A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000: 1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Secretion of a constitutive endopectinase was detected in cultures of Venturia inaequalis grown in complex and defined media. Pectinylase or pectinesterase activity was not present. The attainable level of extracellular pectinase was present after 5–10 d post inoculation, when only about 5–10% of mycelial mass had developed. Time course culture experiments with conidial and mycelial inoculum indicated that enzyme secretion did not depend on physiological effects within or shortly after conidial germination. Cellulosic substrates did not affect pectinase production. Catabolite repression or repression by glucose was not detected and there was no stimulation of enzyme production in media supplemented with pectic substrates. Inhibition of enzyme activity by galacturonic acid was not detected. Nineteen isolates of V. inaequalis were tested, all of which displayed constitutive pectinase production. All isolates were able to grow in the defined media with galacturonic acid or the pectic substrates as the sole carbon source. Mycelial pectinolytic activity was only detectable in substantial quantities when the fungus was grown in media supplemented with 1% pectin. The pectinase preparations displayed a long-term stability at temperatures up to 20°C and within a pH-range of 5–7·5. Activity was very low at 5 and 10° and there was a distinct increase at 15 and 20°. Enzyme activity was highest at pH 4 and decreased with increasing pH. Time course viscometric assays and hplc analyses of liberated galacturonic acid indicated an endo-type degradation of pectin and polygalacturonic acid. Di- and trigalacturonic acid were released from pectin with high enzyme concentration and long incubation. Pectin with a degree of esterification of 93% showed reduced degradability. Pectinase preparations had a macerating activity on solvent extracted apple leaves and caused galacturonic acid release. Isoelectric focusing followed by a zymogram technique revealed a single activity band at pI 10. The pectinase preparations from culture filtrates or mycelia of 19 isolates did not show any differences in characteristics of degradative activity or the activity band detected after isoelectric focusing.

The susceptibility of mustard beetle, Phaedon cochleariae, larvae to the insect pathogenic fungus Metarhizium anisopliae, was influenced by the topography of the host plant, the dosage and the larval instar. At high doses (>109 conidia ml−1) more insects were likely to acquire conidia. Most of the inoculum adhered to the mid-ventral region of the abdomen. Inoculum applied to the leaf became diluted during leaf expansion and this decreased beetle mortality. Leaf expansion was slower at 10°C than at alternating temperatures of 23/10° or when constant at 23°. Slightly more larvae acquired conidia when fed on oilseed rape than Chinese cabbage or turnip. Most conidia adhered to the legs and the mid-ventral region of the abdomen. Mortality was usually less on oilseed rape than Chinese cabbage and turnip. These observations suggest that fungistatic compounds of oilseed rape were interfering with the infection process.

The presence of the xylariaceous genus Entonaema in Thailand is confirmed. One collection from Chiang Mai Province, E. siamensis, is described as new.

Asterina hopiicola, Asterinella pterigotae, Echidnodella memecyli and Lembosia humboltiae are described and illustrated as new species.

Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf24. Sense (Inf24-S), 5′-ATG AGT GCT TCA TCG CAG CAG CAG-3′ fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense (Inf24-RC), 5′-CTG CTG CTG CGA TGA AGC ACT CAT-3′ blocked gene transcription and appressoria were rarely formed. Injection of Inf24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.

A region of about 800 bp of ribosomal DNA has been sequenced in 10 isolates of Polymyxa and one isolate each of Plasmodiophora brassicae and the chytrid Olpidium brassicae. This region consists of about 330 bp at the 3′ end of the 18S gene, the 5·8S DNA and the two internal transcribed spacers. A shorter region was also sequenced from one isolate each of Spongospora subterranea and a Ligniera sp. The results supported the earlier division of P. graminis into three subgroups and the recognition of P. betae as a distinct species. In phylogenetic analyses, the Ligniera and Spongospora isolates grouped with the other plasmodiophorids and the Olpidium isolate with the Mycota. The plasmodiophorids appeared as a distinct group and were not closely related to any other eukaryotes (including a range of fungi and protozoa). The DNA sequences were used to design primers for specific amplification of either Polymyxa or P. graminis DNA.

Intraspecific variation of 25 Metarhizium anisopliae var. anisopliae isolates from various insect hosts and geographical origins was studied using RFLP analysis of the rDNA gene complex and the mtDNA. Heterologous and homologous mitochondrial and ribosomal probes were used to produce RFLP profiles of the isolates. The rDNA analysis allowed the differentiation of only seven isolates but provided evidence for the possible presence of introns in different sites of the nuclear rRNA-gene-complex. PCR amplification of the ITS1-5.8S-ITS2 rDNA region and the mtDNA regions corresponding to the SrRNA and the LrRNA, followed by restriction analysis of the PCR products underlined the conserved nature of these regions. By contrast, digestion with any one of endonucleases Hae III, Cfo I, Hpa II and subsequent RFLP analysis of the mtDNA, allowed the classification of the 25 isolates in 10 distinct groups. Double digestions using the restriction endonucleases Kpn I and Sac I led to maximum isolate differentiation, producing unique patterns for 16 isolates and subdividing the remainder into four groups of two or three isolates. This indicates the potential of mtDNA RFLP analysis as an excellent tool for fingerprinting M. anisopliae isolates and for studying genetic polymorphism.

We conducted a series of experiments to determine if extraradical hyphae of VAM fungi acclimate to low temperatures. Blocks of field soil containing VAM fungi were either slowly cooled or held at room temperature prior to freezing. Infectivity of VAM fungi was greater in soil that was slowly cooled. We hypothesized that greater infectivity following freezing resulted from cold acclimation of extraradical hyphae. This hypothesis was tested using in vitro mycorrhizas cultured in two-compartment Petri plates, in which hyphae grew into a separate compartment. Metabolic activity of these hyphae following freezing was assessed using a vital stain. The majority of cultures that were slowly cooled prior to freezing contained active hyphae, whereas hyphal activity was almost completely eliminated by freezing in non-precooled cultures. Freezing temperature influenced survival and activity of hyphae. To our knowledge this is the first report of acclimation to cold temperatures by VAM fungi.

The following foliicolous fungi on Tabebuia species are described and illustrated: Anhelia tabebuiae sp. nov. and Dictyonella tabebuiae sp. nov. (ascomycetes), Fumagospora tabebuiae sp. nov., Polychaeton tabebuiae sp. nov. and Septoria tabebuiae-impetiginosae sp. nov. (coelomycetes), Cercospora tabebuiae-impetiginosae sp. nov. and Pseudocercospora tabebuiae-roseo-albae sp. nov. (hyphomycetes). Uncinula peruviana was found and described on T. impetiginosa as a first record for Distrito Federal, Brazil.

The characterization of type cultures of the S. thermophilum complex remains unclear and morphological features have been relied upon to recognize such isolates. The aim of this study was to compare morphological and thermal methods, in order to identify relationships between cell wall and cultural characteristics. The differences in the proportions of structural and non-structural fractions and the peak decomposition temperatures of the test isolates were significant. The thermograms produced can be grouped into two distinct types, which can be further sub-divided, in agreement with microscopic classification. The relationship between the various morphological parameters including extension rate, sporulation, microscopic type and DTG parameters are discussed briefly.

RFLP, DNA fingerprinting and VCG methods were used to characterize fourteen Verticillium dahliae isolates collected from southwestern Ontario, Canada. The isolates were typed as not pathogenic to tomato (NP), race 1 (avirulent on cvs carrying the Ve resistance gene) or race 2 (virulent on Ve cvs). On the basis of RFLPs, RAPDs, and DNA fingerprints detected by hybridization to a dispersed, repetitive genomic DNA probe, the isolates were classified into five DNA types. Type I included two NP isolates. Type II included four race 2, and three NP isolates. Types III and V were represented by single race 2 and race 1 isolates, respectively. Type IV included one race 2, and two race 1 isolates. Vegetative compatibility was determined for selected NP, race 1, and race 2 isolates of each race type/DNA type combination. Isolates of the same DNA type were compatible, as were type II and III isolates (VCG 4B), and type IV (VCG 2A) and V isolates (VCG 2B). These data show a level of genetic diversity not previously identified in the V. dahliae tomato pathogen population, and suggest multiple origins for the Ontario race 2 pathotype.

The thiophene 5-(4-hydroxy-1-butinyl)2,2′-bithienyl (BBTOH) strongly inhibited in vitro eight different dermatophytes. Epidermophyton floccosum proved most sensitive to all doses of BBTOH when applied in conjunction with uv-A irradiation. BBTOH also proved quite active against Nannizia cajetani, the only dermatophyte which was also strongly inhibited when treated (50 μg ml−1) and kept in the dark. For this reason, N. cajetani was chosen as the test organism for TEM and SEM aimed at determining what treatment-induced ultrastructural and morphological modifications had occurred. TEM revealed that the photoactive mechanism of BBTOH was similar to that of 2,2′[ratio ]5′,2″-terthienyl (α-T). SEM, on the other hand, showed that early culture aging resulted from treatment.

Random Amplified Microsatellite (RAMS) markers were used to detect variation among Phlebiopsis gigantea isolates from seven European countries. The scoring of 26 bands revealed five markers common to all isolates and 21 variable markers, of which six occurred in only one strain. A considerable amount of variation was observed among the progeny isolates of single strains. The analysis of variable markers resulted in 75 different banding patterns among the 86 isolates studied. The high degree of polymorphism and heterozygosity observed in RAMS markers suggests that the amount of genetic variation within this fungus is considerable. The equal distribution of markers in strains from different locations and the lack of country-specific markers suggests that genetic differentiation among the populations is low. The lack of distinct groups of similar banding patterns supports the idea of P. gigantea being a true biological species consisting of a single intersterility group.

A fluorometric-based method for the kinetic measurement of conidia growth of Botrytis cinerea in liquid culture is described. The method involves an oxido-reduction dye, Alamar blue, as an indicator of growth. The effect of nystatin, a non-specific antifungal agent, was studied on conidial germination. An excellent correlation was found between the IC50 obtained by the fluorometric method, and by microscopic measurement. The method is rapid and quantitative, and requires only a small amount of chemicals and fungal material. Since the measurements were performed in a 96-well plate, it is possible to test many different parameters in parallel.

The utilization profile of 49 carbohydrates, based on API 50 CH biochemical tests, was used for the identification and the discrimination of 75 isolates of Beauveria and Tolypocladium. The data were used to construct a most parsimonious tree in which Beauveria and Tolypocladium were separated into two main branches. Although the Beauveria isolates could not be separated at the species level, eight different clusters corresponding to the eight Tolypocladium species studied were revealed. The reproducible carbohydrate utilization profiles of B. bassiana, B. brongniartii, T. cylindrosporum, T. niveum, T. geodes and T. tundrense have permitted the construction of an identification key that could serve as a tool to complement the morphological description of selected Beauveria and Tolypocladium isolates for their correct identification at the species level. The API 50 CH method is time-saving and can complement effectively more classical techniques for the discrimination between Beauveria and Tolypocladium and for the taxonomy of Tolypocladium at the species level.

Aquaphila albicans gen. and sp. nov. from submerged wood in the tropics is described and illustrated. It produces hyaline, multiseptate, fusoid or falcate conidia resembling the macroconidia of Fusarium species, but differs by its conidiogenesis and the aquatic habitat. The conidiophores of A. albicans are septate, hyaline, sympodially proliferating with conidiogenous denticles. Conidial development in A. albicans has been studied in culture and results are presented. The genus is compared with similar genera.

Pestalotiopsis microspora is an endophyte associated with many plants including Taxus wallachiana. Isolates commonly produce taxol in liquid culture. Defining culture amendments to optimize taxol production by P. microspora is a critical step toward the realization of fungal taxol for treating human cancers. The lowering of phosphate and the addition of sodium benzoate in the medium increased taxol production. Sterol biosynthesis inhibitors, such as tebuconazole and triadimefon, dramatically increased taxol yields.

Endogone maritima sp. nov. is described and illustrated. This fungus was recovered from around the roots of endomycorrhizal Ammophila arenaria, Corynephorus canescens, Juncus balticus, J. articulatus, and ectomycorrhizal Pinus sylvestris colonizing a deflation hollow of the Słowiński National Park in Poland. Endogone maritima produces sunflower to brownish yellow zygosporangia enveloped by a separable hyphal mantle consisting of interwoven hyphae appearing in section as a dense net. The zygosporangia occur both singly in the soil and in small, non-peridial sporocarps typically containing 2–7 zygosporangia.

The occurrence of an undescribed ergot species, Claviceps citrina, on the halophytic chloridoid grass Distichlis spicata, in the Texcoco region of central Mexico, is reported. RAPD and ITS1 sequences of this fungus were compared with other Claviceps species and the morphological uniqueness of the fungus was reinforced. Its sclerotia do not contain any alkaloids of the ergoline type. Germination of sclerotia occurred immediately after placing them on humid sand. After removal of the capitula, the remaining stipe was able to regenerate the capitulum.