Orlando Petrini

Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus

Trichoderma aggressivum sp. nov. and T. aggressivum f. europaeum f. nov. are described. These forms cause the green mold epidemic in commercially grown Agaricus bisporus in North America and Europe, respectively. In the literature they have been reported as T. harzianum biotypes Th 4 and Th 2, respectively. They are strongly separated from their closest relative, T. harzianum, in sequences of the ITS-1 region of nuclear rDNA and an approximately 689 bp fragment of the protein coding translation elongation factor gene (EF-1α). They are distinguished from the morphologically similar T. harzianum and T. atroviride (the latter also known as biotype Th 3) most readily by rate of growth. Of these, only T. harzianum grows well and sporulates at 35 C, while T. atroviride is the slowest growing. Trichoderma aggressivum f. aggressivum and f. europaeum are effectively indistinguishable morphologically although they have subtly different growth rates at 25 C on SNA and statistically significant micromorphological differences. Based on findings of this study, descriptions of T. harzianum and T. atroviride are expanded. A key to Trichoderma species commonly found associated with commercially grown A. bisporus is provided.

Endophytic fungi associated with palms.

Endophytic fungi were isolated from three unidentified Licuala sp. palms in Brunei Darussalam and from three L. ramsayi palms in Australia. Endophytes were very common in both species, with overall colonisation rates of 81–89%. Taking into account a lower sampling frequency in Australia, endophyte diversity was similar in the two Licuala species. The endophyte assemblages examined were very diverse, consisting of 75 fertile species and 60 sterile morphospecies. The endophyte communities of both palms were composed of a single, dominant xylariaceous species, approximately ten less common but equally ubiquitous species and a large number of species occurring at very low frequencies. Differences were observed between the endophytic mycotas of different palm tissues and of tissues of different ages. The results presented suggest that most of the endophytes entered the petiole via the leaf and that transmission of palm endophytes is likely to be horizontal (via airborne propagules) rather than vertical (via seed). Seasonal differences were not observed in Brunei. Increased sampling effort could be expected to yield more endophyte taxa in both species investigated.

The Trichoderma koningii aggregate species

The morphological concept of Trichoderma koningii is found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Phylogenetic analysis utilizing partial sequences of the translation-elongation factor 1 alpha (tef1), as well as fragments of actin and calmodulin genes, indicate that phenotypic characters typical of T. koningii evolved independently in three well-separated main lineages. Combined molecular and phenotype data lead to the development of a taxonomy with the recognition of twelve taxonomic species and one variety within the three lineages. These lineages include: (1) T. koningii and T. ovalisporum and the new species T. caribbaeum var. caribbaeum, T. caribbaeum var. aequatoriale, T. dorotheae, T. dingleyae, T. intricatum, T. koningiopsis, T. petersenii and T. taiwanense; (2) the new species T. rogersonii and T. austrokoningii, and (3) the new anamorph T. stilbohypoxyli. Trichoderma koningii s. str. is an uncommon species restricted to Europe and eastern North America; T. caribbaeum var. aequatoriale, T. koningiopsis, and T. ovalisporum were isolated as endophytes of trunks of Theobroma species in tropical America, and T. ovalisporum from the woody liana Banisteropsis caapi in Ecuador; T. koningiopsis is common in tropical America but was isolated also from natural substrata in East Africa, Europe and Canada, and from ascospores in eastern North America, and as an endophyte in Theobroma species; T. stilbohypoxyli, originally described as a parasite of Stilbohypoxylon species in Puerto Rico, is found to be more common in the tropics, besides an endophytic isolate from Fagus in U.K. The additional new species are known almost exclusively from their teleomorphs. Isolates of T. ovalisporum and T. koningiopsis may have biological control potential. A morphophenetic key and a set of tools for molecular species identification were developed.

PATTERNS OF SUBSTRATE UTILIZATION BY SOME FUNGAL ENDOPHYTES FROM CONIFEROUS FOLIAGE

Fifty-six strains of endophytic fungi isolated from coniferous foliage in Europe and in Oregon, U.S.A., were tested for their ability to utilize various complex substrates including cellulose, xylan, mannan, several other hemicelluloses, pectin, and two lignin derivatives. Cultures were also assayed for lipolytic activity, laccase production, growth on gallic acid, and production of darkly pigmented reaction products on gallic acid. A majority of isolates utilized xylan and pectin, showed lipolytic activity, and grew on gallic acid. Fewer isolates (54%) utilized cellulose, and only a small proportion utilized hemicelluloses other than xylan. Among isolates from conifers with distinct needles, fungi occurring only in the petioles showed a broad range of substrate utilization capabilities, while those from the needle blades were more restricted in their abilities. Isolates from cuppressaceous foliage utilized xylan but varied greatly with respect to other capabilities. Different isolates of the same fungus often varied in their substrate utilization capacities. Substrate utilization patterns of all the common endophytes from a single host suggested that several endophytes may co-exist within a single leaf by biochemical partitioning of resources.

Trichoderma asperellum sensu lato consists of two cryptic species

Analysis of a worldwide collection of strains of Trichoderma asperellum sensu lato using multilocus genealogies of four genomic regions (tef1, rpb2, act, ITS1, 2 and 5.8s rRNA), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) of the proteome and classical mycological techniques revealed two morphologically cryptic sister species within T. asperellum, T. asperellum, T. asperelloides sp. nov. and a third closely related but morphologically distinct species. T. yunnanense. Trichoderma asperellum and T. asperelloides have wide sympatric distribution on multiple continents; T. yunnanense is represented by a single strain from China. Several strains reported in the literature or represented in GenBank as T. asperellum are re-identified as T. asperelloides. Four molecular SPD typing patterns (I–IV) were found over a large geographic range. Patterns I–III were produced only by T. asperellum and pattern IV by T. asperelloides and T. yunnanense. Pattern I was found in North America, South America, Africa and Europe and Asia (Saudi Arabia). Pattern III was found in Africa, North America, South America and Asia, not in Europe. Pattern II was found only in Cameroon (central Africa) and Peru. Pattern IV was found in all continents. All SPD II pattern strains formed a strongly supported subclade within the T. asperellum clade in the phylogenetic tree based on rpb2 and MLS (combined multilocus sequence). The diversity of DNA sequences, SPD markers and polypeptides in T. asperellum suggests that further speciation is under way within T. asperellum. MALDI-TOF MS distinguished T. yunnanense from related taxa by UPGMA clustering, but separation between T. asperellum and T. asperelloides was less clear.

Endophytic Fungi of Stylosanthes: a first report

Stylosanthes Sw. (Leguminosae, Papilionoideae, Aeschinomeneae) is a leguminous genus widely distributed in the tropical and subtropical regions of the South American continent that has become increasingly important over the last two decades as a forage plant. The two most impor? tant species are S. guianensis Sw. and S. humilis H. B. K., both used as pasture legumes in Brazil and Australia. Some cultivars of S. guianensis are tolerant to drought and heavy grazing and have been adapted to grow over a wide range of tropical regions, thus reflecting the high genetic variability of this species.

Isozyme subgroups in Trichoderma section Longibrachiatum

Seventy-eight representatives of Trichoder? ma sect. Longibrachiatum, Hypocrea jecorina, and H. schweinitzii were compared using ten enzyme systems. The results essentially supported Bissetts morpholog? ically based taxonomy of Trichoderma sect. Longibra? chiatum, in that the species T longibrachiatum, T ci- trinoviride, T parceramosum, and T pseudokoningii could be distinguished. Trichoderma reesei was closely related to H. jecorina and more distantly linked to T longibrachiatum, thus the synonymy of T reesei with T longibrachiatum was not supported. The possibility that T. reesei is a clonal derivative of H. jecorina is discussed. Strains of Trichoderma citrinoviridewere in?

Isozyme polymorphism in six endophytic Phyllosticta species

Isozyme variation of 70 endophytic Phyllosticta isolates representing four species from coniferous hosts and two species from angiospermous hosts was examined using starch gel electrophoresis of ten enzymes. All enzymes were polymorphic among species and each electromorph was restricted to a single host. Strains of Phyllosticta pseudotsugae were most diverse and separated into seven phenotypes. In Ph. cryptomeriae five phenotypes and in Ph. abietis two phenotypes were detected. The remaining species (Ph. multicorniculata, Ph. ampelicida, Ph. pyrolae) were monomorphic. Interspecific variation was always very high, with differences in at least 70% of the electromorphs. Within some species the same amount of variation was present, suggesting the possibility of splitting these taxa in subspecific entities. Cluster and principal components analysis of phenotype frequencies revealed two clearly separated groups that corresponded to species from different continents. The two species from angiospermous hosts were also distinct from the other species.

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

ABSTRACT The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory.

Colonization of beech leaves (Fagus sylvatica) by the endophyte Discula umbrinella (teleomorph: Apiognomonia errabunda)

Kinetics of the colonization process of beech leaves by Discula umbrinella have been studied. D. umbrinella can either form an appressorium at the end of the germ-tube or penetrate subcuticularly. Penetration through the host cell wall is achieved by a comparatively fine penetration hypha that pierces the cuticular membrane and the epidermal cell wall; the extracellular matrix usually present around both external fungal hyphae and appressoria is absent in subcuticular and subepidermal hyphae. Cuticular and epidermal cell-wall degradation was evident around the penetration hypha. Subcuticularly the fungus was found in the reticulate part of the cuticle, underneath the amorphous layer of the cuticular membrane. The epidermal cell wall underneath the subcuticular hyphae was at least partly digested, and the cytoplasm contained electron-dense compounds that may be of phenolic origin and are probably related to the infection process. At this stage the parenchyma cells were morphologically still intact. At later stages of infection D. umbrinella grew through the epidermal cell wall into the parenchyma and colonized mainly the intercellular spaces of the mesophyll, although intracellular growth was occasionally seen. Transmission electron microscopy studies of appressoria and subcuticular hyphae suggest that enzymic activities could be mainly responsible for fungal penetration. Enzymic digestion of cuticle and epidermis cell walls is also suggested by the presence of electron-transparent material in beech leaf sections, which results probably from the digestion of the cell wall in the vicinity of penetrating hyphae. These results indicate that endophytes penetrate into their host in ways similar to those described for biotrophic and hemibiotrophic pathogenic fungi. Under in vitro conditions, D. umbrinella is able to colonize host tissues, causing conspicuous necroses. The time between infection and onset of the first symptoms varied from 48 h to several days, depending on the infection conditions. During the latent period the fungus is mainly located at the subcuticular or subepidermal level.