Potassium Phosphonate Induces Resistance in Sweet Chestnut against Ink Disease Caused by Phytophthora Species.

Ink disease, caused by Phytophthora spp., represents a serious threat to sweet chestnuts throughout their distribution area. Among the control strategies, new perspectives have been offered by using potassium phosphonate, which indirectly controls Phytophthora diseases by acting on both host physiology and host-pathogen interactions. In this study, we tested in planta the effectiveness of trunk injection with K-phosphonate against seven different Phytophthora species associated with ink disease. For the two most aggressive species, P. cinnamomi and P. ×cambivora, the treatments were repeated at two different environmental conditions (a mean temperature of 14.5 °C vs. 25 °C) and tree phenology stages. The results obtained in this study demonstrated that K-phosphonate could contain the development of Phytophthora infection in phloem tissues. However, its effectiveness varied based on the concentration applied and the Phytophthora species tested. A concentration of 280 g/L of K-phosphonate was the most effective, and in some cases, callus formation around the necrotic lesion was detected. Overall, this study broadens the knowledge of endotherapic treatments with K-phosphonate as an effective measure for managing chestnut ink disease. Interestingly, the increase in mean temperature had a positive impact on the development of P. cinnamomi lesions on chestnut phloem tissues.

Physiological and cannabinoid responses of hemp (Cannabis sativa) to rock phosphate dust under tropical conditions.

Growing a high-value crop such as industrial hemp (Cannabis sativa L.) in post-mining environments is economically and environmentally attractive but faces a range of biotic and abiotic challenges. An opportunity to investigate the cultivation of C. sativa presented itself as part of post-mining activities on Christmas Island (Australia) to profitably utilise disused phosphate (PS) quarries. Challenges to plant growth and cadmium (Cd) uptake were addressed in this study using potted plants under fully controlled conditions in a growth chamber. A complete nutritional spectrum, slow-release fertiliser was applied to all plants as a control treatment, and two levels of rock PS dust, a waste product of PS mining that contains 35% phosphorus (P) and 40ppm of naturally occurring Cd, were applied at 54 and 162gL-1 . After 12weeks, control plants (no PS dust) significantly differed in phenological development, with no flower production, lower aboveground biomass and reduced photosynthesis efficiency than those with P applied as rock dust. Compared with the controls, the 54gL-1 level of P dust increased shoot biomass by 38%, while 162gL-1 increased shoot biomass by 85%. The concentration of Δ9 -tetrahydrocannabinol also increased with the higher P levels. Cd uptake from PS dust by C. sativa was substantial and warrants further investigation. However, there was no increase in Cd content between the 54 and 162gL-1 application rates in seed and leaf. Results indicate that hemp could become a high-value crop on Christmas Island, with the readily available rock PS dust providing a source of P.

Semi-quantitative analysis of cannabinoids in hemp (Cannabis sativa L.) using gas chromatography coupled to mass spectrometry.

BACKGROUND: Hemp (Cannabis sativa L.) is a producer of cannabinoids. These organic compounds are of increasing interest due to their potential applications in the medicinal field. Advances in analytical methods of identifying and quantifying these molecules are needed. METHOD: This study describes a new method of cannabinoid separation from plant material using gas chromatography-mass spectrometry (GC-MS) as the analytical tool to detect low abundance cannabinoids that will likely have implications for future therapeutical treatments. A novel approach was adopted to separate trichomes from plant material to analyse cannabinoids of low abundance not observed in raw plant extract. Required plant sample used for analysis was greatly reduced compared to other methods. Derivatisation method was simplified and deconvolution software was utilised to recognise unknown cannabinoid compounds of low abundance. RESULTS: The method produces well-separated spectra and allows the detection of major and minor cannabinoids. Ten cannabinoids that had available standards could be identified and quantified and numerous unidentified cannabinoids or pathway intermediates based on GC-MS spectra similarities could be extracted and analysed simultaneously with this method. CONCLUSIONS: This is a rapid novel extraction and analytical method from plant material that can identify major and minor cannabinoids using a simple technique. The method will be of use to future researchers seeking to study the multitude of cannabinoids whose values are currently not understood.

qPCR Assays for Sensitive and Rapid Detection of Quambalaria Species from Plant Tissues.

Several species from the genus Quambalaria (order Microstromatales) cause diseases on eucalypts (Eucalyptus and related genera) both in plantations and natural ecosystems. We developed real-time quantitative PCR (qPCR) assays to rapidly detect and distinguish five Quambalaria species. The design of the species-specific qPCR assay for each species, Q. pitereka (PIT), Q. coyrecup (COR), Q. cyanescens (CYN), Q. pusilla (PUS), and Q. eucalypti (EUC), was based on the ITS region and was evaluated for specificity and sensitivity. The PIT, COR, and CYN qPCR assays could amplify as little as 10 fg µl-1 from pure cultures, whereas PUS and EUC qPCR assays could amplify 100 fg µl-1 of their target species. The PIT, COR, and CYN qPCR assays were further validated using naturally and artificially infected samples of their plant host Corymbia calophylla. These assays will be used for rapid diagnostics and future experiments on the infection process.

Timing and abundance of sporangia production and zoospore release influences the recovery of different Phytophthora species by baiting.

Analysis of soil samples using High Throughput Sequencing (HTS) frequently detects more Phytophthora species compared with traditional soil baiting methods. This study investigated whether differences between species in the timing and abundance of sporangial production and zoospore release could be a reason for the lower number of species isolated by baiting. Stems of Eucalyptus marginata were inoculated with ten Phytophthora species (P. nicotianae, P. multivora, P. pseudocryptogea, P. cinnamomi, P. thermophila, P. arenaria, P. heveae, P. constricta, P. gondwanensis and P. versiformis), and lesioned sections for each species were baited separately in water. There were significant differences between species in timing of sporangia production and zoospore release. P. nicotianae, P. pseudocryptogea, P. multivora and P. thermophila released zoospores within 8-12 h and could be isolated from lesioned baits within 1-2 days. In contrast, P. constricta did not produce zoospores for over 48 h and was only isolated 5-7 days after baiting. P. heveae and P. versiformis did not produce zoospores and were not recovered from the baits. When species were paired in the same baiting tub, those that produced zoospores in the shortest time were isolated most frequently, while species slow to produce zoospores, or which produced them in lower numbers, were isolated from few baits or not at all. Thus, species differences in the timing of sporangia production and zoospore release may contribute to the ease of isolation of some Phytophthora species when they are present together with other Phytophthora species in an environmental sample.

Global meta-analysis of tree decline impacts on fauna.

Significant portions of the world's forests have been impacted by severe and large-scale tree declines characterised by gradual but widespread loss of vigour and subsequent death of either single or several tree species. Tree deaths represent a threat for fauna that are dependent on forest habitats for their survival. Although tree declines have received considerable scientific attention, surprisingly, little is known about their impacts on fauna. In total, we calculated 631 effect sizes across 59 studies that quantified the impact of tree declines on animal abundance. Data representing 186 bird species indicated an overall increase in bird abundance in response to tree declines (meta-analysis mean ± estimation g = 0.172 ± 0.053 [CI 0.069 to 0.275], P = 0.001); however, there was substantial variability in responses (significant heterogeneity P < 0.001) with a strong influence of diet as well as nesting guild on bird responses. Granivores (especially ground-foraging species, e.g. Passerellidae species), bark-foraging insectivores (e.g. woodpeckers), as well as ground- and cavity-nesting species apparently benefitted from tree declines, while nectarivorous birds [and, although not significant, aerially foraging insectivores (e.g. flycatchers) and leaf-gleaning insectivores (canopy-feeding)] were less common in the presence of tree declines. Data representing 33 mammal species indicate a tendency for detrimental effects of tree declines on mammals that use trees as refuges, while aerial foragers (i.e. bats) may benefit from opening up the canopy. Overall the average effect for mammals was neutral (meta-analysis mean estimation g = -0.150 ± 0.145 [-0.433 to 0.134], P = 0.302). Data representing 20 reptile species showed an insufficient range of responses to determine any diet or foraging effect on their responses. Data for 28 arthropod taxa should be considered with caution, as we could not adequately separate taxa according to their specialisations and reliance on key habitat. The data broadly suggest a detrimental effect of tree declines (meta-analysis mean estimation g = -0.171 ± 0.072 [-0.311 to -0.031], P = 0.017) with ground-foraging arthropods (e.g. detritivores and predators such as spiders and centipedes) more likely to be detrimentally impacted by tree declines. The range of responses to tree declines signifies substantially altered animal communities. In many instances, altered ecosystem function due to loss of key animal services will represent a significant threat to forest health.

Underappreciated plant vulnerabilities to heat waves.

With climate change, heat waves are becoming increasingly frequent, intense and broader in spatial extent. However, while the lethal effects of heat waves on humans are well documented, the impacts on flora are less well understood, perhaps except for crops. We summarize recent findings related to heat wave impacts including: sublethal and lethal effects at leaf and plant scales, secondary ecosystem effects, and more complex impacts such as increased heat wave frequency across all seasons, and interactions with other disturbances. We propose generalizable practical trials to quantify the critical bounding conditions of vulnerability to heat waves. Collectively, plant vulnerabilities to heat waves appear to be underappreciated and understudied, particularly with respect to understanding heat wave driven plant die-off and ecosystem tipping points.

A qPCR Assay for the Detection of Phytophthora cinnamomi Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples.

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

Carbon consequences of drought differ in forests that resprout.

Prolonged drought and intense heat-related events trigger sudden forest die-off events and have now been reported from all forested continents. Such die-offs are concerning given that drought and heatwave events are forecast to increase in severity and duration as climate change progresses. Quantifying consequences to carbon dynamics and storage from die-off events are critical for determining the current and future mitigation potential of forests. We took stand measurements five times over 2+ years from affected and unaffected plots across the Northern Jarrah Forest, southwestern Australia, following an acute drought/heatwave in 2011. We found a significant loss of live standing carbon (49.3 t ha-1 ), and subsequently a significant increase in the dead standing carbon pool by 6 months post-die-off. Of the persisting live trees, 38% experienced partial mortality contributing to the rapid regrowth and replenishment (82%-88%) of labile carbon pools (foliage, twigs, and branches) within 26 months. Such regrowth was not substantial in terms of net carbon changes within the timeframe of the study but does reflect the resprouting resilience of this forest type. Dead carbon generated by the die-off may persist for centuries given low fragmentation and decay rates resulting in low biogenic emission rates relative to other forest types. However, future fire may threaten persistence of both dead and live pools via combustion and mortality of live tissue and impaired regrowth capacity. Resprouting forests are commonly regarded as resilient systems, however, a changing climate could see vulnerable portions of forests become carbon sources rather than carbon sinks.

Tree host-pathogen interactions as influenced by drought timing: linking physiological performance, biochemical defence and disease severity.

There is increasing concern about tree mortality around the world due to climatic extremes and associated shifts in pest and pathogen dynamics. Yet, empirical studies addressing the interactive effect of biotic and abiotic stress on plants are very rare. Therefore, in this study, we examined the interaction between drought stress and a canker pathogen, Quambalaria coyrecup, on the eucalypt - Corymbia calophylla (marri), which is experiencing increasing drought stress. We hypothesized that drought stress would increase marri's susceptibility to canker disease, and cankers would have the largest negative effect on plants that are already drought stressed before pathogen inoculation. To test the hypotheses, in a glasshouse, marri saplings were exposed to drought either before or after pathogen inoculation, or were well-watered or droughted throughout the experiment either with or without inoculation. Canker development was greater in well-watered saplings than in droughted saplings, with the fastest development occurring in well-watered saplings that had experienced drought stress before inoculation. Irrespective of water treatments, marri saplings employed phenol-based localized biochemical defence against the pathogen. Drought reduced photosynthesis and growth, however, a negative effect of canker disease on saplings' physiological performance was only observed in well-watered saplings. In well-watered saplings, canker-induced loss of sapwood function contributed to reduced whole-plant hydraulic conductance, photosynthesis and growth. The results provide evidence that timing of drought stress influences host physiology, and host condition influences canker disease susceptibility through differences in induced biochemical defence mechanisms. The observations highlight the importance of explicitly incorporating abiotic and biotic stress, as well as their interactions, in future studies of tree mortality in drought-prone regions worldwide.

Subcontinental heat wave triggers terrestrial and marine, multi-taxa responses.

Heat waves have profoundly impacted biota globally over the past decade, especially where their ecological impacts are rapid, diverse, and broad-scale. Although usually considered in isolation for either terrestrial or marine ecosystems, heat waves can straddle ecosystems of both types at subcontinental scales, potentially impacting larger areas and taxonomic breadth than previously envisioned. Using climatic and multi-species demographic data collected in Western Australia, we show that a massive heat wave event straddling terrestrial and maritime ecosystems triggered abrupt, synchronous, and multi-trophic ecological disruptions, including mortality, demographic shifts and altered species distributions. Tree die-off and coral bleaching occurred concurrently in response to the heat wave, and were accompanied by terrestrial plant mortality, seagrass and kelp loss, population crash of an endangered terrestrial bird species, plummeting breeding success in marine penguins, and outbreaks of terrestrial wood-boring insects. These multiple taxa and trophic-level impacts spanned >300,000 km2-comparable to the size of California-encompassing one terrestrial Global Biodiversity Hotspot and two marine World Heritage Areas. The subcontinental multi-taxa context documented here reveals that terrestrial and marine biotic responses to heat waves do not occur in isolation, implying that the extent of ecological vulnerability to projected increases in heat waves is underestimated.

eDNA from roots: a robust tool for determining Phytophthora communities in natural ecosystems.

Proper isolation and identification of Phytophthora species is critical due to their broad distribution and huge impact on natural ecosystems throughout the world. In this study, five different sites were sampled and seven methods were compared to determine the Phytophthora community. Three traditional isolation methods were conducted (i) soil baiting, (ii) filtering of the bait water and (iii) isolation from field roots using Granny Smith apples. These were compared to four sources of eDNA used for metabarcoding using Phytophthora-specific primers on (i) sieved field soil, (ii) roots from field, (iii) filtered baiting water and (iv) roots from bait plants grown in the glasshouse in soil collected from these sites. Six Phytophthora species each were recovered by soil baiting using bait leaves and from the filtered bait water. No Phytophthora species were recovered from Granny Smith apples. eDNA extracted from field roots detected the highest number of Phytophthora species (25). These were followed by direct DNA isolation from filters (24), isolation from roots from bait plants grown in the glasshouse (19), and DNA extraction from field soil (13). Therefore, roots were determined to be the best substrate for detecting Phytophthora communities using eDNA.

Phytophthora Contamination in a Nursery and Its Potential Dispersal into the Natural Environment.

A detailed site investigation of a eucalypt nursery suffering disease losses revealed the causal agent to be Phytophthora boodjera. The pathogen was detected in vegetation surrounding the nursery production area, including the lawn, under the production benches during the growing season, and, most importantly, from plant debris in used trays. However, it was not found in the container substrate, water supplies, or production equipment or on the workers themselves. The sterilization methods used by the nursery were shown to be ineffective, indicating that a more rigorous method was required. Boiling trays for 15 min or steaming at 65°C for 60 min eradicated P. boodjera. This pathogen was more pathogenic to the eucalypts tested in their early seedling stage than P. cinnamomi. Tracing of out-planting to revegetation sites showed that P. boodjera was able to spread into the environment. Dispersal via out-planting to native vegetation may affect seedling recruitment and drive long-term shifts in native plant species. Inadequate nursery hygiene increases the risk of an outbreak and can limit the success of biosecurity efforts as well as damage conservation efforts.

Transitioning from phosphate mining to agriculture: Responses to urea and slow release fertilizers for Sorghum bicolor.

Globally, land-use transition from mining to agriculture is becoming increasingly attractive and necessary for many reasons. However, low levels of necessary plant nutrients, and high levels of heavy metals, can hamper plant growth, affecting yield, and potentially, food safety. In post-phosphate mining substrates, for example, nitrogen (N) is a key limiting nutrient, and, although legumes are planted prior to cereals, N supplementation is still necessary. We undertook two field trials on Christmas Island, Australia, to determine whether Sorghum bicolor could be grown successfully in a post-phosphate mining substrate. The first trial investigated N (urea) demand (amount of N required for adequate crop growth) for S. bicolor, and whether N addition could reduce the naturally occurring cadmium (Cd) concentrations in the crop. The second trial examined whether slow release nitrogen fertilizers (SRF) could replace urea to increase biomass and reduce Cd concentrations. Our first trial demonstrated that S. bicolor has a high N demand, with the highest biomass being recorded in the 160kg/ha urea treatment. However, plants treated with 80, 120 and 160kg/ha were not significantly different from one another. After 7weeks of growth, leaf Cd concentrations were significantly lower for all urea treatments compared with the control plants. However, after 23weeks, seed Cd concentrations did not differ across treatments. Our second trial demonstrated that the application of SRF (Macracote® and Sulsync®) and 160kg/ha urea significantly increased biomass above the control plants. There was, however, no treatment response in terms of Cd or N concentrations in the seed at final harvest. Thus, we have shown that N is currently critical for S. bicolor, even following legume cropping, and that high biomass and a significant reduction in Cd can be attained with appropriate levels of urea. Our work has important implications for cereal growth and food safety in post-mining agriculture.

Contemporary Remotely Sensed Data Products Refine Invasive Plants Risk Mapping in Data Poor Regions.

Invasive weeds are a serious problem worldwide, threatening biodiversity and damaging economies. Modeling potential distributions of invasive weeds can prioritize locations for monitoring and control efforts, increasing management efficiency. Forecasts of invasion risk at regional to continental scales are enabled by readily available downscaled climate surfaces together with an increasing number of digitized and georeferenced species occurrence records and species distribution modeling techniques. However, predictions at a finer scale and in landscapes with less topographic variation may require predictors that capture biotic processes and local abiotic conditions. Contemporary remote sensing (RS) data can enhance predictions by providing a range of spatial environmental data products at fine scale beyond climatic variables only. In this study, we used the Global Biodiversity Information Facility (GBIF) and empirical maximum entropy (MaxEnt) models to model the potential distributions of 14 invasive plant species across Southeast Asia (SEA), selected from regional and Vietnam's lists of priority weeds. Spatial environmental variables used to map invasion risk included bioclimatic layers and recent representations of global land cover, vegetation productivity (GPP), and soil properties developed from Earth observation data. Results showed that combining climate and RS data reduced predicted areas of suitable habitat compared with models using climate or RS data only, with no loss in model accuracy. However, contributions of RS variables were relatively limited, in part due to uncertainties in the land cover data. We strongly encourage greater adoption of quantitative remotely sensed estimates of ecosystem structure and function for habitat suitability modeling. Through comprehensive maps of overall predicted area and diversity of invasive species, we found that among lifeforms (herb, shrub, and vine), shrub species have higher potential invasion risk in SEA. Native invasive species, which are often overlooked in weed risk assessment, may be as serious a problem as non-native invasive species. Awareness of invasive weeds and their environmental impacts is still nascent in SEA and information is scarce. Freely available global spatial datasets, not least those provided by Earth observation programs, and the results of studies such as this one provide critical information that enables strategic management of environmental threats such as invasive species.

Pathways to false-positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study.

Phytophthora cinnamomi is one of the world's most invasive plant pathogens affecting ornamental plants, horticultural crops and natural ecosystems. Accurate diagnosis is very important to determine the presence or absence of this pathogen in diseased and asymptomatic plants. In previous studies, P. cinnamomi species-specific primers were designed and tested using various polymerase chain reaction (PCR) techniques including conventional PCR, nested PCR and quantitative real-time PCR. In all cases, the primers were stated to be highly specific and sensitive to P. cinnamomi. However, few of these studies tested their primers against closely related Phytophthora species (Phytophthora clade 7). In this study, we tested these purported P. cinnamomi-specific primer sets against 11 other species from clade 7 and determined their specificity; of the eight tested primer sets only three were specific to P. cinnamomi. This study demonstrated the importance of testing primers against closely related species within the same clade, and not just other species within the same genus. The findings of this study are relevant to all species-specific microbial diagnosis.

Current and projected global distribution of Phytophthora cinnamomi, one of the world's worst plant pathogens.

Globally, Phytophthora cinnamomi is listed as one of the 100 worst invasive alien species and active management is required to reduce impact and prevent spread in both horticulture and natural ecosystems. Conversely, there are regions thought to be suitable for the pathogen where no disease is observed. We developed a climex model for the global distribution of P. cinnamomi based on the pathogen's response to temperature and moisture and by incorporating extensive empirical evidence on the presence and absence of the pathogen. The climex model captured areas of climatic suitability where P. cinnamomi occurs that is congruent with all available records. The model was validated by the collection of soil samples from asymptomatic vegetation in areas projected to be suitable by the model for which there were few records. DNA was extracted, and the presence or absence of P. cinnamomi was determined by high-throughput sequencing (HTS). While not detected using traditional isolation methods, HTS detected P. cinnamomi at higher elevations in eastern Australia and central Tasmania as projected by the climex model. Further support for the climex model was obtained using the large data set from south-west Australia where the proportion of positive records in an area is related to the Ecoclimatic Index value for the same area. We provide for the first time a comprehensive global map of the current P. cinnamomi distribution, an improved climex model of the distribution, and a projection to 2080 of the distribution with predicted climate change. This information provides the basis for more detailed regional-scale modelling and supports risk assessment for governments to plan management of this important soil-borne plant pathogen.

Species from within the Phytophthora cryptogea complex and related species, P. erythroseptica and P. sansomeana, readily hybridize.

During a study on the phylogenetic relationships between species in the Phytophthora cryptogea complex and related species, Phytophthora erythroseptica and Phytophthora sansomeana, 19 hybrid isolates with multiple polymorphisms in the nuclear sequences were observed. Molecular characterization of hybrids was achieved by sequencing three nuclear (internal transcribed spacers, ß-tubulin (TUB), heat shock protein 90) and two mitochondrial (cytochrome c oxidase subunit I (coxI), NADH dehydrogenase subunit I (NADH)) gene regions and cloning of the single-copy nuclear gene, TUB. Based on the molecular studies the hybrid isolates belonged to six distinct groups between P. cryptogea, P. erythroseptica, Phytophthora pseudocryptogea, P. sansomeana, and Phytophthora sp. kelmania. In all cases, only a single coxI and NADH allele was detected and nuclear genes were biparentally inherited, suggesting that the hybrids arose from sexual recombination events. Colony morphology, growth rate, cardinal temperatures, breeding system, and morphology of sporangia, oogonia, oospores, and antheridia were also determined. Some morphological differences between the hybrids and the parental species were noted; however, they were not sufficient to reliably distinguish the taxa and DNA markers from nuclear and mitochondrial genes will to be necessary for their identification. The parental species are all important pathogens of agricultural fields that have been transported globally. With the apparent ease of hybridization within this group there is ample opportunity for virulent hybrids to form, perhaps with extended host ranges.

Spatial Configuration of Drought Disturbance and Forest Gap Creation across Environmental Gradients.

Climate change is increasing the risk of drought to forested ecosystems. Although drought impacts are often anecdotally noted to occur in discrete patches of high canopy mortality, the landscape effects of drought disturbances have received virtually no study. This study characterized the landscape configuration of drought impact patches and investigated the relationships between patch characteristics, as indicators of drought impact intensity, and environmental gradients related to water availability to determine factors influencing drought vulnerability. Drought impact patches were delineated from aerial surveys following an extreme drought in 2011 in southwestern Australia, which led to patchy canopy dieback of the Northern Jarrah Forest, a Mediterranean forest ecosystem. On average, forest gaps produced by drought-induced dieback were moderate in size (6.6 ± 9.7 ha, max = 85.7 ha), compact in shape, and relatively isolated from each other at the scale of several kilometers. However, there was considerable spatial variation in the size, shape, and clustering of forest gaps. Drought impact patches were larger and more densely clustered in xeric areas, with significant relationships observed with topographic wetness index, meteorological variables, and stand height. Drought impact patch clustering was more strongly associated with the environmental factors assessed (R2 = 0.32) than was patch size (R2 = 0.21); variation in patch shape remained largely unexplained (R2 = 0.02). There is evidence that the xeric areas with more intense drought impacts are 'chronic disturbance patches' susceptible to recurrent drought disturbance. The spatial configuration of drought disturbances is likely to influence ecological processes including forest recovery and interacting disturbances such as fire. Regime shifts to an alternate, non-forested ecosystem may occur preferentially in areas with large or clustered drought impact patches. Improved understanding of drought impacts and their patterning in space and time will expand our knowledge of forest ecosystems and landscape processes, informing management of these dynamic systems in an uncertain future.

Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry for Analysis of VOCs Produced by Phytophthora cinnamomi.

Volatile organic compounds (VOCs) from Phytophthora cinnamomi-infected lupin seedlings were collected by headspace solid-phase microextraction (HS-SPME). The sampling was done 28 to 44, 52 to 68, and 76 to 92 h after inoculation (HAI). The HS-SPME samples were analyzed by gas chromatography-flame ionization detector (GC-FID) to assess the differences in volatile compounds between the P. cinnamomi-infected lupin seedlings and the control. Three specific peaks were identified after 52 to 68 h with the infected lupin seedlings, at which time there were no visible aboveground symptoms of infection. Subsequently, the VOCs of five different substrates (V8A, PDA, lupin seedlings, soil, and soil + lupin seedlings) infected with P. cinnamomi and the corresponding controls were analyzed by gas chromatography-mass spectrometry (GC/MS). A total of 87 VOCs were identified. Of these, the five most abundant that were unique to all five inoculated substrates included: 4-ethyl-2-methoxyphenol, 4-ethylphenol, butyrolactone, phenylethyl alcohol, and 3-hydroxy-2-butanone. Therefore, these metabolites can be used as markers for the identification of P. cinnamomi in different growing environments. Some VOCs were specific to a particular substrate; for example, 2,4,6-rrimethyl-heptanes, dl-6-methyl-5-hepten-2-ol, dimethyl trisulfide, 6,10-dimethyl- 5,9-undecadien-2-ol, and 2-methoxy-4-vinylphenol were specific to P. cinnamomi + V8A; heptanes and 5-methyl-3-heptaneone were specific to P. cinnamomi + PDA; 3-methyl-1-butanol, ethyl acetate, 2-methyl-propanoic acid, ethyl ester, and ethyl ester 2-methyl-butanoic acid were specific to P. cinnamomi-inoculated lupin seedlings; and benzyl alcohol and 4-ethyl-1, 2-dimethoxybenzene were only detected in the headspace of inoculated soil + lupin seedlings. Results from this investigation have multiple impacts as the volatile organic profiles produced by the pathogen can be utilized as an early warning system to detect the pathogen from contaminated field soil samples. Data from this investigation have also illuminated potential metabolic pathways utilized by the oomycete during infection which may serve as potential targets for the development of specific control strategies.