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This issue of Mycological Research News features the evolutionary origins of ryegrass endophytes, and implications of the cyclosporin experience for bioprospecting.
This month's Mycological Research includes 17 papers. The first demonstrates the successful and stable transformation of the genome in Fusarium circinatum using the Agrobacterium tumifaciens Ti plasmid. Molecular studies include ones on six species of Agaricus to elucidate their breeding systems, further research on the ascomycete order Agyriales, Alternaria alternata isolates associated with pistachio late blight, 16 species of Claviceps, the Entomophthora muscae complex, Hebeloma velutipes, Mycocalicium subtile, Ophiostoma piceae and O. quercus, genera in the Thelebolaceae, isolates identified as Trichoderma aureoviride, Tuber magnatum, and a Venturia associated with aspen blight.
Alternaria alternata, A. gaisen and A. longipes are shown to be distinct species, and the infection process of A. cirsinoxia on Canada thistle is documented. A xylanase gene of Cochliobolus sativus (anamorph Bipolaris sorokiniana) has been characterized, as has an hydrophobin in Metarhizium anisopliae.
The following new scientific names are introduced: Ainoa, and Antarctomyces gens. nov.; Antarctomyces psychrotrophicus sp. nov.; and Ainoa geochroa (syn. Biatora geochroa), and A. mooreana (syn. Lecidea mooreana) combs. nov.
The development of a transformation system is a significant technical hurdle in the study of many filamentous fungi. Recently
Agrobacterium tumefaciens was shown to transform several filamentous fungi, but until now only one independent confirmation of this
promising finding has been published. Fusarium circinatum (teleomorph Gibberella circinata) is an important pathogen of pine that has
not been transformed previously. A. tumefaciens strain AGL1(pPK2) transformed three different isolates of F. circinatum with an
efficiency of 2–150 transformants 10−5
conidia. The T-DNA was integrated into the genome and was stable through mitotic and
meiotic cell divisions. Most F. circinatum transformants contained a single T-DNA copy. Half of the tested transformants also
contained non-T-DNA vector sequences. These findings should facilitate future analysis of F. circinatum pathogenicity and stimulate
wider use of this valuable transformation method in fungal research.
The phylogeny of the Agyriales was investigated using nucleotide sequences of ITS1, 5.8S, and ITS2 rDNA. Sequences of these
regions from 15 new sequences of 13 agyrialean fungi were aligned to those of four representatives of Ostropales used as outgroup.
Six different alignments were analysed cladistically using maximum parsimony. The Agyriales were supported as a monophyletic
group in the strict consensus tree and the synonymy of the Rimulariaceae and Saccomorphaceae was confirmed. The Schaereriaceae is
also shown to belong to the Agyriaceae. The distribution of selected characters in the Agyriales is investigated and it is shown that
the ascus type is variable within the group, while the ascoma ontogeny is invariant within the Agyriaceae. Trapelia as usually
circumscribed is polyphyletic. The T. geochroa group is not closely related to Trapelia s. str. and the new genus Ainoa is described to
accommodate T. geochroa and T. mooreana.
Phylogenetic trees of 16 Claviceps species were constructed based on alignments of 5.8S rDNA and the adjacent ITS1 and ITS2
spacers. Two highly supported clades were found: (1) C. paspali, C. zizaniae, C. grohii, C. sulcata, C. fusiformis, and C. purpurea; and
(2) C. citrina, C. phalaridis, two unidentified Claviceps spp. (isolates PM and SG), C. sorghicola, C. gigantea, C. sorghi, C. africana, C.
viridis, and C. pusilla. No relationship was found between the species placement and its morphological markers. The probe from C.
purpurea gene cpd1 for dimethylallyl tryptophan synthase, the first enzyme of alkaloid biosynthesis, was hybridized to Pst I digested
genomes of the above species under non-stringent conditions. Hybridizing DNA was present in all species of clade 1, although the
signal of the C. paspali gene was weaker. In clade 2, only C. africana, C. gigantea, and C. pusilla gave weak positive signals.
Colorimetric detection found small amounts of alkaloids in cultures of Claviceps sp. SG and PM but despite that, no cpd1 hybridizing
bands were found. The occurrence of two major clades of Claviceps and their biogeography suggests, that the genus originates from
South America and that the evolution of its species was influenced by comigration with their hosts and with the global climatic
changes that influenced spreading of grass subfamilies.
The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was
found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in
monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci
in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There
was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced
intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic
analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group
together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17
showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of
frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in
allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential
problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are
possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.
Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to
predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to
inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were
grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were
extracted and analysed by HPLC–DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin I, which
has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component
analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the
diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the
39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The
results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically,
physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is
unwarranted and pathotypes should not be used.
Genetic variation in the pistachio late blight fungus, Alternaria alternata, was investigated by restriction fragment length
polymorphism (RFLP) in the rDNA region. Southern hybridization of EcoRI, HindIII, and XbaI digested fungal DNA with a RNA
probe derived from Alt1, an rDNA clone isolated from a genomic library of the Japanese pear pathotype of A. alternata, revealed
34 different rDNA haplotypes among 56 isolates collected from four central valley locations in California. Analysis of molecular
variation revealed a significant amount of genetic diversity within populations (85.8%), with only marginal variation accounting for
differentiation among populations (14.2%, ϕST =0.142). All isolates examined were highly pathogenic. The identity of the four
geographic populations sampled was not evident in both cluster and principal component analyses, probably indicating either the
selectively neutral nature of rDNA variation or prevalence of widespread gene flow among populations combined with uniform
The ITS II and the first part of the LSU rDNA were amplified from 26 isolates within the genus Entomophthora. The specificity of
the primers allowed the use of both in vivo and in vitro material. Size polymorphism and long amplification of the ITS II regions,
ranging from 1200 to 2000 bp, were observed. The PCR-products were cut with eight different restriction endonucleases and
analysed by UPGMA, one analysis from each of the two regions. Conidial morphology was of predictive value for the overall
taxonomy of the genus Entomophthora, as the genus clustered together in the analysis of the LSU rDNA. In both analyses the
E. muscae complex clustered into three different clades, which support the validity of E. schizophorae and E. syrphi as separate species.
Considerable variation was detected in the E. muscae clade, but it could not be grouped by host, geographic origin or conidial
morphology, though the E. muscae s. str. isolates in both analyses grouped together. One isolate with E. muscae-like conidia found on
Hymenoptera clustered out within the E. muscae clade, widening the host range for E. muscae significantly.
The identity of strains identified as Trichoderma aureoviride/Hypocrea aureoviridis was reconsidered. Trichoderma aureoviride was isolated
originally from a specimen identified as H. aureoviridis and thus is H. aureoviridis. The morphological and molecular characters of
most strains identified as T. aureoviride differ from those of the ex-type but are more typical of T. harzianum, a member of sect.
Pachybasium. Molecular data do not support inclusion of T. aureoviride in sect. Trichoderma, nor was there strong phenotypic
similarity between H. aureoviridis and H. rufa. In the ITS phylogeny the T. aureoviride ex-type and other collections of H. aureoviridis
form a strongly supported clade that is separate from any other recognized section of Trichoderma. Hypocrea vinosa, which was
originally included in the T. aureoviride aggregate species concept, is distinct from T. aureoviride, but closely allied with H. rufa/T.
viride. Trichoderma aureoviride/H. aureoviridis is a rare species, restricted to the UK and the Netherlands. We redefine T. aureoviride,
limiting it to strains with very slow growth rate, effuse conidiation, and the ITS-1 and 2 sequence type D.
The aim of the present paper is to investigate if Mycocalicium subtile as presently circumscribed is a morphologically highly variable
species, or in fact represents two or more cryptic species, and further to investigate the status of M. minutellum. The morphological
variation of 19 specimens of Mycocalicium subtile from five continents was investigated and the ITS1-5.8S-ITS2 region of their rDNA
sequenced. Sequences from closely related taxa, 4 specimens of M. albonigrum, 2 of Mycocalicium sp., and Chaenothecopsis nana were
also included. For comparison the corresponding sequence of M. victoriae and C. pusilla were also determined. We used the sequence
of Monascus purpureus as the outgroup for the analysis. The sequences were used for phylogenetic inference using parsimony and
distance methods. All the specimens assigned to M. subtile with the exception of two, form a well-supported monophyletic group.
Those two specimens represent a morphologically cryptic, but genetically distinctive taxon. The infraspecific clades of M. subtile in
the molecular phylogeny did not correspond to geographical origin. Only a weak correlation with geographical origin was found
in the morphological analysis. The molecular analysis supports the suggestion that Mycocalicium minutellum is a taxonomic
synonym of M. subtile.
To determine the feasibility of using internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA sequences as genetic
markers in molecular systematic and phylogenetic studies of Ophiostoma, the presence of multiple types of ITS in the two sibling
sapstain fungi, Ophiostoma piceae and O. quercus were examined in 22 isolates from diverse geographic regions. PCR and sequence
analysis revealed that two different versions of ITS sequences were present in the PCR-products amplified with the ITS primers,
ITS1-ITS4 or ITS5-ITS4 or ITS1-F-ITS4, in every isolate of the two Ophiostoma species tested. Surprisingly, the two ITS sequences
were shared by both species. One ITS sequence which was major in O. piceae was designated as the OPC type, and the other ITS
sequence which was minor in O. piceae was designated as the OPH type. In O. quercus the OPH type was major and the OPC type
was minor. The major ITS comprised more than 99% of the amplicons in both species. The results suggest that common ITS gene
pools have been maintained at different levels within these sibling species.
Morphological characters and genetic markers from 35 single-ascospore isolates of a Venturia sp. collected from Populus tremula were
analysed. The morphological data were compared with the literature, and genetic markers were amplified for comparison with
isolates of V. populina, V. macularis and V. ditricha. According to ascospore morphology the fungus resembled V. populina, but the
morphology of the conidia was closer to Pollaccia radiosa (the anamorph of V. macularis). Analysis of 49 RAMS markers, variation
within the internal transcribed spacer (ITS) sequence of the ribosomal gene cluster and a single marker locus derived from RAMS
fingerprint suggested that the Venturia is a previously undescribed species.
The infection process of Alternaria cirsinoxia was studied on Canada thistle (Cirsium arvense) in the controlled environment and in the
field. In the controlled environment, germination of conidia began at 2 h and appressoria formation at 4 h after inoculation.
Approximately 75% of appressoria formed at the anticlinal wall junctions of the epidermis. Leaf penetration occurred between 6 and
24 h, most commonly in between adjoining anticlinal walls. Penetration through stomata was rare. After penetration, large,
intracellular infection hyphae formed and branched within epidermal cells, ramifying throughout the leaf tissues inter- and
intracellularly by 24 h. In field conditions, infection coincided with prolonged rainfall and conidia remained viable on the leaf surface
for 8–9 days (d) before causing infection on leaves. A host response occurred after penetration, involving deposition of lignin and
callose in the infected epidermal and mesophyll cell walls. High levels of silicon were detected in epidermal cells directly below
appressoria, often appearing to form entirely silicified infected cells which were resistant to collapse after air-drying. This study
shows that A. cirsinoxia has the potential for rapid invasion of the leaf tissues of Canada thistle under good moisture conditions. The
implications of the host responses, in terms of defence against infection, are discussed.
A xylanase gene, XYL2, was identified and characterised in Cochliobolus sativus (anamorph Bipolaris sorokiniana), a necrotrophic cereal
pathogen that attacks both shoots and roots. The fungus was grown on a xylanase inducing medium containing mineral salts, oat
spelt xylan, cellulose, and peptone, RNA was isolated, and a complementary DNA (cDNA) library constructed. The library was
screened with a xylanase (XYL1) cDNA clone from the maize pathogen Cochliobolus carbonum. Xylanase cDNA clones, all
representing a single gene, were identified. Corresponding genomic DNA was amplified by PCR. Sequencing of the cDNA and the
PCR products gave a nucleotide sequence of 2211 bp containing two introns in an open reading frame of 693 bp that codes for a
xylanase from glycosyl hydrolase family 11. The most similar sequences to this gene in nucleotide sequence databases are the XYL2
gene of C. carbonum and a xylanase (XYL1) cDNA from a saprophytic fungus, Humicola insolens. Northern blot analysis and reverse
transcription PCR (RT-PCR) showed expression of the gene when the fungus was grown on xylan or cellulose, but not when
peptone or sucrose was the only carbon source. Expression of XYL2 in inoculated barley seedlings was detected by RT-PCR.
A 909 bp region containing a genomic clone encoding for hydrophobin (ssgA) from the entomopathogenic fungus Metarhizium
anisopliae has been sequenced and the regulatory motifs analysed against those recognized in other fungi. The genomic clone was
also compared with the open reading frame of the hydrophobin ssgA (starvation stress gene) cDNA sequence. The genomic clone
contained a 291 bp coding sequence with one intron of 64 nucleotides. From this sequence primers were established that could be
used to amplify the hydrophobin. Restriction fragment polymorphism analysis of hydrophobin amplified by the polymerase chain
reaction from 80 isolates of M. anisopliae showed no variability. Analysis of the potential regulatory elements 313 bp upstream from
the transcriptional start site revealed typical TATAA and CCAAT boxes. CT or GC motifs were not found. Upstream regulatory
elements were also found with sequence homologies to the AREA, CREA, CRE (cAMP response element) and BRLA regions of
Aspergillus nidulans as well as the CYS3 and AmyB regions of Aspergillus oryzae. The promoter regions of other fungal hydrophobins
were also assessed for the presence of regulatory elements. Upstream regulatory elements are also present for the gene encoding a
cuticle-degrading protease (Pr1) from M. anisopliae. We suggest that nutrient levels and cAMP mediation of thigmotropic signals in
the entomopathogenic fungus, M. anisopliae, co-ordinate the regulation of the gene products required for morphological development
and secretion of ‘penetration’ enzymes.
Eleven enzymes from 139 specimens of thirteen Italian populations of Tuber magnatum were analysed with multilocus horizontal
starch gel electrophoresis. The research was carried out to obtain insight into the genetic variability of this species across its
geographic range. All the gene-enzyme systems scored appear to be fixed in homozygosity in accordance with what is hitherto
known in the genus Tuber; nine seemed monomorphic, whereas two, that is MDH-1 and ME-2, showed three alleles each. These
results indicated a self-reproductive system and the low genetic variability is in agreement with the restricted endemism of the white
truffle. The distribution of the electrophoretic types is discussed as a basis for further molecular applications.
The genus Agaricus encompasses the cultivated mushroom A. bisporus, and includes both unifactorial heterothallic and homothallic
species. The intractable nature of their life cycles and an absence of diagnostic morphological features, hinder analysis of breeding
systems in Agaricus and some other homobasidiomycete species. We utilised RAPD markers to assess genetic variation in single
spore progeny from different Agaricus species and to confirm heterokaryon formation in matings. Single spore progeny from
heterothallic species should be more heterogeneous than those from homothallics. Homokaryotic progeny from the secondarily
homothallic A. bisporus were less variable (12%) with fewer segregating loci than the known heterothallic species, A. bitorquis (50%)
and A. nivescens (32 %). Variation in the progeny of A. campestris (36%) was similar to that for the heterothallics. Heterothallism in
the field mushroom was confirmed by RAPD analysis of putative matings; the first time A. campestris heterokaryons have been
constructed. No variation was observed in progeny from two collections of A. subfloccosus. Homogeneity in RAPD profiles, absence
of matings and self-fertility of single spores shows that A. subfloccosus is homothallic. In progeny from A. arvensis very low variation
(5%) occurred and proportions of segregating loci were around half of those for A. bisporus. However, matings were observed and
RAPD characterised within the A. arvensis progeny. RAPD analysis of genetic variation within spore progeny should have general
utility in the characterisation of fungal breeding systems.
A new ascomycete, Antarctomyces psychrotrophicus gen. et sp. nov., characterised by naked asci, hyaline, thick-walled, ellipsoidal to
fusiform, echinulate ascospores, and blastoconidia, isolated from Antarctican soil samples, is described and illustrated. Analysis of the
nuclear rDNA ITS region sequences showed that this taxon is related to Thelebolaceae.