Calculating minimum sample sizes for taxonomic measurements : examples using Gäumann’s Peronospora Spore Data

Methods are presented for calculating minimum sample sizes necessary to obtain precise estimates of fungal spore dimensions. Using previously published spore-length data sets for Peronospora species, we demonstrate that 41—71 spores need to be measured to estimate the mean length with a reasonable level of statistical precision and resolution. This is further progressed with examples for calculating the minimum number of spore lengths to measure when matching an undetermined specimen to a known species. Although applied only to spore-length data, all described methods can be applied to any morphometric data that satisfy certain statistical assumptions.

Environmental influences on akinete germination and development in Nodularia spumigena (Cyanobacteriaceae), isolated from the Gippsland Lakes, Victoria, Australia

This study was carried out to investigate the genesis of N. spumigena blooms by specifically studying the effects of environmental variables (salinity, nitrogen, phosphorus and light) on the germination of N. spumigena akinetes. Optimal conditions for maximum germination and germling growth were determined by exposing akinetes to a range of salinities and nutrient (nitrogen and phosphorus) concentrations under two different irradiances. At pre-determined time periods, treatments were sampled and the percent germination and length of germlings assessed. The results indicated that akinete germination and germling growth were optimal at salinities from 5 to 25 and significantly reduced outside this range. A positive correlation in germination was observed with increasing nutrient (phosphorus and nitrate) concentration. Similarly, germling growth increased with increasing concentrations of both nutrients. Irradiance significantly influenced both germination and growth during salinity experiments, whereas in nutrient addition experiments, irradiance had no effect on germination; however, growth was significantly influenced during phosphorus addition experiments. Consequently, salinity and light appeared to be most critical in the germination process for N. spumigena akinetes, with phosphorus most important for germling growth. The study showed that N. spumigena may be able to germinate under environmental conditions outside its optimal range, but the growth of the germling is significantly reduced, which in turn suggests that its ability to form a bloom outside its optimal environmental conditions would also be greatly reduced.

Germination ecology and seed dispersal of a critically endangered plant: a case study of Pomaderris vacciniifolia (Round-leaf Pomaderris)

Change in ecosystem disturbance regimes from human land-use poses a worldwide problem for management of rare species. Two important types of disturbance influencing the persistence of species in Australian ecosystems are habitat fragmentation and fire. In this study, seed dispersal and the germination ecology of Pomaderris vacciniifolia-a critically endangered, rare endemic Australian shrub-were examined to identify likely influences of fire and fragmentation on the decline of populations. The response of seed germination to simulated effects of wildfire and canopy openings was investigated, as was the unaided dispersal capability of seeds from parent plants. A significant increase in germination rate was observed following 100°C heat treatment to seeds, while smoke and light exposure had little influence. Seed imbibition was strongly influenced by heat treatment. The findings indicate a likely positive post-fire germination response, with implications for recruitment success determined by moisture availability following fire. Unaided seed dispersal was limited, which partly explains the apparent decline of populations. Understanding disturbance requirements for threatened species, and subsequent management of landscapes for disturbance, will aid conservation of rare species throughout the world.

Reduced triacylglycerol mobilization during seed germination and early seedling growth in Arabidopsis containing nutritionally important polyunsaturated fatty acids

There are now several examples of plant species engineered to synthesize and accumulate nutritionally important polyunsaturated fatty acids in their seed triacylglycerols (TAG). The utilization of TAG in germinating seeds of such transgenic plants was unknown. In this study, we examined the TAG utilization efficiency during seed germination in transgenic Arabidopsis seeds containing several examples of these fatty acids. Seed TAG species with native fatty acids had higher utilization rate than the TAG species containing transgenically produced polyunsaturated fatty acids. Conversely, quantification of the fatty acid components remaining in the total TAG after early stages of seed germination revealed that the undigested TAGs tended to contain elevated levels of the engineered polyunsaturated fatty acids (PUFA). LC-MS analysis further revealed asymmetrical mobilization rates for the individual TAG species. TAGs which contained multiple PUFA fatty acids were mobilized slower than the species containing single PUFA. The mobilized engineered fatty acids were used in de novo membrane lipid synthesis during seedling development.

Zostera nigricaulis seed germination experiment

Data from seed germination experiment to determine the impact of temperature, salinity, sediment type and sediment depth on seed germination.

Reworked Devonian (Givetian-Frasnian) spores from a Permian glacially-derived sequence at Bacchus Marsh, Victoria

Emphanisporites rotatus McGregor emend. McGregor 1973 is a distinctive Devonian spore with a known primary source age range in Australia spanning the upper Givetian to early Frasnian (Middle to Late Devonian). This is the first record of the species from Victoria. It occurs as a reworked element in an Early Permian assemblage belonging to the Granularisporites confluens Zone derived from glacigene diamictite in the Bacchus Marsh area. As the predominant direction of Permian ice movement recorded in the Bacchus Marsh district was south-west to north-east, it is possible that the reworked spores were transported from Antarctica.

Small-scale temporal variation in propagule supply of an intertidal red alga

Information on the variability in supply of algal propagules is scarce, hindered by the difficulty in identifying propagules, but this variability may affect the distribution and abundance of algal assemblages. This study examined the small-scale (½ hourly to hourly) temporal variation in propagule supply of Chondrus verrucosus (Gigartinaceae, Rhodophyta) over a dense, isolated bed in south-eastern Japan in summer and winter of 1999. Either 0.5 litre scoop samples or 5 litre pump samples were collected ½ hourly to hourly over 13, 22.5, and 30 h on three occasions in summer (June & July) and 32 h on one occasion in winter (December). Sampling was conducted around either the new moon (two occasions in summer) or full moon (one occasion in both summer and winter) and incorporated full tidal sequences including daytime (summer) and nighttime (winter) low-low (LL) tides. Chondrus verrucosus was the only red alga with spores within the size range of 15–20 μm that was fertile in the study area and surrounds at the time of sampling facilitating identification of spores. Spores in scoop samples were settled onto Petri dishes and identified on the basis of cell shape, colour and size. Pump samples were filtered onto transparent membrane filters and identified using epifluorescence microscopy: C. verrucosus spores fluoresced bright yellow and were easily distinguished from other micro-organisms of similar size, which fluoresced red or green. Results showed that while propagules could be found in the water column at most times, propagule supply of C. verrucosus was greatest during the 1–2 h period following LL tides. Variability in propagule supply was less than in previous studies examining surface or offshore waters. Spore release is thought to be stimulated by either desiccation or salinity changes associated with periods of emersion at low tide followed by re-immersion on incoming tides.

Contribution of fungi to primary biogenic aerosols in the atmosphere: wet and dry discharged spores, carbohydrates, and inorganic ions

Biogenic aerosols play important roles in atmospheric chemistry physics, the biosphere, climate, and public health. Here, we show that fungi which actively discharge their spores with liquids into the air, in particular actively wet spore discharging Ascomycota (AAM) and actively wet spore discharging Basidiomycota (ABM), are a major source of primary biogenic aerosol particles and components. We present the first estimates for the global average emission rates of fungal spores.

Measurement results and budget calculations based on investigations in Amazonia (Balbina, Brazil, July 2001) indicate that the spores of AAM and ABM may account for a large proportion of coarse particulate matter in tropical rainforest regions during the wet season (0.7–2.3 μg m⁻³). For the particle diameter range of 1–10 μm, the estimated proportions are ~25% during day-time, ~45% at night, and ~35% on average. For the sugar alcohol mannitol, the budget calculations indicate that it is suitable for use as a molecular tracer for actively wet discharged basidiospores (ABS). ABM emissions seem to account for most of the atmospheric abundance of mannitol (10–68 ng m⁻³), and can explain the observed diurnal cycle (higher abundance at night). ABM emissions of hexose carbohydrates might also account for a significant proportion of glucose and fructose in air particulate matter (7–49 ng m⁻³), but the literature-derived ratios are not consistent with the observed diurnal cycle (lower abundance at night). AAM emissions appear to account for a large proportion of potassium in air particulate matter over tropical rainforest regions during the wet season (17–43 ng m⁻³), and they can also explain the observed diurnal cycle (higher abundance at night). The results of our investigations and budget calculations for tropical rainforest aerosols are consistent with measurements performed at other locations.

Based on the average abundance of mannitol reported for extratropical continental boundary layer air (~25 ng m⁻³), we have also calculated a value of ~17 Tg yr⁻¹ as a first estimate for the global average emission rate of ABS over land surfaces, which is consistent with the typically observed concentrations of ABS (~10³–10⁴ m⁻³; ~0.1^–1 μg m⁻³). The global average atmospheric abundance and emission rate of total fungal spores, including wet and dry discharged species, are estimated to be higher by a factor of about three, i.e. 1 μg m⁻³ and ~50 Tg yr⁻¹. Comparisons with estimated rates of emission and formation of other major types of organic aerosol (~47 Tg yr⁻¹ of anthropogenic primary organic aerosol; 12–70 Tg yr⁻¹ of secondary organic aerosol) indicate that emissions from fungi should be taken into account as a significant global source of organic aerosol. The effects of fungal spores and related chemical components might be particularly important in tropical regions, where both physicochemical processes in the atmosphere and biological activity at the Earth's surface are particularly intense, and where the abundance of fungal spores and related chemical compounds are typically higher than in extratropical regions.

Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.

Investigation of compounds causing water repellency in the rhizosphere of sandy soils from a wide range of locations.

Although soils are generally considered to wet readily, some are actually water repellent at the surface and in the rhizosphere. This phenomenon occurs at low to moderate moisture contents and has been reported from soils under a range of vegetation types and from many regions around the globe. Water repellency in soils can have serious environmental implications including reduced seed germination and plant growth as well as irrigation efficiency, accelerated soil erosion, and enhanced leaching of agrochemicals through preferential flow. it has been proposed that water repellency is caused by the accumulation of hydrophobic organic compounds released as root exudates, microbial byproducts or from decomposing organic matter, which are deposited on mineral or aggregate surfaces, or are present as interstitial matter, Few studies to date have attempted to isolate and characterize these compounds and their structure is therefore only poorly understood, These studies have generally focussed on only a single soil or a small range of samples, have not included non-repellent soils as a control and have not always been able to demonstrate that the substances isolated are indeed responsible for repellency formation.

This study reports on the first part (extraction procedures) of a research programme addressing these gaps in current knowledge by investigating a wide range of severely repellent and wettable ‘control’ samples from different countries, and by including assessments of extraction efficiency and ability of extracts to cause repellency. Analytical methods include DRIFT (Diffuse Reflectance Infrared Fourier Transform Spectroscopy) of soils and IR (Infrared) analysis of extracts.

Key findings are that (i) soil sample heating after extraction is valuable in assessing the effectiveness of the extraction procedure, (ii) Soxhlet extraction using isopropanol/ ammonia (70/30 v/v) was the most effective method in extracting hydrophobic compounds, while leaving the ability of extracted compounds to induce water repellency virtually unaffected, (iii) wettable control soils also contain hydrophobic substances capable of inducing water repellency, (iv) the amount of organic compounds extracted was poorly related to sample repellency, indicating that compounds responsible for repellency may only represent a small fraction of the extract, (v) differences in extraction efficiency between different samples indicate that the compounds responsible may differ generically and/or in terms of their bonding to minerals, and (vi) the combination of repellency assessments with DRIFT on soils and JR on extracts used with internal standards has considerable potential to allow quantification of CH bearing organic matter in the soil, the efficiency of extraction processes for its removal, and its significance in causing water repellency in soils.

Variation in gametophyte dominance in populations of Chondrus verrucosus (Gigartinaceae, Rhodophyta)

We describe the abundance, including spatial and temporal variability, of phases of the isomorphic Chondrus verrucosus Mikami from Japan. Chondrus verrucosus occurred in a dense (∼90% cover) and temporally stable bed on a small, isolated rocky outcrop (Oyakoiwa) in Shizuoka Prefecture. Small vegetative fronds were always much more abundant than large vegetative and fertile fronds over the spring to late summer periods in 1999 and 2000. Over the same period, fertile carposporophytic fronds were generally more abundant than fertile tetrasporophytic fronds, and fertile male fronds appeared infrequently at low densities. Using the resorcinol-acetal test, we determined the proportion of gametophytes and tetrasporophytes in three populations of C. verrucosus: Oyakoiwa and Noroshi (Shizuoka) in the summers of 1999 and 2000 and Kamehana Point (Miyagi) in autumn 2000. All populations had a significantly higher proportion of gametophytes than tetrasporophytes in both years, although gametophytic proportions were lower at Noroshi (∼70%) than at Oyakoiwa (∼80%) and Kamehana Point (∼97%). However, examination of all isolated individuals sampled on Noroshi showed equal proportions of each phase in 1999, but gametophyte dominance (74%) in 2000. Differences in dispersal and spore production between phases are discussed as mechanisms potentially contributing to variation in gametophyte dominance.

Development and use of a model system to monitor clubroot disease progression with an Australian field collection of Plasmodiophora brassicae

A modified sand–liquid culture method facilitated easy visualisation of the primary life cycle stages of Plasmodiophora brassicae within clean root hairs of the Arabidopsis host. Pathogen penetration occurred from day 4 onwards and then primary plasmodia developed within the host root. Several Arabidopsis ecotypes tested in varying growth conditions showed differences in disease expression. Defined growth cabinet conditions were found most suitable for studying disease progression in the ecotypes and for achieving uniform infection and disease development. Arabidopsis ecotypes Ta-0 and Tsu-0 known to be partially resistant to a German single-spore isolate of P. brassicae were susceptible to an Australian (Victorian) field population of P. brassicae. The European clubroot differential test was used to confirm virulence and describe the pathotype of the Victorian field population. Knowledge of the interaction of an Australian population of P. brassicae with its host will provide valuable information on a disease which is very difficult to control.

Heterotrimeric G proteins facilitate arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gbg dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Ga or Gb subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gb-deficient mutants while Ga-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gb-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gb-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Ga-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by Ga. We hypothesize that Gbg acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Heterotrimeric G protein γ subunits provide functional selectivity in Gβγ dimer signaling in arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

Ecology of Pittosporum undulatum Vent. (Pittosporaceae) an environmental weed in south east Australia

Pittosporum undulatum Vent. (Sweet Pittosporum) is a densely foliaged tall shrub or small tree, native to the wet forests of south east Australia, This species now functions as a serious environmental weed in a range of habitats in Australia and on other continents and islands throughout the temperate, sub-tropical and tropical zones. This study investigated some of the ecological causes and consequences of P. undulatum invasion across a range of habitat types in south east Australia. Key aspects of P. undulatum biology and ecology investigated in the current study include; patterns of morphological variation across the range of habitats occupied (as a measure of the species’ plasticity), dispersal ecology and seed germinability, population structure and spatial pattern, community relationships and the ecological impacts of invasion. Phenotypic plasticity is considerable in P. undulatum. No clear patterns of geographic variation emerged from a study of leaf morphological attributes across the current range of this species on mainland south east Australia. The pattern of morphological variation is particularly complex in Victoria, where the invasion of this species is most advanced. The species’ adaptability to a range of environments and environmental conditions will likely promote further range expansion. The abundant winter fruit crop produced by functionally female P. undulatum plants attracts a suite of generalist opportunistic frugivores, which feed on P. undulatum fruits and seeds at various stages of fruit dehiscence, thereby enhancing dispersal opportunities for this species. P. undulatum seed collected from natural and invasive populations, at two stages of fruit maturity and from the scats and pellets of dispersal agents, displayed high germinability. European Blackbirds and Pied Currawongs are implicated as the main avian dispersal agents of P undulatum in south east Australia. The broader ecological implications of developing relationships between invasive fleshy-fruited bird-dispersed plant species and adaptive frugivores are likely to be considerable. The distribution of P. undulatutn seedlings was significantly negatively correlated with adult conspecifics and significantly positively correlated with trees and shrubs of other genera. This pattern reflects the importance of both firugivorous dispersal agents and the species’ germination and establishment requirements, in shaping the contagious distribution pattern typical of this species. These analyses suggest that recruitment opportunities for conspecific seedlings are limited beneath the canopy of adult conspecifics. Densities of P. undulatum were on average, 2.7 times higher in invaded populations, compared to the natural populations sampled. A male-bias was evident in all populations and no relationships between reproductive activity and the density of seedlings and juveniles were evident. Invading populations of P. undulatum impose substantial changes on ecosystem-level properties and functions. Mean species richness and cover-abundance declined notably once P. undulatum cover-abundance exceeded 20% at the invaded sites and 60% at the natural sites sampled. The natural communities sampled displayed comparatively greater resilience to the competitive effects of P. undulatum, but community attributes were affected at high densities and cover-abundance of this species. The cover-abundance of herbs and grasses declined most substantially with increasing P. undulatum at invaded sites, whereas, at the natural sites sampled, the species’ structural analogues appeared to be most affected by increasing P. undulatum cover-abundance. This study has demonstrated that the ecological consequences of P. undulatum population expansion are substantial and contribute to changes in the composition and successional trajectory of affected communities. These processes ultimately lead to the loss and simplification of biodiversity values and the homogenisation of affected habitats. P. undulatum has the potential to emerge as one of south east Australia's most serious environmental weed species.