Three Clonal Lineages of Phytophthora cinnamomi in Australia Revealed by Microsatellites.

ABSTRACT The genetic structure of populations of Phytophthora cinnamomi, a pathogen of an enormous variety of woody plants, was investigated using microsatellites. Three intensively sampled disease sites in southwest Australia were analyzed along with a large culture collection of Austra-lian isolates and some isolates from elsewhere in the world. The mutation in the four microsatellite loci analyzed revealed spatial patterns at the disease sites that correlated with the age of the infestation. Only three clonal lineages were identified in Australian populations and these same clonal lineages were present in worldwide populations, where it is suggested that a limited number of clonal lineages have spread in most regions. No evidence for sexual reproduction between these clonal lineages in Australia has been found even though the pathogen has the opportunity. Instead, mitotic recombination is frequent within the clonal lineages. The implications of this are discussed.

Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests?

A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiss and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata.

Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers.

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.

Nature's ground force.

Some of the world's oldest and richest commercial biotechnologies are based on fungi, for example the production of bread, wine, beer and medicines. But they have a far greater, yet largely unrecognised, influence on human existence--they are crucial to some of the processes which ensure that major ecosystems world-wide keep ticking over.

Microsatellites in the mitochondrial genome of phytophthora cinnamomi failed to provide highly polymorphic markers for population genetics.

Microsatellites were evaluated as genetic markers for the mitochondrial genome (mtDNA) of Phytophthora cinnamomi for population studies. Two (A)n microsatellite loci were cloned from the mtDNA of P. cinnamomi. Amplification products from these loci showed little polymorphism among Phytophthora isolates due to their location in coding regions of mtDNA. A further three (A)n microsatellite loci obtained from the complete mtDNA sequence of P. infestans were also not highly polymorphic, although located in non-coding mtDNA. The presence of the (A)n microsatellites was not conserved in the genus Phytophthora. Unlike those of the chloroplast genome of plants, (A)n microsatellite loci of mtDNA do not have potential as highly polymorphic markers in Phytophthora.

Airstream fractionation of vesicular-arbuscular mycorrhizal fungi: concentration and enumeration of propagules.

Spores and fragments of vesicular-arbuscular mycorrhizal fungi in dry soils were concentrated up to 100-fold when the soils were partitioned by fluidization and elutriation with a series of upward airstreams at progressively increasing velocities. The propagules were transported with the finer soil particles according to their equivalent spherical diameters. The system was used to predict the transport of propagules by wind. Concentrated propagules were rapidly separated from the soil particles in each soil fraction by an aqueous flotation method. The technique is proposed as a quantitative method for estimating the numbers of spores and fragments of mycorrhizae. The scheme includes a viability test that was used to differentiate between potentially infective propagules and those that were either dormant or incapable of regrowth.

Production of aseptic spores of vesicular-arbuscular endophytes and their viability after chemical and physical stress.

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30 degrees C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30 degrees C for Glomus caledonius or at 35 degrees C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 mug of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 mug/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40 degrees C for 80 min, 55 degrees C for 10 min, and 60 degrees C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 x 10 ergs cm from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.

Preservation of spores of vesicular-arbuscular endophytes by L-drying.

The spores of four species of vesicular-arbuscular endophytes were L-dried at 22 degrees C, and their viability was tested after heating at 80 degrees C for up to 40 min. L-drying of spores in the soil in which they developed was a very effective method of preservation of all spore types examined. Slow L-drying of spores separated from soil and supported on glass fiber filters also gave high viability for spores of some species. A scheme for the long-term preservation of vesicular-arbuscular endophyte spores is proposed.