Paradevriesia (incl. Paradevriesiaceae) (based on P. americana) from air (USA); Phaeoseptoriella (based on P. zeae) on leaves of Zea mays (South Africa); Piniphoma (based on P. wesendahlina) on wood debris of Pinus sylvestris (Germany); Pseudoconiothyrium (based on P. broussonetiae) on branch of Broussonetia papyrifera (Italy); Sodiomyces (based on S. alkalinus) from soil (Mongolia), and Turquoiseomyces (incl. Turquoiseomycetales and Turquoiseomycetaceae) (based on T. eucalypti) on leaves of Eucalyptus leptophylla (Australia); Typhicola (based on T. typharum) on leaves of Typha sp. (Germany); Xenodevriesia (incl. Xenodevriesiaceae) (based on X. strelitziicola) on leaves of Strelitzia sp. (South Africa). New species include: Bacillicladium clematidis on branch of Clematis vitalbae (Austria); Cercospora gomphrenigena on leaves of Gomphrena globosa (South Africa); Cyphellophora clematidis on Clematis vitalba (Austria); Exophiala abietophila on bark of Abies alba (Norway); Exophiala lignicola on fallen decorticated trunk of Quercus sp. (Ukraine); Fuscostagonospora banksiae on Banksia sp. (Australia); Gaeumannomycella caricicola on dead leaf of Carex remota (Germany); Hansfordia pruni on Prunus persica twig (Italy) (incl. Hansfordiaceae); Microdochium rhopalostylidis on Rhopalostylis sapida (New Zealand); Neocordana malayensis on leaves of Musa sp. (Malaysia); Neocucurbitaria prunicola on fallen twigs of Prunus padus (Ukraine); Neocucurbitaria salicis-albae on Salix alba twig (Ukraine); Neohelicomyces deschampsiae on culm base of dead leaf sheath of Deschampsia cespitosa (Germany); Pararoussoella juglandicola on twig of Juglans regia (Germany); Pezicula eucalyptigena on leaves of Eucalyptus sp. (South Africa); Phlogicylindrium dunnii on leaves of Eucalyptus dunnii (Australia); Phyllosticta hagahagaensis on leaf litter of Carissa bispinosa (South Africa); Phyllosticta austroafricana on leaf spots of unidentified deciduous tree host (South Africa); Pseudosigmoidea alnicola on Alnus glutinosa leaf litter (Germany); Pseudoteratosphaeria africana on leaf spot on unidentified host (Angola); Porodiplodia vitis on canes of Vitis vinifera (USA); Sodiomyces alkalinus from soil (Mongolia), Sodiomyces magadiensis and Sodiomyces tronii from soil (Kenya), Sympodiella quercina on fallen leaf of Quercus robur (Germany) and Zasmidium hakeicola on leaves of Hakea corymbosa (Australia). Epitypes are designated for: Cryptostictis falcata on leaves of E. alligatrix (Australia), Hendersonia phormii on leaves of Phormium tenax (New Zealand), Sympodiella acicola on needles of Pinus sylvestris (Netherlands), and Sphaeria scirpicola var. typharum on leaf of Typha sp. (Germany). Several taxa originally described from rocks are validated in this study. New taxa include: Extremaceae fam. nov., and new genera, Arthrocatena, Catenulomyces, Constantinomyces, Extremus, Hyphoconis, Incertomyces, Lapidomyces, Lithophila, Monticola, Meristemomyces, Oleoguttula, Perusta, Petrophila, Ramimonilia, Saxophila and Vermiconidia. New species include: Arthrocatena tenebrosa, Catenulomyces convolutus, Constantinomyces virgultus, C. macerans, C. minimus, C. nebulosus, C. virgultus, Exophiala bonariae, Extremus adstrictus, E. antarcticus, Hyphoconis sterilis, Incertomyces perditus, Knufia karalitana, K. marmoricola, K. mediterranea, Lapidomyces hispanicus, Lithophila guttulata, Monticola elongata, Meristemomyces frigidus, M. arctostaphyli, Neodevriesia bulbillosa, N. modesta, N. sardiniae, N. simplex, Oleoguttula mirabilis, Paradevriesia compacta, Perusta inaequalis, Petrophila incerta, Rachicladosporium alpinum, R. inconspicuum, R. mcmurdoi, R. monterosanum, R. paucitum, Ramimonilia apicalis, Saxophila tyrrhenica, Vermiconidia antarctica, V. calcicola, V. foris, and V. flagrans.

First Report of Soft Rot of Potatoes Caused by Dickeya dadantii in Zimbabwe.

A survey was carried out in the potato- (Solanum tuberosum L.) growing regions of Zimbabwe in April 2009 to assess the prevalence of bacterial soft rot. A total of 125 tubers with soft rot symptoms were collected. The disease caused severe economic losses ranging from 20 to 60% on tubers in the field and in storage. Affected tubers had symptoms that ranged from light vascular discoloration to complete seed piece decay. Infected tuber tissue was often cream colored and soft to the touch. In the field, plants showed severe wilting, often accompanied by a slimy, brown necrosis of the lower stems. Seventy-five of 125 isolations from diseased tubers yielded pectolytic bacteria on crystal violet pectate (CVP) medium and colonies were characterized after purification on King's B medium. All 75 isolates were gram-negative rods, oxidase negative, facultatively anaerobic, able to degrade pectate, and rot potato slices. They grew at 37°C, were sensitive to erythromycin, positive for phosphatase, indole production, cis-aconitate, lactose, d-arabinose, meso-tartrate, casein, d-melibiose, myo-inositol, and malonate utilization, while negative for acid production from trehalose, inuline, and α-methyl glucose. Dickeya dadantii (Erwinia chrysanthemi 3937 from the Scottish Research Institute) was included in all biochemical and pathogenicity tests. These characteristics are typical for two species, D. zeae and D. dadantii (2). Thus, the 75 isolates were further identified by PCR amplification with BOX and REP primers (3) and five isolates by gyrB sequence analysis (1). These analyses give support for the isolates being D. dadantii. Partial gyrB sequence analysis showed that the analyzed isolates had 96% sequence identity with the D. dadantii type strain Ech 586T (GenBank Accession No. CP001836.1). One-microliter suspensions (108 CFU per ml) of 20 samples were injected into the stolon end of potato tubers (S. tuberosum L.) cv. BP1. Each isolate was inoculated into three tubers, which were maintained at 25°C. Three control tubers were inoculated with sterile distilled water. Soft rot symptoms identical to those observed in the field and in storage appeared on all inoculated tubers 1 to 2 days after inoculation but not on the control tubers. A bacterium with identical characteristics to those described above was consistently reisolated from the rotted tissue of inoculated tubers. To our knowledge, this is the first report of soft rot on potato in Zimbabwe caused by D. dadantii, formerly referred to as E. chrysanthemi. This finding has implications for import and export of potato material into and out of Zimbabwe. Zimbabwe imports seed from various countries because of the current seed shortage and exports table potatoes to other African states. References: (1) C. Brady et al. Int. J. Syst. Evol. Micobiol. 59:2339, 2009. (2) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (3) J. Versalovic et al. 1991. Nucleic Acids Res. 19:6823, 1991.

Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay.

AIMS: This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay. METHODS AND RESULTS: Several molecular techniques including 16S rRNA and rpoB gene sequencing and DNA-DNA hybridization were used to characterize the gram-negative, facultatively anaerobic, slime-producing bacterial strains isolated along with Pantoea species from Eucalyptus. Hypersensitivity reactions (HR) and pathogenicity tests were performed on tobacco and Eucalyptus seedlings, respectively. The isolates clustered closely with the type strain of Enterobacter cowanii in both phylogenetic trees constructed. The DNA-DNA similarity between the isolates and the type strain of Ent. cowanii ranged from 88% to 92%. A positive HR was observed on the tobacco seedlings, but no disease symptoms were visible on the inoculated Eucalyptus seedlings. CONCLUSIONS: Enterobacter cowanii was isolated from trees with symptoms of bacterial blight although strains of this bacterial species do not appear to be the causal agent of the disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first report of Ent. cowanii isolated from Eucalyptus. Its presence in Eucalyptus tissue suggests that it is an endophyte in trees showing symptoms of blight.

Isolation and Identification of the Causal Agent of Brown Stalk Rot, A New Disease of Maize in South Africa.

During 2004 to 2005, an unreported disease of maize (Zea mays) was observed on commercial fields in the Northwest and Mpumalanga Provinces of South Africa. Infected plants were stunted, with a vertical crack at the first internode. Inside the stem, a dark-brown, narrow lesion was present along the crack. Internal browning inside the stem extended upward, reaching the top internode in some plants. Seed cobs were underdeveloped. Diseased plants were scattered in the fields and 10 to 70% of the crop was affected. Gram-negative, facultatively anaerobic bacteria were consistently isolated from diseased tissues. Pathogenicity tests established that representative strains induced disease symptoms similar to those observed on maize plants in the field. Physiological and biochemical characterization using the API 20E and API 50CHE systems and 16S rRNA gene sequence analyses showed that the strains belonged to the genus Pantoea. The results of these tests also separated the strains into two groups. The first group, giving a positive reaction in the indole test, was similar to Pantoea ananatis. The second group of strains was indole negative and resembled P. agglomerans. The fluorescent amplified fragment length polymorphism (F-AFLP) genomic fingerprints generated by the indole-positive strains and P. ananatis reference strains were similar and clustered together in the dendrogram, confirming that the indole-positive bacteria causing brown stalk rot on maize were P. ananatis. The F-AFLP fingerprints produced by the indole-negative strains were distinctly different from those generated by P. ananatis, P. agglomerans, P. dispersa, P. citrea, P. stewartii subsp. stewartii, and P. stewartii subsp. indologenes. The results indicated that indole-negative bacteria causing brown stalk rot on maize might belong to a previously undescribed species of the genus Pantoea. This is the first report of a new disease on maize, brown stalk rot, caused by two bacterial species, P. ananatis and an undescribed Pantoea sp.

DNA sequence and RFLP data reflect geographical spread and relationships of Amylostereum areolatum and its insect vectors.

The white rot fungus, Amylostereum areolatum (Basidiomycetes), is best known for its symbiotic relationship with various siricid wood wasp species. In this study, the relationship between isolates of A. areolatum associated with two wood wasp species, Sirex noctilio and S. juvencus, are considered to identify possible intraspecific groups. Isolates from the northern (native) and southern (exotic) hemispheres are included to determine patterns of geographical spread and origin of introductions into the southern hemisphere. The phylogenetic relationships of these isolates to authentic isolates of A. chailletii, A. laevigatum and A. ferreum were also investigated. Sequence and restriction fragment length polymorphism (RFLP) analyses of the variable nuc-IGS-rDNA region provided markers to distinguish intraspecific groups within A. areolatum. Isolates of A. areolatum associated with S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These sequences occurred in one of five combinations in each isolate. Some of these sequences were unique to isolates of A. areolatum from either wasp species, while others were present in both groups. This shows the ancient and specialized evolutionary relationship that exists between these insects and fungi. Isolates from the southern hemisphere all share the same sequence group. This supports previous hypotheses that S. noctilio has spread between countries and continents of this region. At the interspecific level, the IGS-rDNA sequence analysis showed that A. ferreum and A. laevigatum are closely related to each other, and they in turn are related to A. chailletii. Amylostereum areolatum was the most distinctly defined species in the genus. This can be attributed to the obligate relationship between A. areolatum and its insect vectors. Polymerase chain reaction-RFLP analysis was also shown to be an effective tool to distinguish between the different species of Amylostereum.

Bacterial Blight and Dieback of Eucalyptus Species, Hybrids, and Clones in South Africa.

During 1998, a new disease appeared on trees representing a Eucalyptus grandis × E. nitens (GN) hybrid in a nursery in KwaZulu/Natal. The disease has subsequently spread to other Eucalyptus species, hybrids, and clones in nurseries and plantations throughout South Africa. Typical symptoms of the disease include dieback of young shoots and leaf blight. This ultimately leads to stunting of trees. The objective of this study was to isolate and identify the causal agent of the disease. A bacterium was consistently isolated from infected tissue. Pathogenicity tests were undertaken with a range of bacterial strains. Four pathogenic strains were selected from different geographical regions and Eucalyptus hosts for further study. The bacterium causing Eucalyptus leaf and shoot blight is gram negative and rod-shaped, varying in size from 0.5 to 0.75 µm wide and 1.0 to 2.0 µm long. Colonies of this bacterium have a yellow pigment. The results from the Biolog tests identified the bacterium as Pantoea agglomerans with a similarity index of 0.315. The 16S rDNA sequences of the purported Pantoea sp. were compared with those of other related Enterobacteriaceae from GenBank/EMBL. Phylogenetic analysis using PAUP revealed that the isolates group together with P. agglomerans, P. ananatis, and P. stewartii subsp. stewartii. The fatty acid profiles and phenotypic characteristics of the new pathogen are similar to P. ananatis, and % G + C is within the range of this species. DNA:DNA hybridization between the four strains and the type strain of P. ananatis conclusively showed that the bacterium causing blight and dieback of Eucalyptus in South Africa belongs to this species. This is the first report in which P. ananatis has been found as a causal agent of a disease on Eucalyptus.

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

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

Gibberella fujikuroi mating population E is associated with maize and teosinte.

Summary Isolates of Fusarium subglutinans mating population E are usually found on maize. This fungus forms part of the so-called Gibberella fujikuroi species complex. Previously, F. subglutinans has been associated with two additional mating populations (B and H) and a variety of plant hosts. This was mainly due to a lack of diagnostic morphological characters, but the use of DNA sequence information showed that the strains making up mating populations B, E and H, as well as those associated with the different plant hosts, represent separate species. Recently, another putative mating population has been reported on the wild teosinte relatives of maize. Based on sexual compatibility studies, these isolates were apparently closely related to the pitch canker fungus, F. subglutinans f. sp. pini (= F. circinatum;G. fujikuroi mating population H). The aim of the current study was to determine whether the population of F. subglutinans from teosinte constitutes a new or an existing lineage within the G. fujikuroi complex. For this purpose, portions of the mitochondrial small subunit ribosomal DNA, calmodulin and beta-tubulin genes from the fungi were sequenced. Phylogenetic analyses and comparison with sequences from public domain databases indicated that the F. subglutinans isolates from teosinte are most closely related to strains of G. fujikuroi mating population E. These results were confirmed using sexual compatibility studies. The putative mating population from the wild relatives of maize therefore forms part of the existing E-mating population and does not constitute a new lineage in the G. fujikuroi species complex.

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an approximately 800-bp fragment from MAT-2 or an approximately 200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.

Genotypic Diversity of Sphaeropsis sapinea from South Africa and Northern Sumatra.

Sphaeropsis sapinea is the most important pathogen of Pinus spp. in South Africa. The fungus, which reproduces only asexually, occurs on exotic Pinus spp. In this study, the diversity of the S. sapinea population in South Africa was compared with a population from Northern Sumatra. The populations for both countries were obtained from exotic Pinus patula plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percent maximum genotypic diversity, based on Stoddart and Taylor's index, for the South African population was 30.5% compared with 1.5% for the Northern Sumatran population. Based on the number of phenotypes, the South African S. sapinea population was significantly more diverse (P = 0.05) than that of the Northern Sumatran population. The results indicate that the population of S. sapinea in South Africa has, in all likelihood, arisen as a result of introductions of the fungus on pine seeds imported from various parts of the world during the last century.

Differentiation of Fusarium subglutinans f. sp. pini by histone gene sequence data.

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.

Eucalyptus Rust: A Disease with the Potential for Serious International Implications.

Eucalyptus spp. are propagated extensively as non-natives in plantations in many parts of the tropics and sub-tropics. A number of diseases result in serious losses to this economically important forest resource. Eucalyptus rust, caused by Puccinia psidii, is one such example. The economic losses due to this disease are the result of infections of seedlings, young trees, and coppice. P. psidii occurs predominately in Central and South America, but reports of a similar rust are known from other areas. Eucalyptus rust is a remarkable disease in that the pathogen is not known on eucalypts in their centers of origin. It has apparently originated on native Myrtaceae in South America and is highly infective on some Eucalyptus spp. planted there. P. psidii causes one of the most serious forestry diseases in Brazil and is considered to be the most serious threat to eucalypt plantations worldwide. Advances in eucalyptus rust research are reviewed here, with a focus on topics such as distribution, host range, pathogen specialization, symptomatology, etiology, epidemiology, and control.

A serious canker disease of Eucalyptus in South Africa caused by a new species of Coniothyrium.

Eucalyptus spp. are being propagated extensively as exotics in plantations in South Africa, and many other parts of the world. In South Africa, a number of diseases result in serious losses to this resource. This paper describes a new and very damaging stem canker disease, which has recently appeared on plantation-grown eucalyptus in South Africa. The disease, first noted in an isolated location in Zululand is now common in other parts of the country, and is typified by discrete necrotic lesions on stems. These lesions coalesce to form large, gum-impregnated cankers and malformed stems. The causal agent of the disease, as inferred from pathogenicity tests, is a new species of Coniothyrium described here as C. zuluense. This fungus is a serious impediment to eucalypt propagation in South Africa, and is most likely a threat to similar forest industries elsewhere in the world.