Changes in orchid populations and endophytic fungi with rainfall and prescribed burning in Pterostylis revoluta in Victoria, Australia.

Background and Aims: Wildfires are common in seasonally dry parts of the world with a Mediterranean climate. Prescribed burning is used to reduce fuel load and fire risk, but often without reliable information on its effects. This study investigated the effects of prescribed burns in different seasons on Pterostylis revoluta, an autumn-flowering Australian terrestrial orchid, and its orchid mycorrhizal fungi (OMFs) to find the least damaging season for a prescribed burn. Methods: Burns were conducted mid-season in spring and summer 2011 and autumn and winter 2012. Orchids were enumerated and measured during their flowering season in autumn 2011-2014 and mycorrhizal fungi were isolated before and after the burns in autumn 2011, 2012 and 2014. Micro-organisms isolated were characterized. DNA was extracted from the OMFs, and the internal transcribed spacer region was amplified by PCR. Amplicons were clustered by restriction fragment length polymorphism (RFLP), and representative amplicons were sequenced. OMF were tested for sensitivity to smoke water. Key Results: The number of plants increased up to 4-fold and 90 % of plants became vegetative during this study. Isolation of mycorrhizal fungi increased and isolation of bacteria decreased. Before the burns, the main OMF isolated was unexpectedly Tulasnella calospora (Boud.) Juel. By 2014, after the burns, the expected Ceratobasidium sp. D.P. Rogers was the only OMF isolated in most burnt quadrats, whereas T. calospora was confined to a minority of unburnt 'control' and the 'spring burn' quadrats, which were also the only ones with flowering plants. Conclusions: The decline in rainfall during 2010-2012 probably caused the switch from mainly flowering to mainly vegetative plants and the change in OMFs. Burning in spring to summer was less damaging to this orchid than burning in autumn to winter, which should be noted by authorities in fire management plans for fire-prone areas in which this orchid occurs.

Differences in carbon source utilisation by orchid mycorrhizal fungi from common and endangered species of Caladenia (Orchidaceae).

Terrestrial orchids depend on orchid mycorrhizal fungi (OMF) as symbionts for their survival, growth and nutrition. The ability of OMF from endangered orchid species to compete for available resources with OMF from common species may affect the distribution, abundance and therefore conservation status of their orchid hosts. Eight symbiotically effective OMF from endangered and more common Caladenia species were tested for their ability to utilise complex insoluble and simple soluble carbon sources produced during litter degradation by growth with different carbon sources in liquid medium to measure the degree of OMF variation with host conservation status or taxonomy. On simple carbon sources, fungal growth was assessed by biomass. On insoluble substrates, ergosterol content was assessed using ultra-performance liquid chromatography (UPLC). The OMF grew on all natural materials and complex carbon sources, but produced the greatest biomass on xylan and starch and the least on bark and chitin. On simple carbon sources, the greatest OMF biomass was measured on most hexoses and disaccharides and the least on galactose and arabinose. Only some OMF used sucrose, the most common sugar in green plants, with possible implications for symbiosis. OMF from common orchids produced more ergosterol and biomass than those from endangered orchids in the Dilatata and Reticulata groups but not in the Patersonii and Finger orchids. This suggests that differences in carbon source utilisation may contribute to differences in the distribution of some orchids, if these differences are retained on site.

Isolation and screening of natural organic matter-degrading fungi.

Drinking water quality and its treatment are negatively impacted by the presence of coloured natural organic matter (NOM) derived from the breakdown of animal and plant materials. Ligninolytic fungi (i.e., white rot fungi - WRF) secrete non-specific oxidative enzymes that can oxidise a wide range of recalcitrant organic compounds. The potential for these organisms to decolourise concentrated aquatic NOM was investigated. Twenty-one isolates from diverse fungal genera were screened using NOM plate assays. Four WRF strains: Trametes sp., Polyporus sp., Trametes versicolor ATCC 7731 and Bjerkandera adusta, which displayed good NOM decolourisation on solid medium were further investigated in shake-flask culture using concentrated NOM as the only source of nutrients. Of these, B. adusta demonstrated the greatest decolourisation (65% for 100 mg C L(-1) NOM). NOM decolourisation coincided with ligninolytic enzyme activity and decrease in average molecular weight of NOM. The expression of the oxidative enzymes (manganese peroxidase (MnP), lignin peroxidase and laccase (Lac)) varied with fungal strain. The enzyme activities of Polyporus sp. and the two Trametes strains were significantly greater than those of B. adusta, although their decolourisation was less. For the Trametes and Polyporus sp., Lac activity was greatest, whereas for B. adusta MnP activity was greatest, suggesting its predominant role in the decolourisation process. This research demonstrates the significant potential for WRF in NOM removal so long as the enzyme activity can be controlled.

Specific Polymerase Chain Reaction Primers for the Detection of Plasmodiophora brassicae in Soil and Water.

ABSTRACT The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10(-15) g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil.

Relationships among rhizobia from native Australian legumes.

Isolates from 12 legumes at three sites in Victoria showed a wide range of morphological, cultural, symbiotic, and serological properties. Isolates from Acacia longifolia var. sophorae and Kennedia prostrata were fast growing but nodulated ineffectively Macroptilium atropurpureum and all native legumes except Swainsonia lessertiifolia. Isolates from S. lessertiifolia showed anomalous properties intermediate between fast- and slow-growing rhizobia. All isolates from the other two sites were slow-growing "cowpea" rhizobia. Symbiotic effectiveness was usually poor, and there was no relationship between effectiveness and host taxonomy or serological affinities of the isolates. This is the first report of fast-growing rhizobia from temperate Australian woody legumes and the first report of the symbiotic effectiveness of native Australian legumes with indigenous rhizobia.

Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.

The incorporation of nitrogen into products of recent photosynthesis in Anabaena cylindrica Lemm.

In vivo tracer studies with 14C have been performed to help determine pathways of incorporation of newly assimilated nitrogen into N2-fixing cells of Anabaena cylindrica. After photosynthesis in Ar: O2:14CO2 for 30 min, the addition of N2 or NH+4 resulted in increased rates of 14CO2-incorporation both in the light and dark, and in increased incorporation of 14C into amino acids at the expense of sucrose and sugar phosphates. Evidence of enhanced sucrose catabolism and increased pyruvate kinase activity was obtained on adding nitrogen, and, of the 14C-labelling entering the tricarboxylic acid cycle, more appeared in citrate and 2-oxoglutarate than in malate and oxaloacetate. The kinetics of 14C-incorporation into various amino acids suggest that in the light and dark the most important route of primary ammonia assimilation involves glutamine synthetase and that glutamate, aspartate, glycine and probably alanine are formed secondarily from glutamine.