P.A. O'Brien

Analysis of variation in isolates of Rhizoctonia solani by random amplified polymorphic DNA assay

Genetic variation in Australian isolates of Rhizoctonia solani was analyzed by random amplified polymorphic DNA (RAPD) assay. Isolates originated from different geographic locations in Australia and belong to a number of different anastomosis and pectic zymogram groups. Using four different oligonucleotide primers fingerprint patterns were generated for each isolate. All of the anastomosis and pectic zymogram groups (including subgroups) tested could be distinguished. For some groups there was considerable variation in the fingerprint patterns between isolates. This variation was more marked between isolates from different geographic locations. Other groups showed very little variation between isolates. RAPD-PCR analysis is a very useful alternative to anastomosis grouping for identification of isolates of R. solani.

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

Abstract. 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.

Reliability of RAPD fingerprinting of three basidiomycete fungi, Laccaria, Hydnangium and Rhizoctonia

Randomly amplified polymorphic DNA (RAPD) profiles are currently being developed for Laccaria and Hydnangium species and Rhizoctonia solani. The technique is increasingly being used to differentiate fungal isolates. As for the polymerase chain reaction (PCR) from which it was derived, the conditions necessary for reproducible RAPD products have received attention. However, in contrast to the PCR reaction, the technique relies on non-specific primers and as a consequence the reaction conditions are not necessarily as specific as they are in PCR. Compared with PCR products, RAPD fingerprints were therefore more sensitive to reaction and thermocycle conditions. RAPD products produced using 10 and 17–23 mer primers were visualized on ethidium bromide stained polyacrylamide electrophoresis gels. Factorial experiments showed RAPD patterns were altered by changes in various reaction mixture components including the concentration of non-DNA impurities, the number and concentration of primers and the DNA polymerase enzyme type, source and concentration. These factors, particularly the enzyme source, reacted differently with the magnesium chloride concentration. Fluorescent dye labelled primers were used with internal lane standards in a DNA sequencing system to assess accurately the molecular weights and relative amounts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on patterns visualized on ethidium bromide stained non-denaturing polyacrylamide gels. The temperature at which DNA was denatured in the first cycles altered the fingerprints. Reaction mixture temperatures of 94° or higher, compared with 91–93°, caused loss of visual yield of some products, particularly those greater than 500 base pairs, and increased the yield of others. Reproducibility of RAPD patterns, when the reaction mixture and temperature profile factors were varied, was facilitated by cross reference to fluorescently labelled bands separated with internal lane standards in a DNA sequencing system. Reproducibility of fingerprint patterns in a standard reaction mixture was achieved, with different thermocycle programmes and in different thermocyclers when the temperature profile was reproduced, suggesting that particular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used.

Nitrogen dynamics in an Australian semiarid grassland soil

We conducted a four-week laboratory incubation of soil from a Themeda triandra Forsskal grassland to clarify mechanisms of nitrogen (N) cycling processes in relation to carbon (C) and N availability in a hot, semiarid environment. Variation in soil C and N availability was achieved by collecting soil from either under tussocks or the bare soil between tussocks, and by amending soil with Themeda litter. We measured N cycling by monitoring: dissolved organic nitrogen (DON), ammonium (NH4+), and nitrate (NO3-) contents, gross rates of N mineralization and microbial re-mineralization, NH4+ and NO3- immobilization, and autotrophic and heterotrophic nitrification. We monitored C availability by measuring cumulative soil respiration and dissolved organic C (DOC). Litter-amended soil had cumulative respiration that was eightfold greater than non-amended soil (2000 compared with 250 microg C/g soil) and almost twice the DOC content (54 compared with 28 microg C/g soil). However, litter-amended soils had only half as much DON accumulation as non-amended soils (9 compared with 17 microg N/g soil) and lower gross N rates (1-4 compared with 13-26 microg N x [g soil](-1) x d(-1)) and NO3- accumulation (0.5 compared with 22 microg N/g soil). Unamended soil from under tussocks had almost twice the soil respiration as soil from between tussocks (300 compared with 175 microg C/g soil), and greater DOC content (33 compared with 24 microg C/g soil). However, unamended soil from under tussocks had lower gross N rates (3-20 compared with 17-31 microg N x [g soil](-1) d(-1)) and NO3- accumulation (18 compared with 25 microg N/g soil) relative to soil from between tussocks. We conclude that N cycling in this grassland is mediated by both C and N limitations that arise from the patchiness of tussocks and seasonal variability in Themeda litterfall. Heterotrophic nitrification rate explained >50% of total nitrification, but this percentage was not affected by proximity to tussocks or litter amendment. A conceptual model that considers DON as central to N cycling processes provided a useful initial framework to explain results of our study. However, to fully explain N cycling in this semiarid grassland soil, the production of NO3- from organic N sources must be included in this model.

Interspecific and intraspecific variation of ectomycorrhizal fungi associated with Eucalyptus ecosystems as revealed by ribosomal DNA PCR–RFLP

Gondwanan vegetation, and the Australian region in particular, is species rich for ectomycorrhizal fungi in epigeous and hypogeous forms with over 100 species recorded in small (1 ha) patches of forests. Distinguishing co-occurring ectomycorrhizal fungi as root associations in native (natural or wildlands) vegetation or plantations and discriminating them from other larger basidiomycetes, e.g. wood and leaf litter decomposer fungi, places large demands on molecular identification, especially if interspecific similarities and intraspecific variation occur in target sequences. One hundred and nine species of larger basidiomycetes from a single forest location were characterised by PCR-RFLP profiles of two genomic regions (nuclear rDNA ITS and mtLSU). Over one-third of the species for which multiple isolates were tested showed intraspecific variation in either one or both genomic regions. This remarkably high variation questions previous assumptions about intraspecific ITS sequence variation and highlights the value of integrated molecular and morphological databases including voucher specimens. It also emphasises the value of molecular investigations that use more than one genomic region. Interspecific similarities were common among the Cortinariaceae, especially in the ITS region. Discrimination of most Cortinariaceae species was achieved using variation in the mtLSU region in conjunction with the ITS. This new information raises the possibilities that the ITS sequence is more conserved and the mtLSU more variable than among species of the other 23 families. In the other families, interspecific ITS variation was greater and the mtLSU profiles grouped species within families. The high variation in the two genomic regions indicated possible differences in the fungal population structure between two adjacent, differently managed blocks of Eucalyptus marginata forest. The significance of this variation to ecology, biodiversity assessment and ecosystem management are discussed.

Stimulation of synthesis and release of swainosonine from transformed roots of Swainsona galegifolia.

Transformed root cultures of Swainsona galegifolia were established for swainsonine production. Stimulation of production of swainsonine and its release into the culture medium was achieved using copper sulphate, reduction of medium pH and supply of swainsonine precursors. The yield of control cultures (0.3 g of roots grown in 15 ml medium for 30 days) was 79 micrograms swainsonine with only a trace of swainsonine in the medium. After treatment with 1 mM copper sulphate for two days before harvest on day 30, 155 micrograms of swainsonine was produced, of which 14.3 micrograms was in the medium. Reduction of medium pH from 5.7 to 2.7 for one day before harvest resulted in 159 micrograms of swainsonine (47 micrograms in the medium). Supplementation with 2 mM malonic acid for 12 days before harvest resulted in 187 micrograms total swainsonine (34 micrograms in the medium), while 2 mM pipecolic acid for six days before harvest gave the highest swainsonine yield (220 micrograms total, 43 micrograms in the medium). The increased yields were achieved through small increases in biomass, as well as increases in the level of swainsonine synthesis.

Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations

Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR-RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR-RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR-RFLP tool for identifying basidiomycetes associated with plants from field samples.

Heterokaryon formation with homokaryons derived from protoplasts ofRhizoctonia solani anastomosis group eight

Homokaryons were successfully recovered by regenerating protoplasts prepared from vegetative hyphae of field isolates ofRhizoctonia solani anastomosis group (AG) 8, the causal pathogen of bare-patch disease of cereals. A mating type incompatibility system, which is similar to that reported in AG 1 and AG 4, was demonstrated in AG 8. All homokaryons obtained in AG 8 were able to form tufts with their parent isolates and other heterokaryotic field isolates of AG 8 tested. Heterokaryons were readily recovered from tufts of pairing of certain homokaryon combinations. The synthesized heterokaryons formed tufts with both of the contributing homokaryons. The majority of hyphal tip cultures isolated from tufts resembled one of the contributing homokaryons. These nonheterokaryotic hyphae in tufts are attributed to transient heterokaryon effects.

Molecular markers in Australian isolates of Rhizoctonia solani

Isolates of Rhizctonia solani from different locations within Australia and Japan were analysed for restriction fragment length polymorphisms (RFLPs). These isolates belong to different anastomosis and pectic zymogram groups. Southern blots of restriction enzyme digested DNA were probed with a cloned 18S ribosomal RNA (rRNA) gene, or random cloned fragments of R. solani DNA. The patterns obtained with the rRNA probe revealed significant variation within some of the groups whereas other groups were more uniform. Group specific patterns could not be identified for all groups. There was less variation in the RFLP patterns when random cloned fragments of DNA were used as probes and group specific patterns could be identified for all groups.