Phytopathogenic Fungus Botrytis Cinerea Research Articles

Novel Aminocoumarin-Based Schiff Bases: High Antifungal Activity in Agriculture.

Structural modification is an effective way to improve the antifungal activity of natural products and has been widely used in the development of novel fungicides. In this work, a series of aminocoumarin-based Schiff bases were synthesized and characterized by 1H-NMR, 13C NMR and HR-MS spectra. The in vitro inhibition activity of all compounds was tested against four phytopathogenic fungi (Alternaria solani, Fusarium oxysporum, Botrytis cinerea, and Alternaria alternata) using the mycelial growth rate method. The results showed that most of the target compounds exhibited significant antifungal activities. In particular, compounds 5b, 5c, 5d, 5h, 5n, 7c, 7n, and 7p exhibited more effective antifungal activity than commercially available fungicides, chlorothalonil and azoxystrobin. The structure-activity relationship revealed that the electron-withdrawing groups with more electronegativity introduced at the C-3 position were effective in improving the inhibitory activity and that halogenated benzaldehydes would be necessary in the preparation of Schiff bases. The compound 5n against Fusarium oxysporum (EC50=8.73 μg/mL) and the compound 7p against Alternaria alternata (EC50=26.25 μg/mL) were much better than the positive controls (chlorothalonil and azoxystrobin). Therefore, compounds 5n and 7p could serve as promising lead compounds for the development of novel broad-spectrum fungicides, which could be useful for applications in the agriculture.

An asymptomatic geminivirus activates autophagy and enhances plant defenses against diverse pathogens

Plant viral diseases cause great losses in agricultural production. Virus cross-protection is a strategy in which a mild virus is employed to shield plants against subsequent infections by severe viral strains. However, this approach is restricted to protection against the same viruses. In this study, we observed that pre-inoculation with apple geminivirus (AGV) reduced the accumulation of secondarily infected heterologous viruses, such as cucumber mosaic virus, potato virus X, and tobacco mosaic virus in Nicotiana benthamiana, tomato, and pepper plants. Transcriptional expression analysis showed that autophagy-related genes were transcriptionally up-regulated upon AGV inoculation at an early stage of infection. Accordingly, autophagic activity was observed to be elevated following AGV infection. Interestingly, AGV accumulation was reduced in autophagy-deficient plants, suggesting that autophagy activation promotes AGV infection in the plant. Moreover, pre-inoculation with AGV provided cross-protection against infection with a phytopathogenic bacterium (Pseudomonas syringae) and fungus (Botrytis cinerea) in Nicotiana species. In summary, our study showed that AGV, an asymptomatic virus, could protect plants against severe viral, fungal, and bacterial diseases to some extent through the activation of autophagy pathways, highlighting its potential as a biocontrol agent for managing a wide range of plant crop diseases in the field.

Evaluation of the biocide activity of tomatine-rich extracts from tomato cannery residues against fungi and bacteria

Synthetic pesticides are discouraged for their environmental and health impacts, making research into alternatives essential. Several solutions of vegetal origin are being evaluated. The use of residual biomass from the agri-food system is particularly suitable due to its abundance and often unexplored potential. This study focuses on characterizing and assessing the activity of extracts obtained from wastes of the tomato cannery industry (including green fruit, stems, and leaves), which are rich in steroidal glycoalkaloids such as α-tomatine and tomatidine in different proportion. The antimicrobial activity of these extracts was tested on three bacterial strains belonging to the Escherichia coli (EC), Xanthomonas campestris (XC), and Bacillus pumilus (BP) species, as well as the phytopathogenic fungus Botrytis cinerea (BC). In particular, the mechanism of action of the extracts in relation to their surfactant properties was investigated, with the effect of the analytical standard serving as a reference. Both extracts showed strong inhibition of bacterial and fungal growth in vitro, with values reaching 100 %.The inhibitory effect was mainly due to the presence of α-tomatine in the extracts, which reached its aggregated state of micelle at the critical micelle concentration (CMC). Tomatidine, although known for its biocidal properties, did not contribute significantly due to its limited solubility. However, exceptions to this pattern were observed for extract rich in tomatidine, which exhibited efficacy at doses below the CMC. A possible explanation could be the enhanced solubility of tomatidine (which corresponds to enhanced bioactivity) in the presence of surfactant secreted by BP or as a consequence of the interaction between tomatidine and α-tomatine at the pre-micellar state for BC. In vivo assays with BC showed a reduction in symptoms comparable to that of a commercial fungicide available for organic agriculture, particularly at low concentrations. The relative content of α-tomatidine and tomatidine in the extracts modulated their bioactivity. An excess of tomatidine relative to α-tomatine led to a decrease in biocidal effect due to the chemical interactions among these species.

Antifungal Potential of Carnosic Acid from Salvia somalensis against Phytopathogenic Fungi

The aims of the present study were (i) to characterize the dichloromethane extract of the fresh aerial parts of S. somalensis, cultivated in Liguria (Italy), (ii) to quantify carnosic acid production and (iii) to find an eco-friendly alternative approach to control diseases caused by phytopathogenic fungi. The phytochemical investigation yielded several known terpenoids, as well as a diterpene, 4α,9α-epoxy-2H-dibenzo[a,d]cyclohepten-7(5H)-one, not previously described as a plant metabolite before. The results showed a noteworthy quantity of carnosic acid (113.90 µg/mg of dried extract). The potential antifungal activity of the plant surface extract and carnosic acid against five phytopathogenic fungi (Colletotrichum coccodes, Fusarium oxysporum, Sclerotinia sclerotiorum, Botrytis cinerea and Rhizoctonia solani) was considered. A complete inhibition of C. coccodes, S. sclerotiorum and R. solani mycelium growth was observed by carnosic acid at 500 µg/mL. High inhibition values were observed against B. cinerea and F. oxysporum compared to reference active ingredients. Four different B. cinerea strains exhibited a pronounced sensitivity to carnosic acid, as well as those originating from agricultural crop scenarios where a high load of active ingredient for gray mold control was historically adopted. Additionally, the formation and development of the germinative tube in B. cinerea were greatly slowed down.

Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively namedBotrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay.

Biocontrol mechanisms of the Antarctic yeast Debaryomyces hansenii UFT8244 against post-harvest phytopathogenic fungi of strawberries.

The use of yeasts has been explored as an efficient alternative to fungicide application in the treatment and prevention of post-harvest fruit deterioration. Here, we evaluated the biocontrol abilities of the Antarctic yeast strain Debaryomyces hansenii UFT8244 against the post-harvest phytopathogenic fungi Botrytis cinerea and Rhizopus stolonifer for the protection and preservation of strawberry fruit. The strongest inhibition of germination of B. cinerea (57%) was observed at 0 °C, followed by 40% at 25 °C. In addition, germ tubes and hyphae of B. cinerea were strongly surrounded and colonized by D. hansenii. Production of the enzymes β-1,3-glucanase, chitinase and protease by D. hansenii was detected in the presence of phytopathogenic fungus cell walls. The activity of β-1,3-glucanase was highest on day 12 of incubation and remained high until day 15. Chitinase and protease activities reached their highest levels on the day 15 of incubation. D. hansenii additionally demonstrated the ability to resist oxidative stress. Our data demonstrated that the main biocontrol mechanisms displayed by D. hansenii were the control of phytopathogenic fungal spore germination, production of antifungal enzymes and resistance to oxidative stress. We conclude that isolate D. hansenii UFT8422 should be further investigated for use at commercial scales at low temperatures.

Evaluation of Magonia pubescens A. St.-Hill. Roots Extract against Phytopathogens: Searching for Eco-Friendly Crop Protection Products

Botanical biopesticides have emerged as sustainable and eco-friendly alternatives to synthetic pesticides, whose indiscriminate use leads to several drawbacks to human and environmental health. To the best of our knowledge, there have been no reports on M. pubescens’ bioactivity on phytopathogens affecting crops as a potential fungicide or antifeedant. This has encouraged us to investigate the potential of the roots of this plant as a source of biopesticides. The present study reports on the evaluation of the roots extract from Magonia pubescens A. St.-Hill., a species from the Cerrado (Brazilian savannah), on the phytopathogenic fungi Botrytis cinerea, Fusarium oxysporum, and Alternaria alternata. In addition, its insect antifeedant effect was assayed against Chrysodeixis chalcites. Thus, an in vitro test-assay was used to determine the fungicide potential (percentage growth inhibition, % GI) of the ethanolic extract of this plant species, whereas a leaf-disk bioassay on the 5th instar larvae of C. chalcites was performed to evaluate its insecticidal potential. The ethanolic extract was further fractionated by liquid–liquid partition using solvents of increasing polarity. The hexane/dichloromethane fraction exhibited a moderated potency and was similar to the ethanolic extract on the three assayed fungi (around % GI 30 at 1 mg/mL), whereas the n-butanol fraction showed a slight improvement of the fungicide effect against B. cinerea (% GI 39.18 at 1 mg/mL). Moreover, the ethanolic extract exhibited a strong antifeedant activity, with a refusal rate (FR) higher than 90% in both choice and non-choice assays against C. chalcites, while the ethyl acetate and n-butanol fractions behaved as appetite suppressors. These results highlight M. pubescens as a promising source of biopesticides and deserve further investigations to optimize extraction procedures.

The presence of mycoviral infection attenuates the growth and pathogenicity of the phytopathogenic fungus Botrytis cinerea collected from strawberry fields in Pakistan

The presence of mycoviral infection attenuates the growth and pathogenicity of the phytopathogenic fungus Botrytis cinerea collected from strawberry fields in Pakistan

BcTaf14 regulates growth and development, virulence, and stress responses in the phytopathogenic fungus Botrytis cinerea.

TATA box-binding protein (TBP)-associated factor 14 (Taf14), a transcription-associated factor containing a conserved YEATS domain and an extra-terminal (ET) domain, is a multifunctional protein in Saccharomyces cerevisiae. However, the role of Taf14 in filamentous phytopathogenic fungi is not well understood. In this study, the homologue of ScTaf14 in Botrytis cinerea (named BcTaf14), a destructive phytopathogen causing grey mould, was investigated. The BcTaf14 deletion strain (ΔBcTaf14) showed pleiotropic defects, including slow growth, abnormal colony morphology, reduced conidiation, abnormal conidial morphology, reduced virulence, and altered responses to various stresses. The ΔBcTaf14 strain also exhibited differential expression of numerous genes compared to the wild-type strain. BcTaf14 could interact with the crotonylated H3K9 peptide, and mutation of two key sites (G80 and W81) in the YEATS domain disrupted this interaction. The mutation of G80 and W81 affected the regulatory effect of BcTaf14 on mycelial growth and virulence but did not affect the production and morphology of conidia. The absence of the ET domain at the C-terminus rendered BcTaf14 unable to localize to the nucleus, and the defects of ΔBcTaf14 were not recovered to wild-type levels when BcTaf14 without the ET domain was expressed. Our results provide insight into the regulatory roles of BcTaf14 and its two conserved domains in B. cinerea and will be helpful for understanding the function of the Taf14 protein in plant-pathogenic fungi.

The Botrytis cinerea Gene Expression Browser.

For comprehensive gene expression analyses of the phytopathogenic fungus Botrytis cinerea, which infects a number of plant taxa and is a cause of substantial agricultural losses worldwide, we developed BEB, a web-based B. cinerea gene Expression Browser. This computationally inexpensive web-based application and its associated database contain manually curated RNA-Seq data for B. cinerea. BEB enables expression analyses of genes of interest under different culture conditions by providing publication-ready heatmaps depicting transcript levels, without requiring advanced computational skills. BEB also provides details of each experiment and user-defined gene expression clustering and visualization options. If needed, tables of gene expression values can be downloaded for further exploration, including, for instance, the determination of differentially expressed genes. The BEB implementation is based on open-source computational technologies that can be deployed for other organisms. In this case, the new implementation will be limited only by the number of transcriptomic experiments that are incorporated into the platform. To demonstrate the usability and value of BEB, we analyzed gene expression patterns across different conditions, with a focus on secondary metabolite gene clusters, chromosome-wide gene expression, previously described virulence factors, and reference genes, providing the first comprehensive expression overview of these groups of genes in this relevant fungal phytopathogen. We expect this tool to be broadly useful in B. cinerea research, providing a basis for comparative transcriptomics and candidate gene identification for functional assays.

Identification of the main proteins secreted byKluyveromyces marxianusand their possible roles in antagonistic activity against fungi

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

In vitro efficacy of herbal plant extracts on some phytopathogenic fungi

Crops are subject to yield losses caused by the presence of pests whose species and population diversity are changing with climate warming. The use of agrochemicals is still the most effective method of plant protection against diseases and pests. However, the intensive use of pesticides in some EU countries makes it necessary to search for alternative methods that can be applied in plant protection for consumer and environmental safety. The aim of the study was the laboratory evaluation of the fungistatic effect of extracts of herbal plants occurring in natural state in Poland: horseradish (Armoracia rusticana L.), yarrow (Achillea millefolium L.) and tansy (Tanacetum vulgare L.) on selected polyphagic phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Colletotrichum coccodes and Fusarium oxysporum). In this study, 5%, 10% and 20% concentrations of herbal water-extract extracts were applied to evaluate their effects on linear growth of fungi and inhibition of their growth relative to control. Total content of polyphenols and flavonoids was assessed in the extracts using spectrophotometry, and their antioxidant activity by applying the synthetic DPPH radical. The plant extracts of tansy and yarrow leaves were characterized by a higher content of polyphenols and flavonoids compared to horseradish leaf extracts; they also had a higher antioxidant activity. Plant extracts inhibited the growth of fungi to a different extent, depending on the species of fungus, type of extract, its concentration and duration of action. The strongest fungistatic effect was recorded for tansy and yarrow extracts, while the weakest for the extract of horseradish leaves. Plant extracts showed the weakest effect against Botrytis cinerea, inhibiting the development of this fungus only during the first days of the experiment. The present research is a preliminary study that will be used in the further to develop a biological preparation for the protection of agricultural and horticultural plants against fungal pathogens.

Molecular identification and characterization of phytobeneficial osmotolerant endophytic bacteria inhabiting root nodules of the Saharan tree Vachellia tortilis subsp. raddiana.

Nodular endophytes of drought-tolerant legumes are understudied. For this reason, we have isolated and studied non-symbiotic endophytic bacteria from nodules of Vachellia tortilis subsp. raddiana, a leguminous tree adapted to the harsh arid climate of Southern Morocco. Rep-PCR analysis followed by 16S rDNA sequencing revealed two main genera, Pseudomonas and Bacillus. Isolates responded variably to salt and water stresses, and mostly produced exopolysaccharides. Differences concerned also plant growth-promoting activities: phosphate, potassium, and zinc solubilization; biological nitrogen fixation; auxin, siderophore, ammonia, and HCN production; and ACC deaminase activity. Some strains exhibited antagonistic activities against phytopathogenic fungi (Fusarium oxysporum and Botrytis cinerea) and showed at least two enzymatic activities (cellulase, protease, chitinase). Four selected strains inoculated to vachellia plants under controlled conditions have shown significant positive impacts on plant growth parameters. These strains are promising bio-inoculants for vachellia plants to be used in reforestation programs in arid areas increasingly threatened by desertification.

Phosphorus Recycling, Biocontrol, and Growth Promotion Capabilities of Soil Bacterial Isolates from Mexican Oak Forests: An Alternative to Reduce the Use of Agrochemicals in Maize Cultivation

Six bacteria (Bacillus velezensis 13, Bacillus subtillis 42, Pseudomonas fluorescens E221, Pseudomonas Poae EE12, Rahnella sp. EM1, and Serratia sp. EM2) isolated from the soil and litter of Mexican oak forests were characterized by identifying their ability to acquire phosphorus from different sources, analyzed for their biocontrol capabilities against two different phytopathogenic fungi, and finally tested for their ability to stimulate the germination of maize seeds and promotion of maize seedling growth. The greatest capacity to biocontrol the mycelial growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum was found in B. velezensis 13 and B. subtillis 42. P. poae EE12 and P. fluorescens E221 significantly promoted germination and the length of the primary root in Zea mays. Rahnella sp. EM1 and Serratia sp. EM2 could produce indole compounds related to auxin synthesis and increased the fresh weight of the maize seedlings. Together, these isolates represent an alternative to reduce the use of agrochemicals in maize cultivation. In general, soil microorganisms from Mexican oak forests represent a source of genetic resources for the sustainable management and conservation of soils for agricultural use.

Antifungal Potential of Canarian Plant Extracts against High-Risk Phytopathogens.

Phytopathogens are responsible for great losses in agriculture. In particular, Fusarium, Alternaria and Botrytis are fungal diseases that affect crops worldwide. In the search for eco-friendly solutions to pest control, plants and their chemo-biodiversity are promising sources of biopesticides for integrated pest management. The aim of the present study is to report the evaluation of sixteen plant species from the Canary Islands Archipelago against the phytopathogenic fungi Botrytis cinerea, Fusarium oxysporum, and Alternaria alternata. The plants were selected on the basis of their traditional uses in medicine and/or pest control, as well as on scientific studies reporting their uses in crop protection. Their growth inhibition (% I), in an in vitro test-assay on mycelium, was used to identify six ethanolic plant extracts displaying activity (% I > 30% at 1 mg/mL) against at least one of the assayed fungi. The most effective plant extracts were further fractionated by liquid-liquid partition, using solvents of increasing polarity. This procedure led to an improvement of the bioactivity against the phytopathogens, even affecting the hexane fraction from S. canariensis and achieving an 83.93% of growth inhibition at 0.5 mg/mL on B. cinerea. These findings identified five plant-derived extracts as potential candidates for the future development of new biofungicides, which could be applied in integrated pest management.

Isolation and characterization of extracellular vesicles from biotechnologically important fungus Aureobasidium pullulans

Extracellular vesicles (EVs) are increasingly recognized as an important mechanism for cell–cell interactions. Their role in fungi is still poorly understood and they have been isolated from only a handful of species. Here, we isolated and characterized EVs from Aureobasidium pullulans, a biotechnologically important black yeast-like fungus that is increasingly used for biocontrol of phytopathogenic fungi and bacteria. After optimization of the isolation protocol, characterization of EVs from A. pullulans by transmission electron microscopy (TEM) revealed a typical cup-shaped morphology and different subpopulations of EVs. These results were confirmed by nanoparticle tracking analysis (NTA), which revealed that A. pullulans produced 6.1 × 108 nanoparticles per milliliter of culture medium. Proteomic analysis of EVs detected 642 proteins. A small fraction of them had signal peptides for secretion and transmembrane domains. Proteins characteristic of different synthesis pathways were found, suggesting that EVs are synthesized by multiple pathways in A. pullulans. Enrichment analysis using Gene Ontology showed that most of the proteins found in the EVs were associated with primary metabolism. When sequencing the small RNA fraction of A. pullulans EVs, we found two hypothetical novel mil-RNAs. Finally, we tested the biocontrol potential of EVs from A. pullulans. The EVs did not inhibit the germination of spores of three important phytopathogenic fungi—Botrytis cinerea, Colletotrichum acutatum, and Penicillium expansum. However, exposure of grown cultures of C. acutatum and P. expansum to A. pullulans EVs resulted in visible changes in morphology of colonies. These preliminary results suggest that EVs may be part of the antagonistic activity of A. pullulans, which is so far only partially understood. Thus, the first isolation and characterization of EVs from A. pullulans provides a starting point for further studies of EVs in the biotechnologically important traits of the biocontrol black fungus A. pullulans in particular and in the biological role of fungal EVs in general.

Evidencing New Roles for the Glycosyl-Transferase Cps1 in the Phytopathogenic Fungus Botrytis cinerea.

The fungal cell wall occupies a central place in the interaction between fungi and their environment. This study focuses on the role of the putative polysaccharide synthase Cps1 in the physiology, development and virulence of the grey mold-causing agent Botrytis cinerea. Deletion of the Bccps1 gene does not affect the germination of the conidia (asexual spores) or the early mycelial development, but it perturbs hyphal expansion after 24 h, revealing a two-phase hyphal development that has not been reported so far. It causes a severe reduction of mycelial growth in a solid medium and modifies hyphal aggregation into pellets in liquid cultures. It strongly impairs plant penetration, plant colonization and the formation of sclerotia (survival structures). Loss of the BcCps1 protein associates with a decrease in glucans and glycoproteins in the fungus cell wall and the up-accumulation of 132 proteins in the mutant’s exoproteome, among which are fungal cell wall enzymes. This is accompanied by an increased fragility of the mutant mycelium, an increased sensitivity to some environmental stresses and a reduced adhesion to plant surface. Taken together, the results support a significant role of Cps1 in the cell wall biology of B. cinerea.

Redox and Hormonal Changes in the Transcriptome of Grape (Vitis vinifera) Berries during Natural Noble Rot Development.

Noble rot is a favorable form of the interaction between grape (Vitis spp.) berries and the phytopathogenic fungus Botrytis cinerea. The transcriptome pattern of grapevine cells subject to natural noble rot development in the historic Hungarian Tokaj wine region has not been previously published. Furmint, a traditional white Tokaj variety suited to develop great quality noble rot was used in the experiments. Exploring a subset of the Furmint transcriptome redox and hormonal changes distinguishing between noble rot and bunch rot was revealed. Noble rot is defined by an early spike in abscisic acid (ABA) accumulation and a pronounced remodeling of ABA-related gene expression. Transcription of glutathione S-transferase isoforms is uniquely upregulated, whereas gene expression of some sectors of the antioxidative apparatus (e.g., catalases, carotenoid biosynthesis) is downregulated. These mRNA responses are lacking in berries exposed to bunch rot. Our results help to explain molecular details behind the fine and dynamic balance between noble rot and bunch rot development.

The dark side of 5,10,15,20-(tetra-4-sulfonatophenyl)-porphyrin tetra-ammonium (TPPS) on Botrytis cinerea mycelium growth

The powerful inhibitory effect of a photoactivated anionic porphyrin (TPPS) on the mycelium growth of a phytopathogenic fungus Botrytis cinerea, was previously demonstrated, suggesting a new agricultural application of photodynamic treatment. In this context, it was also needed to investigate the effect of non-photoactivated TPPS onB. cinerea. Unexpectedly, TPPS tested at 3.5 [Formula: see text]M, induced a delay of the Botrytis cinerea mycelium growth that was later recovered under dark conditions. This delay, compared to the control, was low and suggested that the stress was induced by TPPS. This hypothesis was also confirmed at phenotypical level by Environmental Scanning Electron Microscopy analysis. Further tests at a biochemical level: total proteins, thiols and proline content determinations; and at cellular level, by monitoring cell leakage, gave more evidence for the presence of this stress. Nevertheless, TPPS induced a very short-lived effect on B. cinerea, suggesting that the mycelium was able to induce a rapid defense mechanism that involved medium acidification. A 4-day-old mycelium acidified the culture medium from a pH of 8 to less than 4. This acidic medium thus induced changes in the TPPS chemical structure that became protonated in its inner core. To our knowledge, this is the first paper that reports a dark effect of TPPS on Botrytis cinerea mycelium growth that could be rapidly counteracted by the protonation of TPPS leading to its no-longer dark toxicity.

Quaternized chitosan as a biopolymer sanitizer for leafy vegetables: synthesis, characteristics, and traditional vs. dry nano-aerosol applications

A series of quaternary dimethyl-(alkyl)-ammonium chitosan derivatives (QACs) was synthesized and studied for physicochemical properties and bioactivity. The QACs tended to spontaneously self-assembly into nanoaggregates. Antimicrobial activity was examined in vitro on Gram-negative Escherichia coli (E. coli) and Gram-positive Listeria innocua (L. innocua) bacteria as well as phytopathogenic fungus Botrytis cinerea. The hexyl chain-substituted QAC-6 demonstrated the highest potency causing 3.0- and 4.5-log CFU mL-1 reduction of E. coli and L. innocua, respectively. QAC-6 was tested for antimicrobial activity on stainless steel coupons and fresh spinach leaves. A traditional ‘wet’ application (spray) and dry Engineered Water Nanostructure (EWNS) approach were used for spinach decontamination. With both approaches, significant reduction of microbial load on the treated produce was achieved. The wet application showed a greater reduction of microbial load, while the advantages of EWNS were reaching the antimicrobial effect with miniscule dose of active agent leaving treated surface visibly dry.