Comparative Characterization of Virulent and Less-Virulent Lasiodiplodia theobromae Isolates.

Lasiodiplodia theobromae attacks over 500 plant species and is an important pathogen of tropical and subtropical fruit. Due to global warming and climate change, the incidence of disease associated with L. theobromae is rising. Virulence tests performed on avocado and mango branches and fruit showed a large diversity of virulence of different L. theobromae isolates. Genome sequencing was performed for two L. theobromae isolates, representing more virulent (Avo62) and less-virulent (Man7) strains, to determine the cause of their variation. Comparative genomics, including orthologous and single-nucleotide polymorphism (SNP) analyses, identified SNPs in the less-virulent strain in genes related to secreted cell wall-degrading enzymes, stress, transporters, sucrose, and proline metabolism, genes in secondary metabolic clusters, effectors, genes involved in the cell cycle, and genes belonging to transcription factors that may contribute to the virulence of L. theobromae. Moreover, carbohydrate-active enzyme analysis revealed a minor increase in gene counts of cutinases and pectinases and the absence of a few glycoside hydrolases in the less-virulent isolate. Changes in gene-copy numbers might explain the morphological differences found in the in-vitro experiments. The more virulent Avo62 grew faster on glucose, sucrose, or starch as a single carbon source. It also grew faster under stress conditions, such as osmotic stress, alkaline pH, and relatively high temperature. Furthermore, the more virulent isolate secreted more ammonia than the less-virulent one both in vitro and in vivo. These study results describe genome-based variability related to L. theobromae virulence, which might prove useful for the mitigation of postharvest stem-end rot. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould.

Pathogenic fungi cause major postharvest losses. During storage and ripening, fruit becomes highly susceptible to fungi that cause postharvest disease. Fungicides are effective treatments to limit disease. However, due to increased public concern for their possible side effects, there is a need to develop new strategies to control postharvest fungal pathogens. Botrytis cinerea, a common postharvest pathogen, was shown to uptake small double-stranded RNA (dsRNA) molecules from the host plant. Such dsRNA can regulate gene expression through the RNA interference system. This work aimed to develop a synthetic dsRNA simultaneously targeting three essential transcripts active in the fungal ergosterol biosynthesis pathway (dsRNA-ERG). Our results show initial uptake of dsRNA in the emergence zone of the germination tube that spreads throughout the fungus and results in down-regulation of all three targeted transcripts. Application of dsRNA-ERG decreased B. cinerea germination and growth in in vitro conditions and various fruits, leading to reduce grey-mould decay. The inhibition of growth or decay was reversed by the addition of ergosterol. While dual treatment with dsRNA-ERG and ergosterol-inhibitor fungicide reduced by 100-fold the required amount of fungicide to achieve the same protection rate. The application of dsRNA-ERG induced systemic protection as shown by decreased decay development at inoculation points distant from the treatment point in tomato and pepper fruits. Overall, this study suggests that dsRNA-ERG can effectively control B. cinerea growth and grey-mould development suggesting its efficacy as a future method for postharvest control of fungal pathogens.

Sustained release varnish containing chlorhexidine for prevention of Streptococcus mutans biofilm formation on voice prosthesis surface: an in vitro study.

OBJECTIVES: In this study, we aimed to develop a novel, sustained release varnish (SRV) for voice prostheses (VP) releasing chlorhexidine (CHX), for the prevention of biofilm formation caused by the common oral bacteria Streptococcus mutans on VP surfaces. METHODS: This study was performed in an in vitro model as a step towards future in vivo trials. VPs were coated with a SRV containing CHX (SRV-CHX) or SRV alone (placebo-SRV) that were daily exposed to S. mutans. The polymeric materials of SRV were composed of ethylcellulose and PEG-400. Biofilm formation was assessed by DNA quantification (qPCR), crystal violet staining, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and kinetics experiments. RESULTS: The amount of DNA in the biofilms formed by S. mutans on VP surfaces coated once with SRV-CHX (1.024 ± 0.218 ng DNA/piece) was 58.5 ± 8.8% lower than that of placebo-SRV-coated VPs (2.465 ± 0.198 ng DNA/piece) after a 48-h exposure to S. mutans (p = 0.038). Reduced biofilm mass on SRV-CHX-coated VPs was visually confirmed by CLSM and SEM. CV staining of SRV-CHX single-coated VPs that have been exposed to S. mutans nine times showed a 98.1 ± 0.2% reduction in biofilm mass compared to placebo-SRV-coated VPs (p = 0.003). Kinetic experiments revealed that SRV-CHX triple-coated VPs could delay bacterial growth for 23 days. CONCLUSIONS: Coating VPs with SRV-CHX has an inhibitory effect on biofilm formation and prevents bacterial growth in their vicinities. This study is a proof-of-principle that paves the way for developing new clinical means for reducing both VPs' bacterial biofilm formation and device failure.

In Pursuit of Optimal Quality: Cultivar-Specific Drying Approaches for Medicinal Cannabis.

A limited number of studies have examined how drying conditions affect the cannabinoid and terpene content in cannabis inflorescences. In the present study, we evaluated the potential of controlled atmosphere drying chambers for drying medicinal cannabis inflorescence. Controlled atmosphere drying chambers were found to reduce the drying and curing time by at least 60% compared to traditional drying methods, while preserving the volatile terpene content. On the other hand, inflorescences subjected to traditional drying were highly infested by Alternaria alternata and also revealed low infestation of Botrytis cinerea. In the high-THC chemovar ("240"), controlled N2 and atm drying conditions preserved THCA concentration as compared to the initial time point (t0). On the other hand, in the hybrid chemovar ("Gen12") all of the employed drying conditions preserved THCA and CBDA content. The optimal drying conditions for preserving monoterpenes and sesquiterpenes in both chemovars were C5O5 (5% CO2, 5% O2, and 90% N2) and pure N2, respectively. The results of this study suggest that each chemovar may require tailored drying conditions in order to preserve specific terpenes and cannabinoids. Controlled atmosphere drying chambers could offer a cost-effective, fast, and efficient drying method for preserving cannabinoids and terpenes during the drying process while reducing the risk of mold growth.

Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology.

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 µL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.

Paper-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the identification of latent Colletotrichum in harvested fruit.

Paper-based analytical devices (PADs) have gained enormous attention because of their low-cost, simple fabrication, and portability. Here, we propose a paper-based device for performing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with real-time simultaneous detection of C. gloeosporioides latent infections in tomatoes. RT-LAMP-based PAD platform comprises a paper substrate on which the DNA amplification reaction occurs. Among different types of tested papers, cellulose membrane (grade 4) enabled effective visualization of the amplification result. The assay was found highly selective for the latent stage of C. gloeosporioides with lower limit of detection (LOD) of 0.5 pg of total extracted RNA. The developed assay generated the results within 40 min and hence can be efficiently employed for identifying C. gloeosporioides in resource-limited settings.

High-Throughput Bioassay for Detection of Latent Fungi in Postharvest Produce.

Enormous fresh agricultural produce is wasted annually due to rots caused by pathogenic microorganisms. Most pathogenic fungi attack the harvested produce by penetrating the fruit at the field and remaining quiescent or latent until the fruit ripens or senescence. In this work, a recently developed simple, cost-effective, and high-throughput 96-well plate-based assay was applied to determine the presence of pathogenic fungi in their latent stage. The surface strands immobilized on the 96-well plate, only with the presence of the complementary RNA marker (enoyl-CoA hydratase (ECH)) of the latent fungal-pathogen Colletotrichum gloeosporioides will create a complex with the target and reporter (labeled with the horseradish peroxidase (HRP) enzyme) strands for positive signal generation. The developed assay demonstrated 3.1-fold higher specificity for the latent marker (ECH) of C. gloeosporioides compared to latent markers of other pathogenic fungi. A 2 nM detection limit of target strands was demonstrated, showing a high plate sensitivity, and was further validated with biological samples extracted from latent infection in tomato fruit. The developed assay provides a new economical tool for detecting the presence of latent RNA markers of pathogenic fungi in agricultural produce, ultimately improving postharvest decision-making and reducing postharvest losses.

Extract from the Macroalgae Ulva rigida Induces Table Grapes Resistance to Botrytis cinerea.

Fungal pathogens are a central cause of the high wastage rates of harvested fruit and vegetables. Seaweeds from the genus Ulva are fast-growing edible green macroalgae whose species can be found on the shore of every continent, and therefore present a resource that can be utilized on a global scale. In this study, we found that the application of ulvan extract, a sulfated polysaccharide extracted from Ulva rigida (1000 mg/L), elicited table grapes defense and reduced the incidence and decay area of Botrytis cinerea by 43% and 41%, respectively. In addition, compared to the control group at two days post-treatment, ulvan extract elicited a variety of defense-related biomarkers such as a 43% increase in the activity of reactive oxygen species, 4-fold increase in the activity of catalase, 2-fold increase in the activity of superoxide dismutase and 1.4-fold increase in the activity of chitinase. No increase was observed in phenylalanine ammonia-lyase activity, and the treatment did not affect fruit quality parameters such as the pH levels, sugar levels, and titratable acidity of grapes. These results illustrate the potential of ulvan extract to naturally induce the plant defense response and to reduce postharvest decay.

eDNA-Mediated Cutaneous Protection Against UVB Damage Conferred by Staphylococcal Epidermal Colonization.

The human skin is a lush microbial habitat which is occupied by a wide array of microorganisms. Among the most common inhabitants are Staphylococcus spp., namely Staphylococcus epidermidis and, in ≈20% of healthy individuals, Staphylococcus aureus. Both bacteria have been associated with cutaneous maladies, where they mostly arrange in a biofilm, thus achieving improved surface adhesion and stability. Moreover, our skin is constantly exposed to numerous oxidative environmental stressors, such as UV-irradiation. Thus, skin cells are equipped with an important antioxidant defense mechanism, the Nrf2-Keap1 pathway. In this work, we aimed to explore the morphology of S. aureus and S. epidermidis as they adhered to healthy human skin and characterize their matrix composition. Furthermore, we hypothesized that the localization of both types of bacteria on a healthy skin surface may provide protective effects against oxidative stressors, such as UV-irradiation. Our results indicate for the first time that S. aureus and S. epidermidis assume a biofilm-like morphology as they adhere to ex vivo healthy human skin and that the cultures' extracellular matrix (ECM) is composed of extracellular polysaccharides (EPS) and extracellular DNA (eDNA). Both bacterial cultures, as well as isolated S. aureus biofilm eDNA, conferred cutaneous protection against UVB-induced apoptosis. This work emphasized the importance of skin microbiota representatives in the maintenance of a healthy cutaneous redox balance by activating the skin's natural defense mechanism.

Efficiency of Bacillus subtilis metabolism of sugar alcohols governs its probiotic effect against cariogenic Streptococcus mutans.

Bacillus subtilis is a Gram-positive probiotic bacterium that successfully colonises plant roots due to its ability to utilise various sugars. The vast probiotic potential of B. subtilis has been recently demonstrated in numerous host organisms under different environmental conditions. We examined the probiotic potential of B. subtilis against the pathogenic bacterium Streptococcus mutans, which is involved in various oral disorders due to its robust biofilm-forming capability. B. subtilis cells attenuated biofilm formation by S. mutans during their dual growth in the presence of sugar alcohols. Transcription of genes encoding key enzymes in the metabolism of sugar alcohols by B. subtilis were highly induced. Moreover, growth-curve analysis suggested that B. subtilis is more efficient at early utilising sugar alcohols than S. mutans, as supported by the bacterial metabolic activity rates. Similarly, a comparison of secondary metabolites of mono and mixed cultures of B. subtilis and S. mutans indicated that B. subtilis is more active metabolically in the dual culture. Finally, knock-out mutations of the genes encoding key enzymes in the central metabolic pathway significantly reduced B. subtilis' ability to mitigate biofilm formation by S. mutans. We conclude that effective metabolism of sugar alcohols by B. subtilis reinforces the probiotic potential of this bacterium against pathogenic species such as S. mutans.

Proteolytic Activity of Bacillus subtilis upon κ-Casein Undermines Its "Caries-Safe" Effect.

Milk is believed to be a relatively "caries-safe" food. This belief relies on the fact that caseins, which constitute around 80% of milk's protein content, were found to inhibit the adhesion of Streptococcus mutans to enamel and, therefore, decrease biofilm formation. While S. mutans is considered a leading cause of dental disorders, Bacillus subtilis is a non-pathogenic foodborne bacterium, frequently contaminating milk and its products. This study aimed to investigate the effects of dairy-associated foodborne bacteria such as B. subtilis on biofilm formation by S. mutans in the presence of casein proteins. Our results indicate that there is a significant decrease in total biofilm formation by S. mutans exposed to a casein protein mixture in a mono-species culture, whereas, in the co-culture with B. subtilis, an inhibitory effect of the caseins mixture on S. mutans biofilm formation was observed. Proteolytic activity analysis suggested that B. subtilis is capable of breaking down milk proteins, especially κ-casein, which enables biofilm formation by S. mutans in the presence of milk caseins. Therefore, these findings may challenge the assumption that milk is "caries-safe", especially in a complex microbial environment.

Harvesting Mango Fruit with a Short Stem-End Altered Endophytic Microbiome and Reduce Stem-End Rot.

Stem-end rot (SER) is a serious postharvest disease of mango fruit grown in semi-dry area. Pathogenic and non-pathogenic microorganisms endophytically colonize fruit stem-end. As fruit ripens, some pathogenic fungi switch from endophytic colonization to necrotrophic stage and cause SER. Various pre/post-treatments may alter the stem-end community and modify SER incidence. This study investigates the effects of harvesting mango with or without short stem-end on fruit antifungal and antioxidant activities, the endophytic microbiome, and SER during fruit storage. Our results show that harvesting mango with short stem significantly reduced SER during storage. At harvest, fruit harvested with or without stem exhibit a similar microorganisms community profile. However, after storage and shelf life, the community of fruit without stem shifted toward more SER-causing-pathogens, such as Lasiodiplodia, Dothiorella, and Alternaria, and separated from the community of fruit with stem. This change correlated to the high antifungal activity of stem extract that strongly inhibited both germination and growth of Lasiodiplodia theobromae and Alternaria alternata. Additionally, fruit that was harvested with stem displayed more antioxidant activity and less ROS. Altogether, these findings indicate that harvesting mango with short stem leads to higher antifungal and antioxidant activity, retaining a healthier microbial community and leading to reduced postharvest SER.

The Adaptive Morphology of Bacillus subtilis Biofilms: A Defense Mechanism against Bacterial Starvation.

Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or "wrinkles" that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels' interiors which supports their possible role in the transport of molecules throughout biofilm structures.

Cell wall associated protein TasA provides an initial binding component to extracellular polysaccharides in dual-species biofilm.

Many bacteria in biofilm surround themselves by an extracellular matrix composed mainly of extracellular polysaccharide (EP), proteins such as amyloid-like fibers (ALF) and nucleic acids. While the importance of EP in attachment and acceleration of biofilm by a number of different bacterial species is well established, the contribution of ALF to attachment in multispecies biofilm remains unknown. The study presented here aimed to investigate the role of TasA, a precursor for ALF, in cell-cell interactions in dual-species biofilms of Bacillus subtilis and Streptococcus mutans. Expression of major B. subtilis matrix operons was significantly up-regulated in the presence of S. mutans during different stages of biofilm formation, suggesting that the two species interacted and modulated gene expression in each other. Wild-type B. subtilis expressing TasA adhered strongly to S. mutans biofilm, while a TasA-deficient mutant was less adhesive and consequently less abundant in the dual-species biofilm. Dextran, a biofilm polysaccharide, induced aggregation of B. subtilis and stimulated adhesion to S. mutans biofilms. This effect was only observed in the wild-type strain, suggesting that interactions between TasA and dextran-associated EP plays an important role in inter-species interactions during initial stages of multispecies biofilm development.

Bacillus subtilis Biofilm Development - A Computerized Study of Morphology and Kinetics.

Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of B. subtilis colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm - a central "core" region and an "expanding" region surrounding it. Our results demonstrate that complex biofilm of B. subtillis grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.

The LuxS Based Quorum Sensing Governs Lactose Induced Biofilm Formation by Bacillus subtilis.

Bacillus species present a major concern in the dairy industry as they can form biofilms in pipelines and on surfaces of equipment and machinery used in the entire line of production. These biofilms represent a continuous hygienic problem and can lead to serious economic losses due to food spoilage and equipment impairment. Biofilm formation by Bacillus subtilis is apparently dependent on LuxS quorum sensing (QS) by Autoinducer-2 (AI-2). However, the link between sensing environmental cues and AI-2 induced biofilm formation remains largely unknown. The aim of this study is to investigate the role of lactose, the primary sugar in milk, on biofilm formation by B. subtilis and its possible link to QS processes. Our phenotypic analysis shows that lactose induces formation of biofilm bundles as well as formation of colony type biofilm. Furthermore, using reporter strain assays, we observed an increase in AI-2 production by B. subtilis in response to lactose in a dose dependent manner. Moreover, we found that expression of eps and tapA operons, responsible for extracellular matrix synthesis in B. subtilis, were notably up-regulated in response to lactose. Importantly, we also observed that LuxS is essential for B. subtilis biofilm formation in the presence of lactose. Overall, our results suggest that lactose may induce biofilm formation by B. subtilis through the LuxS pathway.