Spatial Dependency in Stubble-Borne Pyrenophora teres f. teres and Influence of Sample Support Size on DNA Concentration and Fungicide Resistance Frequency.

Fungicide resistance in foliar fungal pathogens is an increasing challenge to crop production. Yield impacts due to loss of fungicide efficacy may be reduced through effective surveillance and appropriate management intervention. For stubble-borne pathogens, off-season crop residues may be used to monitor fungicide resistance to inform pre-planting decisions; however, appropriate sampling strategies and support sizes for crop residues have not previously been considered. Here, we used Pyrenophora teres f. teres (Ptt) with resistance to demethylase inhibitor fungicides as a model system to assess spatial dependency and to compare the effects of different sampling strategies and support sizes on pathogen density (Ptt DNA concentration) and the frequency of fungicide resistance mutation. The results showed that sampling strategies (hand-picked versus raked) did not affect estimates of pathogen density or fungicide resistance frequency; however, sample variances were lower from raked samples. The effects of differing sample support size, as the size of the collection area (1.2, 8.6, or 60 m2), on fungicide resistance frequency were not evident (P > 0.05). However, measures of pathogen density increased with area size (P < 0.05); the 60 m2 area yielded the highest Ptt DNA concentration and produced the lowest number of pathogen-absent samples. Sample variances for pathogen density and fungicide resistance frequency were generally homogeneous between area sizes. The pattern of pathogen density was spatially independent; however, spatial dependency was identified for fungicide resistance frequency, with a range of 110 m, in one of the two fields surveyed. Collectively, the results inform designs for monitoring of fungicide resistance in stubble-borne pathogens.

Spatiotemporal patterns of wheat response to Pyrenophora tritici-repentis in asymptomatic regions revealed by transcriptomic and X-ray fluorescence microscopy analyses.

Pathogen attacks elicit dynamic and widespread molecular responses in plants. While our understanding of plant responses has advanced considerably, little is known of the molecular responses in the asymptomatic 'green' regions adjoining lesions. Here, we explore gene expression data and high-resolution elemental imaging to report the spatiotemporal changes in the asymptomatic green region of susceptible and moderately resistant wheat cultivars infected with a necrotrophic fungal pathogen, Pyrenophora tritici-repentis. We show, with improved spatiotemporal resolution, that calcium oscillations are modified in the susceptible cultivar, resulting in 'frozen' host defence signals at the mature disease stage, and silencing of the host's recognition and defence mechanisms that would otherwise protect it from further attacks. In contrast, calcium accumulation and a heightened defence response were observed in the moderately resistant cultivar in the later stage of disease development. Furthermore, in the susceptible interaction, the asymptomatic green region was unable to recover after disease disruption. Our targeted sampling technique also enabled detection of eight previously predicted proteinaceous effectors in addition to the known ToxA effector. Collectively, our results highlight the benefits of spatially resolved molecular analysis and nutrient mapping to provide high-resolution spatiotemporal snapshots of host-pathogen interactions, paving the way for disentangling complex disease interactions in plants.

Optimized Sample Processing Pipeline for PCR-Based Fungicide Resistance Quantification of Stubble-Borne Fungal Pathogens.

Globally, yield losses associated with failed crop protection due to fungicide-resistant pathogens present an increasing problem. For stubble-borne pathogens, assessment of crop residues during the off-season could provide early fungicide resistance quantification for informed management decisions to mitigate yield losses. However, stubble assessment is hampered by assay inhibitors that are derived from decaying organic matter. To overcome assay inhibition from weathered stubble samples, we used a systems approach to quantify the frequency of resistance to demethylase inhibitor fungicides of the barley pathogen Pyrenophora teres f. teres. The system canvassed (i) 10 ball-milling conditions; (ii) four DNA extraction methodologies; and (iii) three column purification techniques for the provision of sufficient yield, quality, and purity of fungal DNA for a PCR-based fungicide resistance assay. Results show that DNA quantity and purity differed within each of the above three categories, with the optimized pipeline being (i) ball-milling samples in a 50-ml stainless steel canister for 5 min using a 20-mm ball at 30 revolutions s-1; (ii) a modified Brandfass method (extracted 64% more DNA than other methods assessed); and (iii) use of silica resin columns for the highest DNA concentration with optimal DNA purity. The chip-digital PCR assay, which quantified fungicide resistance from field samples, was unaffected by the DNA extraction method or purification technique, provided that thresholds of template quantity and purity were satisfied. In summary, this study has developed molecular pipeline options for pathogen fungicide resistance quantification from cereal stubbles, which can guide management for improved crop protection outcomes.

Impact of different processing techniques on the key volatile profile, sensory, and consumer acceptance of black truffle (Tuber melanosporum Vittadini).

Fresh truffles which include black truffle (Tuber melanosporum Vittadini) deteriorate and lose aroma rapidly after harvest; therefore, postharvest processing via freeze-drying or encapsulation is an option to preserve truffle aroma for extended supply. However, the aroma profile that directly affects the truffle quality and consumer acceptance is influenced by processing and producers require processing options that balance processing feasibility with retention of a suitable aroma profile. This study aimed to determine the impact of freeze-drying and encapsulation on the profile of key volatiles, consumer discrimination, and overall sensory impression (aroma intensity, liking, and acceptability) of processed truffle products compared to the starting material (positive control). The study combined experimental-scale processing with GC-MS analysis and consumer sensory evaluation to compare and optimize postharvest processing options. Based on the results, some volatile changes were detected in the processed truffle products compared to the positive control which were aligned with the consumer discrimination (triangle test) and the aroma intensity score (consumer sensory test). Despite some chemical and sensory differences detected, the consumer panel did not have any preference for processed truffle products compared to the positive control. The overall finding indicates the potential value of processing truffles into a natural flavoring ingredient for food application via freeze-drying or encapsulation, which should be of great interest for the truffle and food industry. According to the correlation analysis, the consumer acceptance of a truffle product may be increased by retaining 1-octen-3-ol and methional, while reducing the amount of p-cresol in the product. PRACTICAL APPLICATION: The postharvest process of turning truffles into a food flavoring ingredient may cause undesirable volatile changes that would directly impact the aroma quality and consumer acceptance of the processed truffle products. Hence, the impacts of freeze-drying and encapsulation on the chemical and sensory profile of truffles were evaluated in this study. Overall, the results of the concurrent instrument and sensory analysis demonstrated that both freeze-drying and encapsulation are potential options for processing.

Comparative evaluation of encapsulation using ß-cyclodextrin versus freeze-drying for better retention and stabilizing of black Périgord truffle (Tuber melanosporum) aroma.

This study aimed to develop a novel technique to retain and stabilize compounds contributing to truffle aroma by encapsulation using ß-cyclodextrin. Two experiments were conducted. In the first experiment, the key volatile profile and microbial population of products resulting from three different encapsulation methods, namely direct mixing method (M1), direct mixing followed by ethanol addition method (M2), and paste method (M3), were compared with untreated truffles (positive control) over a 90-day period. The M2-derived product was the least optimal for retaining key volatile compounds despite showing the lowest microbial population. There was no significant difference in the volatile profile of products derived from M1 and M3 on day 0. However, it was observed that the M3-derived product could retain its volatile profile better than the M1-derived product by day 90. M3 was compared with freeze-drying in the second experiment. Freeze-dried truffles showed an overall higher relative percentage of volatiles than the M3-derived product on day 0. However, by day 90, some volatile changes occurred in the freeze-dried truffles but not in the M3-derived product. The findings indicate that while freeze-drying could adequately conserve truffle volatiles, the encapsulation of volatile compounds in ß-cyclodextrin could improve the volatile stability of truffle products and allow for longer storage times. Microbes were found in all encapsulated truffle products and freeze-dried truffles on days 0 and 90, suggesting the need to explore the possibility of incorporating a decontamination step in the process prior to either encapsulation or freeze-drying. PRACTICAL APPLICATION: A technique to capture and stabilize compounds responsible for truffle aroma by encapsulation using ß-cyclodextrin was developed and compared with freeze-drying in this study. The overall finding suggests that while freeze-drying of truffle could sufficiently preserve volatiles, encapsulating truffle volatiles with ß-cyclodextrin may improve its stability, extending its shelf life, which can be applied in the development of a natural truffle ingredient that can be applied in food product development.

Methods used for extraction of plant volatiles have potential to preserve truffle aroma: A review.

Truffles are considered one of the world's most highly prized foods mainly due to their desirable organoleptic properties and rarity. However, truffles are seasonal (harvested mostly in winter from June to August in the Southern Hemisphere and from December to February in the Northern Hemisphere) and extremely perishable. Truffles deteriorate rapidly showing undesirable changes within 10 days from harvest in aroma and visual appearance after harvest. The very short postharvest shelf life (about 7-10 days) limits the potential for export and domestic consumption all year round. Several preservation methods have been studied to prolong their shelf life without the loss of aroma. However, all traditional preservation techniques have their own shortcomings and remain challenging. The extraction of natural truffle aroma volatiles for food applications could be a potential alternative to replace the existing synthetic flavoring used for processed truffle products. Four commonly used extraction methods for recovering volatile compounds from plants, namely, supercritical carbon dioxide extraction, Soxhlet extraction, distillation, and cold pressing, are critically analyzed. Up to date, existing research about the extraction of aroma volatiles from truffles is limited in the literature but based on the volatility of the key truffle volatile compounds, supercritical carbon dioxide extraction may offer the best possibility so that a natural truffle-based product that can be used in food applications throughout the year can be made available.

"Wax On, Wax Off": In Vivo Imaging of Plant Physiology and Disease with Fourier Transform Infrared Reflectance Microspectroscopy.

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well-established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non-destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change.

Synchrotron X-ray fluorescence microscopy-enabled elemental mapping illuminates the 'battle for nutrients' between plant and pathogen.

Metal homeostasis is integral to normal plant growth and development. During plant-pathogen interactions, the host and pathogen compete for the same nutrients, potentially impacting nutritional homeostasis. Our knowledge of outcome of the interaction in terms of metal homeostasis is still limited. Here, we employed the X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron to visualize and analyse the fate of nutrients in wheat leaves infected with Pyrenophora tritici-repentis, a necrotrophic fungal pathogen. We sought to (i) evaluate the utility of XFM for sub-micron mapping of essential mineral nutrients and (ii) examine the spatiotemporal impact of a pathogen on nutrient distribution in leaves. XFM maps of K, Ca, Fe, Cu, Mn, and Zn revealed substantial hyperaccumulation within, and depletion around, the infected region relative to uninfected control samples. Fungal mycelia were visualized as thread-like structures in the Cu and Zn maps. The hyperaccumulation of Mn in the lesion and localized depletion in asymptomatic tissue surrounding the lesion was unexpected. Similarly, Ca accumulated at the periphery of the symptomatic region and as microaccumulations aligning with fungal mycelia. Collectively, our results highlight that XFM imaging provides the capability for high-resolution mapping of elements to probe nutrient distribution in hydrated diseased leaves in situ.

Real-Time PCR for Diagnosing and Quantifying Co-infection by Two Globally Distributed Fungal Pathogens of Wheat.

Co-infections - invasions of a host-plant by multiple pathogen species or strains - are common, and are thought to have consequences for pathogen ecology and evolution. Despite their apparent significance, co-infections have received limited attention; in part due to lack of suitable quantitative tools for monitoring of co-infecting pathogens. Here, we report on a duplex real-time PCR assay that simultaneously distinguishes and quantifies co-infections by two globally important fungal pathogens of wheat: Pyrenophora tritici-repentis and Parastagonospora nodorum. These fungi share common characteristics and host species, creating a challenge for conventional disease diagnosis and subsequent management strategies. The assay uses uniquely assigned fluorogenic probes to quantify fungal biomass as nucleic acid equivalents. The probes provide highly specific target quantification with accurate discrimination against non-target closely related fungal species and host genes. Quantification of the fungal targets is linear over a wide range (5000-0.5 pg DNA µl-1) with high reproducibility (RSD ≤ 10%). In the presence of host DNA in the assay matrix, fungal biomass can be quantified up to a fungal to wheat DNA ratio of 1 to 200. The utility of the method was demonstrated using field samples of a cultivar sensitive to both pathogens. While visual and culture diagnosis suggested the presence of only one of the pathogen species, the assay revealed not only presence of both co-infecting pathogens (hence enabling asymptomatic detection) but also allowed quantification of relative abundances of the pathogens as a function of disease severity. Thus, the assay provides for accurate diagnosis; it is suitable for high-throughput screening of co-infections in epidemiological studies, and for exploring pathogen-pathogen interactions and dynamics, none of which would be possible with conventional approaches.

Analysis of the nexus between population, water resources and Global Food Security highlights significance of governance and research investments and policy priorities.

BACKGROUND: Analyses of sensitivity of Global Food Security (FS) score to a key set of supply or demand factors often suggest population and water supply as being the most critical and on which policies tend to focus. To explore other policy options, we characterized the nexus between GFS and a set of supply or demand factors including population, agricultural and industrial water uses, agricultural publications (as a surrogate for investment in agricultural research and development (R&D)) and corruption perception index (CPI), to reveal opportunities for attaining enduring GFS. RESULTS: We found that despite being the primary driver of demand for food, population showed no significant correlation with FS scores. Similarly, agricultural water use was poorly correlated with GFS scores, except in countries where evaporation exceeds precipitation and irrigation is significant. However, FS had a strong positive association with industrial water use as a surrogate for overall industrialization. Recent expansions in cultivated land area failed to yield concomitant improvements in FS score since such expansions have been mostly into marginal lands with low productivity and thus barely compensated for lands retired from cropping in several developed economies. However, FS was positively associated with agricultural R&D investments, as it was with the CPI scores. The apparent and relative strengths of these drivers on FS outcome amongst countries were in the order: industrial water-use ≈ publication rate ≈ corruption perception ≫ agricultural water use > population. CONCLUSIONS: We suggest that to enshrine enduring food security, policies should prioritize (1) increased R&D investments that address farmer needs and (2) governance mechanisms that promote accountability in both research and production value chains. © 2018 Society of Chemical Industry.

Host-Multi-Pathogen Warfare: Pathogen Interactions in Co-infected Plants.

Studies of plant-pathogen interactions have historically focused on simple models of infection involving single host-single disease systems. However, plant infections often involve multiple species and/or genotypes and exhibit complexities not captured in single host-single disease systems. Here, we review recent insights into co-infection systems focusing on the dynamics of host-multi-pathogen interactions and the implications for host susceptibility/resistance. In co-infection systems, pathogen interactions include: (i) Competition, in which competing pathogens develop physical barriers or utilize toxins to exclude competitors from resource-dense niches; (ii) Cooperation, whereby pathogens beneficially interact, by providing mutual biochemical signals essential for pathogenesis, or through functional complementation via the exchange of resources necessary for survival; (iii) Coexistence, whereby pathogens can stably coexist through niche specialization. Furthermore, hosts are also able to, actively or passively, modulate niche competition through defense responses that target at least one pathogen. Typically, however, virulent pathogens subvert host defenses to facilitate infection, and responses elicited by one pathogen may be modified in the presence of another pathogen. Evidence also exists, albeit rare, of pathogens incorporating foreign genes that broaden niche adaptation and improve virulence. Throughout this review, we draw upon examples of co-infection systems from a range of pathogen types and identify outstanding questions for future innovation in disease control strategies.

Smoke-derived taint in wine: the release of smoke-derived volatile phenols during fermentation of Merlot juice following grapevine exposure to smoke.

The release of smoke-derived volatile phenols during the fermentation of Merlot grapes, following grapevine exposure to smoke, has been investigated. The concentrations of guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, and eugenol were determined by gas chromatography-mass spectrometry and found to increase throughout the winemaking process. Only trace levels (< or = 1 microg/L) of guaiacol and 4-methylguaiacol could be detected in free run juice derived from the fruit of smoked vines; the highest levels, 388 microg/L and 93 microg/L, respectively, were observed in the finished wine. Control wine (derived from fruit of unsmoked vines) contained 4 microg/L guaiacol, with the volatile phenols either not detected or detected at only trace levels (< or = 1 microg/L) throughout fermentation. The role of enzyme and acid catalyzed hydrolysis reactions in releasing smoke-derived volatile compounds was also investigated. The volatile phenols were released from smoked free run juice by strong acid hydrolysis (pH 1.0) and enzyme (beta-glucosidase) hydrolysis, but not mild acid hydrolysis (juice pH 3.2-3.7). Guaiacol was again the most abundant smoke-derived phenol, present at 431 microg/L and 325 microg/L in strong acid and enzyme hydrolysates, respectively. Only trace levels of each phenol could be detected in each control hydrolysate. This study demonstrates the potential for under-estimation of smoke taint in fruit and juice samples; the implications for the assessment of smoke taint and quantification of volatile phenols are discussed.

Smoke-derived taint in wine: effect of postharvest smoke exposure of grapes on the chemical composition and sensory characteristics of wine.

Although smoke exposure has been associated with the development of smoke taint in grapes and subsequently in wine, to date there have been no studies that have demonstrated a direct link. In this study, postharvest smoke exposure of grapes was utilized to demonstrate that smoke significantly influences the chemical composition and sensory characteristics of wine and causes an apparent 'smoke taint'. Verdelho grapes were exposed to straw-derived smoke for 1 h and then fermented according to two different winemaking treatments. Control wines were made by fermenting unsmoked grapes. Sensory studies established a perceivable difference between smoked and unsmoked wines; smoked wines were described as exhibiting 'smoky', 'dirty', 'earthy', 'burnt' and 'smoked meat' characteristics. Quantitative analysis, by means of gas chromatography-mass spectrometry, identified guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol, and furfural in each of the wines made from smoked grapes. However, these compounds were not detected in the unsmoked wines, and their origin is therefore attributed to the application of smoke. Increased ethanol concentrations and browning were also observed in wines made from grapes exposed to smoke.

Whole-plant transpiration efficiency of Sultana grapevine grown under saline conditions is increased through the use of a Cl--excluding rootstock.

The aim of this study was to test the influence of salinity (1, 20, 40 and 80 mol m-3) on the transpiration efficiency (W = biomass / water transpired), lamina gas exchange and carbon isotope discrimination (Δ) of grapevine (Vitis vinifera L. cv. Sultana) grown on own roots or grafted to a Cl--excluding rootstock (Ramsey; Vitis champiniL.). Growth of own-rooted and Ramsey-rooted vines irrigated with a salinity of 40 mol m-3 was reduced by 55 and 12%, respectively, compared with vines irrigated with 1 mol m-3. At 1 mol m-3 W of Ramsey-rooted vines was 1.3-fold higher than own-rooted vines (3.9 and 3.0 g L-1, respectively). Salinity resulted in a decrease in W of own-rooted vines (31% reduction at both 40 and 80 mol m-3). In contrast, W of Ramsey-rooted vines increased by up to 1.25-fold under saline conditions. Consequently, at 80 mol m-3 W of Ramsey-rooted vines was 2-fold higher than own-rooted vines. To our knowledge this is the first demonstration of the potential of a rootstock to increase W of a crop species under saline conditions. The rootstock-dependent differences in grapevine W under saline conditions were not determined by differences in lamina gas exchange. Differences in W associated with rootstock may be attributed to differences in ion uptake and the energy requirements associated with ion partitioning and the formation of compatible solutes.

Influence of saline irrigation on growth, ion accumulation and partitioning, and leaf gas exchange of carrot (Daucus carota L.).

Like those of many horticultural crop species, the growth and leaf gas exchange responses of carrot (Daucus carota L.) to salinity are poorly understood. In this study ion accumulation in root tissues (periderm, xylem and phloem tissues) and in leaves of different ages was assessed for carrot plants grown in the field with a low level of salinity (5.8 mM Na(+) and 7.5 mM Cl(-)) and in a glasshouse with salinity ranging from 1-80 mM. At low levels of salinity (1-7.5 mM), in both the field and glasshouse, carrot leaves accumulated high concentrations of Cl(-) (140-200 mM); these appear to be the result of a high affinity for Cl(-) uptake and a low retention of Cl(-) in the root system. However, Cl(-) uptake is under tight control, with an 80-fold increase in external salinity resulting in only a 1.5-fold change in the Cl(-) concentration of the shoot and no increase in the Cl(-) concentration of the root xylem tissue. In contrast to Cl(-), shoot Na(+) concentrations were comparatively low (30-40 mM) but increased by seven-fold when salinity was increased by 80-fold. Growth over the 56-d treatment period in the glasshouse was insensitive to salinity less than 20 mM, but at higher concentrations the yield of carrot tap roots declined by 7 % for each 10 mM increase in salinity. At low levels of salinity the accumulation of high concentrations of Cl(-) (150 mM) in carrot laminae did not appear to limit leaf gas exchange. However, photosynthesis and stomatal conductance were reduced by 38 and 53 %, respectively, for plants grown at a salinity of 80 mM compared with those grown at 1 mM. Salinity-induced reductions in both p(i) and carbon isotope discrimination (delta) were small (2.5 Pa and 1.4 per thousand, respectively, at 80 mM) indicating that the reduction in photosynthesis was only marginally influenced by CO(2) supply. At a salinity of 80 mM the photosynthetic capacity was reduced, with a 30 % reduction in the CO(2)-saturated rate of photosynthesis (A(max)) and a 40 % reduction in both the apparent rate of RuBP-carboxylase-limited CO(2) fixation (V(cmax)) and the electron transport rate limiting RuBP regeneration (J(max)). This study has shown that carrot growth and leaf gas exchange are insensitive to the high leaf Cl(-) concentrations that occur at low levels (1-7 mM) of salinity. However, growth is limited at salinity levels above 20 mM and leaf gas exchange is limited at salinity levels above 8 mM.