Innovative strategies based on the use of biostimulants to manage plant diseases and minimize the application of synthetic fungicides in grapevine and stone fruits

Plant diseases can severely affect the grapevine and stone fruit production, contributing to food losses and food waste. The ordinary way to control plant diseases consists in the use of synthetic fungicides. However, restrictions due to legislation and the request from consumers of food free from pesticide residues triggered the investigation on natural alternatives. Among these, there is a biopolymer extracted from crab shell called chitosan that is able to produce a film on treated commodities. This biopolymer has antimicrobial, eliciting and film-forming activities, contributing for about 40%, 35% and 25%, respectively, to its control of plant diseases. When applied to grapevine canopy, chitosan reduced downy mildew, caused by Plasmopara viticola, at the same magnitude of copper widely used in organic agriculture, but it accumulates in the soil being phytotoxic and harming soil microbiota. Chitosan application on stone fruit decreases the development of brown rot on sweet cherries cold stored and exposed to shelf life. Since consumers are worried about the presence of fungicide residues in fruit, retailers impose to the growers a reduction on the pesticide residues far below the legal threshold. Moreover, copper is a candidate to the substitution, then, in few years, its application in agriculture can be banned in the EU. Therefore, the use of a biostimulants as chitosan, that is a natural compound used in slimming diets then safe for humans and the environment, is a strategy welcomed by organic growers, and applied in several wineries to manage grapevine downy mildew

How Does Chloroplast Protect Chlorophyll Against Excessive Light?

Chlorophylls (Chls) are the most abundant plant pigments on Earth. Chls are located in the membrane of thylakoids where they constitute the two photosystems (PSII and PSI) of terrestrial plants, responsible for both light absorption and transduction of chemical energy via photosynthesis. The high efficiency of photosystems in terms of light absorption correlates with the need to protect themselves against absorption of excess light, a process that leads to the so-called photoinhibition. Dynamic photoinhibition consists of the downregulation of photosynthesis quantum yield and a series of photo-protective mechanisms aimed to reduce the amount of light reaching the chloroplast and/or to counteract the production of reactive oxygen species (ROS) that can be grouped in: (i) the first line of chloroplast defence: non-photochemical quenching (NPQ), that is, the dissipation of excess excitation light as heat, a process that takes place in the external antennae of PSII and in which other pigments, that is carotenoids, are directly involved; (ii) the second line of defence: enzymatic antioxidant and antioxidant molecules that scavenge the generated ROS; alternative electron transport (cyclic electron transport, pseudo-cyclic electron flow, chlororespiration and water-water cycle) can efficiently prevent the over-reduction of electron flow, and reduced ferredoxin (Fd) plays a key role in this context

Genetic transformation of Vitis vinifera via organogenesis

BACKGROUND: Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered. RESULTS: Transgenic grape plants (V. vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis. In vitro proliferating shoots were cultured in the presence of increasing concentrations of N(6)-benzyl adenine. The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots. Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM. After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field. At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth. The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars. CONCLUSIONS: The phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars. Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars

Fine tuning of Real Time PCR as a first tool for the detection of G146A substitution in Venturia inaequalis samples

none5noopenCeren Turan, Irene Maja Nanni, Lucia Landi, Alessandro Pirondi, Marina CollinaTuran, Ceren; Maja Nanni, Irene; Landi, Lucia; Pirondi, Alessandro; Collina, Marin

Effect of auxotrophies on yeast performance in aerated fed-batch reactor

A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae,one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance

Effect of auxotrophies on yeast growth in aerated fed-batch reactor

Mutant and deletion strains of the yeast Saccharomyces cerevisiae having one/several auxotrophies are largely used in the development of recombinant strains for heterologous protein production because they ensure maintenance of plasmids with selectable markers. The production is usually carried out by culturing the recombinant strain in aerated fed-batch, where sugar limitation achieves high yields of biomass and product. In a previous work, it was evidenced that growth of the auxotrophic S. cerevisiae BY4741 (MATa, ura30, leu20, met150, his31) engineered for human IL-1 production, and employed in aerated fed-batch, early arrested even in the presence of a correct nutritional complementation (being specific nutrients for genetically uncomplemented auxotrophies provided in no growthlimiting amounts). It was assumed that this behaviour may depend on the high number of auxotrophies, since the prototrophic S288C, from which BY4741 derives, showed a typical performance under the same cultivation mode. Therefore, a systematic investigation on the effect of auxotrophies on yeast growth in aerated fed-batch was carried out. Four isogenic strains of the CEN.PK family, with a progressively increasing number of auxotrophies (from one to four) were assayed under fed-batch conditions and a proper nutritional complementation. Feeding to the reactor was exponentially increased imposing a specific growth rate below the critical one. The behaviour of the auxotrophic strains was compared with that of the isogenic prototrophic strain. by evaluating the capacity to keep the specific growth rate chosen. A clear correlation among optimum growth and number of auxotrophies has been found. Furthermore we have investigated the possible effect of the type of auxotrophy (ura- or leu-) on the strain performance, monitoring as well cell viability of each strain. The study is a contribution to know the phenotypic effects of auxotrophies in yeast and can have implications for biotechnological applications

Chlorophyll Fluorescence, Photoinhibition and Abiotic Stress: Does it Make Any Difference the Fact to Be a C3 or C4 Species?

Chlorophyll fluorescence analysis is one of the most powerful and widely used techniques to study the effect of stresses on the photosynthetic process. From the first utilization, the Fv/Fm ratio has been largely used as a sensitive indicator of plant photosynthetic performance. Decreases of this index are indicative of the reduction of photosystem II (PSII) efficiency, namely photoinhibition. In the last 20 years, application of chlorophyll fluorescence has been largely improved, and many other informative parameters have been established to detect PSII photochemical efficiency and the partitioning of light energy to alternative dissipative mechanisms (qE, energy-dependent quenching; qZ, zeaxanthin-dependent quenching and qI, photoinhibitory quenching; qH, sustained photoprotective antenna quenching; qM, quenching dependent to chloroplast movement; qT, light harvesting complexes II–I state-transition) such as the recently developed “photoprotective power” of non-photochemical quenching (pNPQ). This review reports a brief description of the main chlorophyll fluorescence parameters and a wide analysis of the current bibliography on the use of different parameters which are useful to detect events of PSII photoinhibition. In addition, in view of the inherent differences in morpho-anatomical, physiological and biochemical features between C3 and C4 metabolism, possible differences in terms of photoinhibition between C3 and C4 plant species under stress conditions are proposed. The attempt is to highlight the limits of their comparison in terms of susceptibility to photoinhibition and to propose direction of future research which, assisted by chlorophyll fluorescence, should improve the knowledge of the different sensitivity of C3 and C4 to abiotic stressors

Occurence of Ochratoxin A (OTA) on grapes in the province of Ancona, Italy

Ochratoxin A (OTA) is a mycotoxin that is naturally produced by some species of filamentous fungi belonging to genera Aspergillus and Penicillium. OTA is among the most harmful of mycotoxins. It has been proven by various studies that OTA has strong toxic properties such as nephrotoxic and neurotoxic effects, and it is classified by the International Agency for Research on Cancer (IARC) as potentially carcinogen for humans and carcinogen for animals. The major problem is the fact that OTA is produced by fungi that are commonly present in a wide variety of agricultural products, including grapes and their derivatives. Due to its toxicity and health hazard provoked by the presence of OTA in food and feed, the European Community has established a limit for toxin concentration of 2 μg/kg in grape and grape derivates. The concentration should be reduced as much as possible, not only below the legal limit, in order to diminish the daily intake of this toxin. A research on the presence of ochratoxigenic fungi on grapes was performed in order to evaluate the risk of contamination of grapes and grape derivates from various vineyards in the province of Ancona, Italy with OTA, and to, eventually, propose a strategy for their control. The mycoflora from grapes harvested in September 2018 was isolated and fungi were determined morphologically. Potential OTA producers were inoculated on OTA conducing media and screened for toxin production. All potential producers and confirmed ochratoxigenic strains will be determined by molecular techniques

On the fermentative behavior of auxotrophic strains of Saccharomyces cerevisiae

Background: The selection of new yeast strains could lead to improvements in bioethanol production. Here, we have studied the fermentative capacity of different auxotrophic mutants of Saccharomyces cerevisiae , which are routinely used as hosts for the production of heterologous proteins. It has recently been found that these strains exhibit physiological alterations and peculiar sensitivities with respect to the parental prototrophic strains from which they derive. In this work the performance of auxotrophic S. cerevisiae CEN.PK strains was compared to the corresponding prototrophic strain, to S. cerevisiae T5bV, a strain isolated from grape must and to another auxotrophic strain, S. cerevisiae BY4741. Results: The results indicate that the fermentative capacity of strains grown in 2% glucose was similar in all the strains tested. However, in 15% initial glucose, the auxotrophic strains exhibited a more than doubled ethanol yield on biomass (10 g g-1dw) compared to the prototrophic strains (less than 5 g g-1dw). Other tests have also evidenced that in medium depletion conditions, ethanol production continues after growth arrest. Conclusions: The results highlight the capacity of auxotrophic yeast strains to produce ethanol per mass unit, in a higher amount with respect to the prototrophic ones. This leads to potential applications for auxotrophic strains of S. cerevisiae in the production of ethanol in both homogeneous and heterogeneous phases (immobilized systems). The higher ethanol yield on biomass would be advantageous in immobilized cell systems, as a reduced yeast biomass could greatly reduce the mass transfer limitations through the immobilization matrix

Parental cancer: mediating and moderating roles of psychological inflexibility in the links between illness severity and parental quality of life and family outcomes

The challenges of parental cancer while caring for young offspring are often neglected by researchers and healthcare providers. Focusing on parents with cancer, this cross-sectional study examines the mediating and moderating roles of a mal- leable risk factor, psychological inflexibility, in the relationships between perceived illness severity and parental quality of life (QoL) and family outcomes. Psychological inflexibility was conceptualized using the acceptance and commitment therapy (ACT) framework. A total of 86 parents with cancer caring for young offspring (aged 11–24 years; M=17.94, SD=3.68) completed a survey that assessed psychological inflexibility, parental mental and physical health QoL, family outcomes (family functioning, parenting concerns, and openness to discuss cancer), socio-demographics, and illness vari- ables. Mediation analyses indicated that higher perceived illness severity was associated with higher levels of psychologi- cal inflexibility, which in turn were related to higher detrimental impacts on parental QoL and family outcomes. Results of moderation analyses were non-significant. Findings highlight the detrimental impacts of illness severity on psychological inflexibility, suggesting that it is a psychosocial risk factor in parents dealing with cancer. ACT-based interventions which foster psychological flexibility are likely to enhance parental QoL and family outcomes