The Influence of Long-Term Storage on the Epiphytic Microbiome of Postharvest Apples and on Penicillium expansum Occurrence and Patulin Accumulation.

Patulin is a secondary metabolite primarily synthesized by the fungus Penicillium expansum, which is responsible for blue mold disease on apples. The latter are highly susceptible to fungal infection in the postharvest stages. Apples destined to produce compotes are processed throughout the year, which implies that long periods of storage are required under controlled atmospheres. P. expansum is capable of infecting apples throughout the whole process, and patulin can be detected in the end-product. In the present study, 455 apples (organically and conventionally grown), destined to produce compotes, of the variety "Golden Delicious" were sampled at multiple postharvest steps. The apple samples were analyzed for their patulin content and P. expansum was quantified using real-time PCR. The patulin results showed no significant differences between the two cultivation techniques; however, two critical control points were identified: the long-term storage and the deck storage of apples at ambient temperature before transport. Additionally, alterations in the epiphytic microbiota of both fungi and bacteria throughout various steps were investigated through the application of a metabarcoding approach. The alpha and beta diversity analysis highlighted the effect of long-term storage, causing an increase in the bacterial and fungal diversity on apples, and showed significant differences in the microbial communities during the different postharvest steps. The different network analyses demonstrated intra-species relationships. Multiple pairs of fungal and bacterial competitive relationships were observed. Positive interactions were also observed between P. expansum and multiple fungal and bacterial species. These network analyses provide a basis for further fungal and bacterial interaction analyses for fruit disease biocontrol.

Effect of high salinity and of priming of non-germinated seeds by UV-C light on photosynthesis of lettuce plants grown in a controlled soilless system.

High salinity results in a decrease in plant photosynthesis and crop productivity. The aim of the present study was to evaluate the effect of UV-C priming treatments of lettuce seeds on photosynthesis of plants grown at high salinity. Non-primed and primed seeds were grown in an hydroponic system, with a standard nutrient solution, either supplemented with 100 mM NaCl (high salinity), or not (control). Considering that leaf and root K+ concentrations remained constant and that chlorophyll fluorescence parameters and root growth were not affected negatively in the high salinity treatment, we conclude that the latter was at the origin of a moderate stress only. A substantial decrease in leaf net photosynthetic assimilation (Anet) was however observed as a consequence of stomatal and non-stomatal limitations in the high salinity treatment. This decrease in Anet translated into a decrease in growth parameters; it may be attributed partially to the high salinity-associated increase in leaf concentration in abscisic acid and decrease in stomatal conductance. Priming by UV-C light resulted in an increase in total photosynthetic electron transport rate and Anet in the leaves of plants grown at high salinity. The increase of the latter translated into a moderate increase in growth parameters. It is hypothesized that the positive effect of UV-C priming on Anet and growth of the aerial part of lettuce plants grown at high salinity, is mainly due to its stimulating effect on leaf concentration in salicylic acid. Even though leaf cytokinins' concentration was higher in plants from primed seeds, maintenance of the cytokinins-to-abscisic acid ratio also supports the idea that UV-C priming resulted in protection of plants exposed to high salinity.

Effect of Regulated Deficit Irrigation on Agronomic Parameters of Three Plum Cultivars (Prunus salicina L.) under Semi-Arid Climate Conditions.

Regulated deficit irrigation (RDI) strategies may greatly contribute to save irrigation water, especially in low water availability conditions. The effects of RDI on the growth process, photosynthesis, fruit yield, and some quality traits were assessed for two years on three plum (Prunus salicina Lindl.) cultivars ('Black Diamond', 'Black Gold' and 'Black Star') grown in Midwest Tunisia. The experiment was conducted during two successive seasons (2011-2012). Two water regimes were applied per cultivar during the phase of fruit growth until fruit ripening. Stressed trees receive 50% of the amount of irrigation compared to controls (CI). Several eco-physiological parameters and some pomological criteria were measured, based on the climatic condition (ETo, ETc, and VDP). Results showed that the three cultivars had an aptitude for tolerance for moderate stress with varying degrees of response time to drought stress. Globally, a slight decrease in the gas exchange rate (AN and gs) and the water status (RWC, Ψmin, and Ψos) was registered. Tree yields, size and weight show a slight decrease under water stress. Nevertheless, there was an improvement in the total soluble solid content (SSC) and firmness in same cultivars. Our results proved that the Black Star cultivar was the most tolerant to deficit irrigation, in reason that it maintains a good water status and a high photosynthetic activity.

Microbiome Status of Cider-Apples, from Orchard to Processing, with a Special Focus on Penicillium expansum Occurrence and Patulin Contamination.

Patulin is a secondary metabolite produced primarily by the fungus Penicillium expansum, responsible for the blue mold disease on apples. It is found in apple products including apple cider when apple juice is added after fermentation. In the present study, two hundred and twenty-five cider-apples of the variety "Bedan", cultivated in Brittany in France, were sampled from the orchard during harvesting until the storage step, right before processing. The patulin analysis on these samples reported a low contamination at the orchard and a significantly higher-level of contamination in the cider-apples starting from the transporting bin. The percentage of positive samples increased from 6% to 47% after 12 h in the harvesting bin before transporting and reached 95% after 24 h of transporting, decreasing then to 69% at the end of the storage. Penicillium expansum was quantified on the surface of apples using real-time PCR and was observed to be mostly consistent between the harvest and post-harvest steps. It was detected on average, on the surface of 85% of all sampled apples with a mean value around 2.35 × 106Penicillium expansum DNA/g of apple. Moreover, the changes in the fungal and bacterial epiphytic microbiota in the different steps were studied using a metabarcoding approach. The alpha and beta diversity analysis revealed the presence of unique and more diverse bacterial and fungal communities on the surface of apples picked from the orchard compared to the rest of the sampling steps. Potential indigenous biological control agents were identified on the surface of sampled apples. Future perspective includes developing actions of prevention and control of the contamination by Penicillium expansum during the harvest and along the various critical post-harvest stages before transformation in a sustainable development concern.

Regulation of sugar metabolism genes in the nitrogen-dependent susceptibility of tomato stems to Botrytis cinerea.

BACKGROUND AND AIMS: The main soluble sugars are important components of plant defence against pathogens, but the underlying mechanisms are unclear. Upon infection by Botrytis cinerea, the activation of several sugar transporters, from both plant and fungus, illustrates the struggle for carbon resources. In sink tissues, the metabolic use of the sugars mobilized in the synthesis of defence compounds or antifungal barriers is not fully understood. METHODS: In this study, the nitrogen-dependent variation of tomato stem susceptibility to B. cinerea was used to examine, before and throughout the course of infection, the transcriptional activity of enzymes involved in sugar metabolism. Under different nitrate nutrition regimes, the expression of genes that encode the enzymes of sugar metabolism (invertases, sucrose synthases, hexokinases, fructokinases and phosphofructokinases) was determined and sugar contents were measured before inoculation and in asymptomatic tissues surrounding the lesions after inoculation. KEY RESULTS: At high nitrogen availability, decreased susceptibility was associated with the overexpression of several genes 2 d after inoculation: sucrose synthases Sl-SUS1 and Sl-SUS3, cell wall invertases Sl-LIN5 to Sl-LIN9 and some fructokinase and phosphofructokinase genes. By contrast, increased susceptibility corresponded to the early repression of several genes that encode cell wall invertase and sucrose synthase. The course of sugar contents was coherent with gene expression. CONCLUSIONS: The activation of specific genes that encode sucrose synthase is required for enhanced defence. Since the overexpression of fructokinase is also associated with reduced susceptibility, it can be hypothesized that supplementary sucrose cleavage by sucrose synthases is dedicated to the production of cell wall components from UDP-glucose, or to the additional implication of fructose in the synthesis of antimicrobial compounds, or both.

Reduced susceptibility of tomato stem to the necrotrophic fungus Botrytis cinerea is associated with a specific adjustment of fructose content in the host sugar pool.

Background and aims: Plant soluble sugars, as main components of primary metabolism, are thought to be implicated in defence against pathogenic fungi. However, the function of sucrose and hexoses remains unclear. This study aimed to identify robust patterns in the dynamics of soluble sugars in sink tissues of tomato plants during the course of infection by the necrotrophic fungus Botrytis cinerea . Distinct roles for glucose and fructose in defence against B. cinerea were hypothesized. Methods: We examined sugar contents and defence hormonal markers in tomato stem tissues before and after infection by B. cinerea , in a range of abiotic environments created by various nitrogen and water supplies. Key Results: Limited nitrogen or water supplies increased tomato stem susceptibility to B. cinerea . Glucose and fructose contents of tissues surrounding infection sites evolved differently after inoculation. The fructose content never decreased after inoculation with B. cinerea , while that of glucose showed either positive or negative variation, depending on the abiotic environment. An increase in the relative fructose content (defined as the proportion of fructose in the soluble sugar pool) was observed in the absence of glucose accumulation and was associated with lower susceptibility. A lower expression of the salicylic acid marker PR1a , and a lower repression of a jasmonate marker COI1 were associated with reduced susceptibility. Accordingly, COI1 expression was positively correlated with the relative fructose contents 7 d after infection. Conclusions: Small variations of fructose content among the sugar pool are unlikely to affect intrinsic pathogen growth. Our results highlight distinct use of host glucose and fructose after infection by B. cinerea and suggest strongly that adjustment of the relative fructose content is required for enhanced plant defence.

Alternation of light/dark period priming enhances clomazone tolerance by increasing the levels of ascorbate and phenolic compounds and ROS detoxification in tobacco (Nicotiana tabacum L.) plantlets.

The effect of the alternation of light/dark periods (AL) (16/8 min light/dark cycles and a photosynthetic photon flux density (PPFD) of 50 µmol photons m(-2) s(-1) for three days) to clarify the mechanisms involved in the clomazone tolerance of tobacco plantlets primed with AL was studied. Clomazone decreased PSII activity, the net photosynthetic rate (Pn), and the ascorbate and total polyphenol contents and increased H2O2 and starch grain accumulation and the number of the cells that underwent programmed cell death (PCD). The pretreatment with AL reduced the inhibitory effect of clomazone on the PSII activity and photosynthesis, as indicated by the decreases in the H2O2 and starch grain accumulation and the PCD levels, and increased the content of ascorbate and certain phenolic compounds, such as chlorogenic acid, neochlorogenic acid and rutin. The AL treatment could promote photorespiration via post-illumination burst (PIB) effects. This alternative photorespiratory electron pathway may reduce H2O2 generation via the consumption of photochemical energy, such as NADH+H(+). At 10 days (D10) of AL treatment, this process induced moderate stress which stimulates H2O2 detoxification systems by increasing the activity of antioxidant enzymes and the biosynthesis of antioxidant components. Therefore, the PCD levels provoked by clomazone were noticeably decreased.

Tolerance to clomazone herbicide is linked to the state of LHC, PQ-pool and ROS detoxification in tobacco (Nicotiana tabacum L.).

In this study, plantlets of two tobacco (Nicotiana tabacum L.) varieties that are clomazone-tolerant (cv. Xanthi) and clomazone-sensitive (cv. Virginie vk51) were subjected to low concentration of clomazone herbicide. The oxygen-evolving rate of isolated chloroplasts, chlorophyll a fluorescence transients, JIP-test responses, hydrogen peroxide contents, antioxidant enzyme activities, cytohistological results and photosynthetic pigment contents were recorded. The results indicated that the carotenoid content was 2-fold higher in Virginie, which had greater clomazone sensitivity than Xanthi. Virginie exhibited noticeable decreases in the LHC content (Chl a/b ratio), the maximum photochemical quantum efficiency of PSII (Fv/Fm), the performance index on the absorption basis (PIabs), and the electron flux beyond the first PSII QA evaluated as (1-VJ) with VJ=(FJ-F0)/(Fm-F0) as well as increases in the rate of photon absorption (ABS/RC) and the energy dissipation as heat (DI0/RC). These results suggest that PSII photoinhibition occurred as a consequence of more reduced PQ-pool and accumulated QA(-). The oxygen evolution measurements indicate that PSI electron transport activity was not affected by clomazone. The more significant accumulation of H2O2 in Virginie compared to Xanthi was due to the absence of ROS-scavenging enzymes, and presumably induced programmed cell death (PCD). The symptoms of PCD were observed by cytohistological analysis, which also indicated that the leaf tissues of clomazone-treated Virginie exhibited significant starch accumulation compared to Xanthi. Taken together, these results indicate that the variable tolerance to clomazone observed between Virginie and Xanthi is independent of the carotenoid content and could be related to the state of the LHC, the redox state of the PQ-pool, and the activity of detoxification enzymes.

Water shortage and quality of fleshy fruits--making the most of the unavoidable.

Extreme climatic events, including drought, are predicted to increase in intensity, frequency, and geographic extent as a consequence of global climate change. In general, to grow crops successfully in the future, growers will need to adapt to less available water and to take better advantage of the positive effects of drought. Fortunately, there are positive effects associated with drought. Drought stimulates the secondary metabolism, thereby potentially increasing plant defences and the concentrations of compounds involved in plant quality, particularly taste and health benefits. The role of drought on the production of secondary metabolites is of paramount importance for fruit crops. However, to manage crops effectively under conditions of limited water supply, for example by applying deficit irrigation, growers must consider not only the impact of drought on productivity but also on how plants manage the primary and secondary metabolisms. This question is obviously complex because during water deficit, trade-offs among productivity, defence, and quality depend upon the intensity, duration, and repetition of events of water deficit. The stage of plant development during the period of water deficit is also crucial, as are the effects of other stressors. In addition, growers must rely on relevant indicators of water status, i.e. parameters involved in the relevant metabolic processes, including those affecting quality. Although many reports on the effects of drought on plant function and crop productivity have been published, these issues have not been reviewed thus far. Here, we provide an up-to-date review of current knowledge of the effects of different forms of drought on fruit quality relative to the primary and secondary metabolisms and their interactions. We also review conventional and less conventional indicators of water status that could be used for monitoring purposes, such as volatile compounds. We focus on fruit crops owing to the importance of secondary metabolism in fruit quality and the importance of fruits in the human diet. The issue of defence is also briefly discussed.

Pretreatment with alternation of light/dark periods improves the tolerance of tobacco (Nicotiana tabacum) to clomazone herbicide.

This work analyses the effects of alternation of light/dark periods pretreatment (AL) in tobacco plantlets (Nicotiana tabacum L. cv.Virginie vk51) growing in solution with low concentration of the clomazone herbicide. The experimentation has been carried out by exposing the plantlets to successive and regulated periods of light (16min light/8min dark cycles, PAR 50µmolm(-2)s(-1)) for three days. The photosynthesis efficiency was determined by mean of the chlorophyll fluorescence and JIP-test. The AL pretreatment improved the clomazone tolerance; this has been observed by the increase in the leaf area of the plant, the maximal photochemical quantum efficiency of PSII (Fv/Fm), the actual PSII efficiency (ФPSII), the performance index (PIabs), the electron flux beyond Quinone A (1-VJ), and also by the diminution of the energy dissipating into heat (DI0/RC). Furthermore, AL pretreatment led to low accumulation of hydrogen peroxide (H2O2) which proves that the scavenging enzymatic system have been activated before clomazone treatment. In the plantlets pretreated with AL, with regard to the ascorbate content, some of antioxidant enzyme whose function is associated with it have continued to scavenge reactive oxygen species (ROS) induced by clomazone, such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). So, the observed photooxidative damages induced by clomazone herbicide were noticeably reduced.

Effect of salt stress on tomato fruit antioxidant systems depends on fruit development stage.

The effects of different levels of salt stress on the oxidative parameters (H2O2 and MDA), the total pool sizes of ascorbate, the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the activities and relative transcript levels of the enzymes of ascorbate-glutathione cycle; ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were studied in fruits of tomato. Plants were treated by three concentrations of NaCl (50, 100 and 150 mM) and fruits at different development stages were harvested after 3 and 6 days of stress. The concentrations of ascorbate (AsA) and dehydroascorbate (DHA) generally changed with salt stress treatments. Moreover, changes in SOD and CAT activities and DHAR, MDHAR, APX and GR activities and relative transcript levels were dependent on the fruit development stage and the intensity and duration of salt stress. These results suggest that the response of antioxidant systems of tomato fruits to oxidative stress induced by salt stress treatments was different depending on the fruit development stage.

High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

Metabolism in orange fruits is driven by photooxidative stress in the leaves.

In plants, stress signals propagate to trigger distant responses and thus stress acclimation in non-exposed organs. We tested here the hypothesis that leaves submitted to photooxidative stress may influence the metabolism of nearby fruits and thus quality criteria. Leaves of orange trees (Citrus sinensis (L.) Osbeck cv. 'Navelate') were acclimated to shade for 1 week and then submitted to full (FL) and medium light (ML) conditions. As expected, photoinhibition was detected in leaves of both FL and ML treatments as revealed by stress indicators (Fv /Fm , Performance Index) for at least 99 h after treatments. In the fruits near the stressed leaves, we then determined the activities of enzymes related to oxidative stress, superoxide dismutase, catalase and the enzymes of the ascorbate (AA)/glutathione cycle, as well as the contents in sugars, organic acids and carotenoids. Ascorbate peroxidase and monodehydroascorbate reductase activities in the pulp of fruits were dramatically higher in both treatments when compared to the control. AA and total sugars were not affected by the photooxidative stress. However, the FL treatment resulted in a 16% increase in total organic acids, with succinic acid being the major contributor, a shift towards less glucose + fructose and more sucrose, and a 15% increase in total carotenoids, with cis-violaxanthin being the major contributor. Our observations strongly suggest the existence of a signal generated in leaves in consequence of photooxidative stress, transmitted to nearby fruits. Exploiting such a signal by agronomic means promises exciting perspectives in managing quality criteria in fruits accumulating carotenoids.

Response of the physiological parameters of mango fruit (transpiration, water relations and antioxidant system) to its light and temperature environment.

Depending on the position of the fruit in the tree, mango fruit may be exposed to high temperature and intense light conditions that may lead to metabolic and physiological disorders and affect yield and quality. The present study aimed to determine how mango fruit adapted its functioning in terms of fruit water relations, epicarp characteristics and the antioxidant defence system in peel, to environmental conditions. The effect of contrasted temperature and light conditions was evaluated under natural solar radiation and temperature by comparing well-exposed and shaded fruit at three stages of fruit development. The sun-exposed and shaded peels of the two sides of the well-exposed fruit were also compared. Depending on fruit position within the canopy and on the side of a well-exposed fruit, the temperature gradient over a day affected fruit characteristics such as transpiration, as revealed by the water potential gradient as a function of the treatments, and led to a significant decrease in water conductance for well-exposed fruits compared to fruits within the canopy. Changes in cuticle thickness according to fruit position were consistent with those of fruit water conductance. Osmotic potential was also affected by climatic environment and harvest stage. Environmental conditions that induced water stress and greater light exposure, like on the sunny side of well-exposed fruit, increased the hydrogen peroxide, malondialdehyde and total and reduced ascorbate contents, as well as SOD, APX and MDHAR activities, regardless of the maturity stage. The lowest values were measured in the peel of the shaded fruit, that of the shaded side of well-exposed fruit being intermediate. Mango fruits exposed to water-stress-induced conditions during growth adapt their functioning by reducing their transpiration. Moreover, oxidative stress was limited as a consequence of the increase in antioxidant content and enzyme activities. This adaptive response of mango fruit to its climatic environment during growth could affect postharvest behaviour and quality.

Effect of water stress on antioxidant systems and oxidative parameters in fruits of tomato (Solanum lycopersicon L, cv. Micro-tom).

The effects of different levels of water stress on oxidative parameters (H2O2 and MDA), the total pool sizes of ascorbate, the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the activities and relative transcript levels of the enzymes of ascorbate-glutathione cycle ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) were studied in the fruit of tomato (Solanum lycopersicum L. cv. Micro-Tom). Plants were subjected to three levels of water stress (S50, S25 and S0) and fruits at different development stages were harvested after 3, 6 and 10 days of stress. Changes in H2O2 and MDA contents indicated that water stress induced oxidative stress in fruits. The concentrations of ascorbate (AsA) and dehydroascorbate (DHA) generally modified with water stress treatments. Moreover, changes in SOD and CAT activities and DHAR, MDHAR, APX and GR activities and relative transcript levels were dependent on the fruit development stage and the intensity and the duration of water stress. These results suggest that the response of antioxidant systems of tomato fruits to oxidative stress induced by water stress treatments was different depending on the fruit development stage.

Microplate quantification of enzymes of the plant ascorbate-glutathione cycle.

Here, we describe microplate assays for determining the specific activities of four enzymes that constitute the ascorbate-glutathione cycle: APX, MDHAR, DHAR, and GR. In plants, these enzymes play a major role in detoxifying reactive oxygen species produced in cells under environmental stress. This work presents the development of plate reader assays to allow rapid analysis of the ascorbate-glutathione cycle activity using tomato fruits subjected to salt stress as a model. With this method, it is possible to analyze easily in one day the activities of the four enzymes for 30 experimental samples, all in triplicate and with blanks.