Mycological Research News features: Numbers of insect and plant species.

Eleven papers are included in this issue. Ectomycorrhizal forests have been discovered in Guyana, and DNA profiles from forest soils demonstrate that ectomycorrhizal fungi are adversely affected by burning. Molecular and morphological studies have led to a reappraisal of the relationships and generic concepts of smuts on certain dicotyledons, and also of ‘subcentric’ Achlya species and allied fungi, leading to the recognition of a new genus.

Five papers concern the molecular characterization and variability of plant pathogens: Alternaria species-groups associated with core rot of apples; the identification of Verticullium longisporum on oilseed rape; Colletotrichum gloeosporioides on Stylosanthes species ; the transposa and vacuma groups of Botrytis cinerea; and Pyrenophora teres. In addition, phytotoxic metabolites have been identified in Sarocladium oryzae.

A revision of Dioicomyces (Laboulbeniales) is also presented and the following new scientific names are introduced: Newbya gen.nov.; Achyla truncatiformis, Dioicomyces denticulatus, D. ladoi, and D. leptalei spp. nov.; Colletotrichum gloeosporioides f. stylosanthis f. nov.; C. gloeosporioides f. sp. stylosanthis f. sp. nov.; N. apiculata (syn. Achyla apiculata), N. braziliensis (syn. A. braziliensis), N. curvicollis (syn. A. curvicollis), N. megasperma (syn. A. megasperma), N. oblongata (syn. A. oblongata), N. oblongata var. gigantea (syn. A. oblongata var. gigantean), N. oligocantha (syn. A. oligocantha), N. pascuicola (syn. Aplanopsis pasuicola), N. polyandra (syn. Achlya polyandra), N. recurva (syn. A. recurva), and N. stellata (syn. A. stellata) combs nov.

Ecologically important ectomycorrhizal (EM) associations are poorly known from equatorial rain forests of South America. Recent field studies in the Pakaraima Mountains of western Guyana revealed previously undocumented forests dominated by EM leguminous trees, with a rich assemblage of EM mycobionts. Along transects, basidiomes from 75 species or morphospecies of putatively EM fungi were spatially associated with leguminous host trees. These fungi belonged to the basidiomycete families Boletaceae, Amanitaceae, Russulaceae, Cortinariaceae, Cantharellaceae, Clavulinaceae, and Entolomataceae, all of which are poorly documented from the lowland neotropics. Ectomycorrhizas were confirmed on D. corymbosa, D. altsonii, and D. jenmanii (Caesalpiniaceae, tribe Amherstieae), and a fourth species, Aldina insignis (Papilionaceae). The tribe Amherstieae contains most of the EM leguminous species forming monodominant forests in Guineo-Congolian Africa. Dicymbe species constituted the first record of EM Amherstieae in the New World. A variety of other co-occurring caesalpiniaceous trees failed to exhibit ectomycorrhizas. Transect surveys indicated that D. corymbosa and D. altsonii were: (1) highly clumped and dominant at specific sites; (2) occurred on soils with widely varying chemical and textural characteristics; and (3) the most important hosts for EM fungi in the local landscape. Dicymbe species have life history attributes, including the ectomycorrhizal habit, which enhance their competitive abilities irrespective of soil conditions. The spatial restriction of EM fungal basidiomes indicated that discrete groves of EM trees harbour an important component of regional macromycete diversity.

DNA was extracted from soil collected from the upper 15 cm of the profile at three sclerophyll forest sites in New South Wales three days before, and 14 d after prescribed burning events. rDNA internal transcribed spacer (ITS) regions were amplified by PCR using fungus-specific primers, and ITS products cloned. Restriction fragment length polymorphism (RFLP) analysis was conducted on the ITS clones using the endonucleases HaeIII, HinfI, MboI and AluI. Cloned ITS products from the three sites were thus separated into 120 RFLP types and the ITS product for each RFLP type was sequenced. Comparison of sequences with those available in the GenBank nucleotide database allowed putative assignment of ITS clones to the following functional groups: ectomycorrhizal (ECM), arbuscular mycorrhizal (AM), ericoid mycorrhizal (ERM), decomposer basidiomycetes (DEC), other soil fungi (OSF). While abundant ECM-like clones were amplified from pre-fire soils at the North Rocks and South Maroota sites, they were largely absent in the post-fire soils at these sites, being replaced mainly by microfungi. In contrast, no decrease in ECM-like ITS clones was observed at the Ridgecrop site following fire. The data indicate that, while the abundance of extraradical ECM fungal propagules in the upper soil profile may be reduced by prescribed burning, the effect is site-specific.

The phylogenetic relationships of several smut fungi parasitic on dicotyledons were analysed. Parsimony analysis was performed based on the sequences of the ITS regions of the rDNA genes. Three genera were considered: Microbotryum, Sphacelotheca and Ustilago. The cladogram showed a dichotomy: the species of Microbotryum and Ustilago parasitic on dicotyledons (dicot Ustilago) were found to be divided into two independent taxa among the Microbotryaceae. A divergent mechanism of evolution of the species of each of these two clades with their respective hosts could be involved in this dichotomy. According to our results, Microbotryum appears monophyletic and restricted to the anthericolous smuts on Caryophyllaceae. However, no morphological characters have yet been found to support this distinction, and we refute the denomination Bauhinus as defined by Moore to describe the group of ‘dicot Ustilago’ as it leads to controversial determinations. Sphacelotheca belongs to Microbotryales, but could not be synonymised with Microbotryum as S. polygoni-persicariae is in an independent clade. Lastly, U. duriaeana is independent of the ‘dicot Ustilago’ clade; the position of this species among Microbotryales is still uncertain.

Analysis of 18S rDNA sequence and morphological data indicate that Achlya s. lat. is polyphyletic. Aplanopsis spinosa and Achlya species with subcentric oospores and laterally applied antheridia are transferred to Newbya gen. nov. The relationship of Calyptralegnia and Aplanes androgynus to Newbya is discussed. Molecular data support morphologically based conclusions concerning the relatedness of Pythiopsis and Aplanopsis terrestris. Achlya truncatiformis nom. nov., is provided for material previously identified as Achlya recurva. The following new combinations within Newbya are made: N. pascuicola (type species), N. apiculata, N. braziliensis, N. curvicollis, N. megasperma, N. oblongata, N. oblongata var. gigantica, N. oligacantha, N. polyandra, N. recurva, N. spinosa, and N. stellata combs nov.

Alternaria core rot of red apple cultivars is a serious post-harvest disease in South Africa. Thirty isolates of Alternaria spp. previously isolated from apple, together with reference isolates of A. alternata and A. infectoria, were characterised and grouped according to their sporulation patterns and conidial morphology. Isolates were identified as belonging to A. arborescens, A. infectoria and A. tenuissima species-groups. The isolates were also analysed for production of mycotoxins and other secondary metabolites and their cultural characteristics on DRYES medium were recorded. Apple fruit were artificially inoculated with the thirty-two Alternaria isolates and the resulting lesion types were recorded. A data matrix was constructed using all these characters and subjected to cluster analysis to show the similarity between different isolates. Isolates classified as A. infectoria species-group based on sporulation patterns, cultural and biochemical data could be easily differentiated from isolates classified as A. arborescens and A. tenuissima species-groups, which clustered close together. Isolates were further subjected to DNA sequence analysis of the internal transcribed spacers (ITS) 1 and 2 of the nuclear rRNA gene. A phylogeny estimated from the ITS data set delineated two clades, one being typified by the A. infectoria species-group, and the other representing both A. tenuissima and A. arborescens species-groups. The ITS data set clearly separated isolates of A. infectoria species-group from the other species-groups, as all isolates had a distinction of 35 base pair insertions and 6 base pair deletions in the ITS regions. The results obtained in the present study showed that the major pathogens associated with core rot disease of Top Red apples in South Africa belong to the A. tenuissima species-group.

Morphological characters and genetic relationships were studied among 36 Verticillium isolates derived from different host species. Conidial length varied from 3.5 μm to 12 μm and the DNA content ranged from 0.028 pg to 0.078 pg with a bias towards large conidia and higher DNA content among the Swedish and German isolates from Brassica napus. Ability to produce nitrate nonutilising (nit) mutants was also assayed. Of all 36 isolates tested, eight V. dahliae isolates from non-Brassica hosts and two from B. napus, V. nigrescens from flax and V. albo-atrum from tomato generated nit mutants. To facilitate disease diagnosis, a species-specific PCR primer pair was developed, aiming towards a PCR-based analysis to confirm Verticillium wilt infection in Brassica plants. To identify useful DNA templates for the PCR primer design, random genomic clones from isolates VD12 and VD17 were used as probes in RFLP analysis. The RFLP results were also utilized in phenetic analysis and data from 29 isolates revealed three major groups. The first group consisted of seven non-Brassica V. dahliae isolates and the V. nigrescens isolate. The second group contained the two V. albo-atrum isolates and the V. dahliae isolate from red clover. Finally, the third group contained all the 18 isolates of Verticillium from B. napus. In the third group, two subgroups were identified, separating the three French isolates from the German and Swedish isolates. The dendrogram indicated that the isolates in the latter subgroup were closely related to each other. Based on the above mentioned criteria, we confirm that the isolates of Verticillium from B. napus in Sweden and Germany should be classified as V. longisporum.

RAPD amplifications of 119 Mexican Colletotrichum gloeosporioides isolates from wild native Stylosanthes plants, of two Australian (internal reference isolates of type A and type B) and of two African (previously demonstrated as different from the A and B types) allowed the generation of four polymorphic clusters. The RAPD cluster I contained the Australian type A and 103 Mexican isolates from other species than S. guianensis. The cluster II contained the Australian type B isolate and 14 Mexican isolates from S. guianensis. The clusters III and IV contained one African and one Mexican isolate each.

A phylogenetic study was performed using the ITS1 sequences from 32 representative Mexican isolates selected in the four RAPD clusters, from the two Australian and the two African used as references, from 13 C. gloeosporioides from various hosts and origins available in the EMBL databank and from three phylogenetically related species of Colletotrichum. Two clusters (S and VHO) were generated. Cluster S contained the 30 isolates clustered in RAPD I and II, and the type A and B isolates from Stylosanthes selected in EMBL. Cluster VHO contained the two isolates belonging to RAPD clusters III and IV as well as the remaining Colletotrichum spp. and C. gloeosporioides isolates from various hosts and origins selected in EMBL.

Severe type A lesions were observed on several Stylosanthes species, including S. guianensis, after inoculation with the 26 Mexican isolates selected in RAPD cluster I. The four Mexican isolates selected in RAPD cluster II induced extensive type B lesions only on S. guianensis genotypes. The virulence spectra of the isolates was not linked to variations in their ITS1 region sequence. A wide range of anthracnose lesions was induced by the Mexican isolates in interactions with the various Stylosanthes genotypes.

On the basis of results obtained from the molecular and pathogenic experiments in the present study, of morphological and molecular data from previous work, as well as from other authors, a taxonomic infraspecific differentiation was proposed, as C. gloeosporioides f. stylosanthis f. sp. stylosanthis and f. sp. guianensis.

This work aimed to evaluate the genetic diversity of Botrytis cinerea in Chile and to determine whether the two genetically different groups transposa and vacuma, described in France, are present in the country. Isolates collected in Chile from grapes, tomato, kiwifruit and blueberry were analysed using molecular markers. We developed a PCR test to identify the two groups of B. cinerea, transposa (with the transposable elements Boty and Flipper) and vacuma (with neither). As described in France, both kind of isolates were found to be present and sympatric in Chile. Isolates containing the transposable element Boty alone (boty isolates) were also detected. The frequencies of transposa, boty and vacuma isolates were significantly different on kiwifruit compared to grapes, tomato and blueberry. RAPD analysis revealed a high degree of genetic diversity, with no widespread clonal lineages. Dendrograms, analysis of molecular variance and Fst values revealed the existence of genetic differentiation in our sample between isolates from the different host plants. PCR-RFLP markers also showed that isolates sampled from grapes and tomato were genetically differentiated. Additional sampling is required to confirm these findings.

The genetic structure of Pyrenophora teres, the causal agent of net blotch of barley, was examined in two fields 30 km apart in the south-western Cape of South Africa. The two fields respectively represented a net- and spot-type population, the two types being distinguished on the basis of symptom expression on differentially susceptible cultivars. The number of isolates sampled from each field was 36 for the net-type population and 29 for the spot-type population. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. Of the 40 10-mer random oligonucleotide primers screened, five produced scorable, reproducible DNA bands suitable for the determination of population structure. A total of 65 loci were produced of which 54 were polymorphic. Genetic analysis of bands produced by one of the primers has revealed single locus segregation in a mating between a net- and spot-type isolate, indicating that RAPD bands can be interpreted as alleles at genetic loci. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spot-type populations. Genetic diversity among the two populations was divided into within- (variation between sampling quadrants) and among population components using Nei's GST. A coefficient of genetic differentiation (GS) of 0.0149 was obtained between quadrants within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis of the two populations together with six progeny from a mating between a net- and spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the net-type population that did not cluster with the other net-type isolates clustered directly next to the cluster containing the sexual progeny. This isolate also contained a unique spot-type DNA band. This suggested that sexual recombination may be occurring between net- and spot-type isolates under field conditions.

The secondary metabolites, helvolic acid and cerulenin were detected in liquid culture of Sarocladiumoryzae, causal agent of sheath rot of rice, and also in diseased sheath tissues. These metabolites were examined by thin-layer chromatography (TLC), column chromatography, and by high-performance liquid chromatography (HPLC). Helvolic acid and cerulenin showed the Rf value of 0.49 and 0.61, respectively using TLC. Cerulenin produced two spots in the TLC chromatogram and produced two major peaks in the HPLC chromatogram due to its instability. External application of helvolic acid and cerulenin produced lesions similar to those caused by the fungus. When infiltrated both of these metabolites caused electrolyte leakage in susceptible cultivar, IR 36 and induced lower levels of leakges in White Ponni, the moderately resistant cultivar to S. oryzae. The concomitant production of helvolic acid and cerulenin by S. oryzae in liquid culture and in planta was confirmed and the role of helvolic acid and cerulenin on the virulence of S. oryzae and sheath rot disease of rice is discussed.

The genus Dioicomyces is reviewed on the basis of the examination of types, authentic material, or personal collections. Three new species from Spanish anthicids are described: D. denticulatus, D. ladoi, and D. leptalei spp. nov. Eleven taxa (including eight species, two forms, and one variety) are synonymized and included under D. anthici, which is considered a very variable species. With additions and changes, Dioicomyces currently includes 23 species. In this synopsis no taxa at infraspecific rank are recognised. For each accepted species synonyms are listed, mostly based on the study of type material. Types for each taxon are included and lectotypes designated when necessary. This monograph also includes descriptions, photographs and/or drawings for all species studied and a key for their determination.