Green Mold Research Articles

Antifungal Action of Metallic Nanoparticles Against Fungicide-Resistant Pathogens Causing Main Postharvest Lemon Diseases

Postharvest fungal diseases are the main cause of economic losses in lemon production. The continued use of synthetic fungicides to control the diseases favors the emergence of resistant strains, which encourages the search for alternatives. The aim of this study was to assess the efficacy of metallic nanoparticles (NPs) as antifungal agents against local isolates of Penicillium digitatum and Penicillium italicum, each of them in a fungicide-sensitive and -resistant version, and a Geotrichum citri-aurantii isolate. NPs of ZnO, CuO, and Ag were synthesized and characterized by spectroscopy and microscopy, presenting average sizes < 25 nm and spherical shapes. ZnO-NPs did not present antifungal activity at the assayed conditions, while the minimum fungicidal concentrations (MFCs) were 1000 and 10 µg mL−1 for CuO-NPs and Ag-NPs, respectively. The NPs’ antimicrobial action included conidial membrane permeability and strong intracellular disorganization. Moreover, the Ag-NPs reduced green mold incidence on inoculated lemons when applied to the fruit. Taken together, Ag-NPs were effective in inhibiting both fungicide-sensitive and -resistant isolates of the main lemon postharvest pathogens, suggesting their potential use as an alternative approach.

Manipulating button mushroom casing affects the disease dynamics of blotch and green mold disease

Productive cultivation of the button mushroom (Agaricus bisporus) relies on the use of selective substrates and effective disease management. In extending our previous work on manipulating the developmental microbiome (devome), this study employs the strategy of substrate passaging to explore its effects on crop outcomes and disease dynamics. Here we subjected the casing substrate to ten cycles of passaging. This manipulated substrate stimulated early pinning (primordia formation) by at least three days. Passaged casing also altered disease dynamics when challenged with two commercially important A. bisporus pathogens, Pseudomonas tolaasii (causing bacterial blotch) and Trichoderma aggressivum f. aggressivum (responsible for green mold). Passaged casing had a suppressive effect on blotch disease and a conducive effect on green mold disease. Blotch suppression resulted in a significantly higher yield of asymptomatic mushrooms in all three mushroom harvests (flushes) and in the overall crop yield. Blotch severity was also significantly reduced in passaged casing compared to standard casing due to a lower yield of mushrooms with the highest degree of blotch disease expression. Green mold disease expression was markedly higher in passaged casing, leading to lower numbers of asymptomatic mushrooms. Zones where no growth of hyphae or mushrooms were also observed in passaged casing due to green mold disease pressure. The stimulating effect of passaged casing on mushroom development and the dynamic outcomes for disease challenge from two distinct, commercially damaging diseases, demonstrates the potential for passaged casing to be used as material to study more sustainable mushroom production and disease management practices.

Carboxymethyl cellulose-induced Cryptococcus laurentii improves disease resistance and regulates phenylpropane and reactive oxygen metabolism in grapefruit

Green mould disease poses a significant threat to the citrus industry. Cryptococcus laurentii can stimulate the fruit defence system, whereas the use of antagonistic yeast alone demonstrates limited efficacy. This study investigated the molecular mechanisms of C. laurentii cultured with carboxymethyl cellulose (CMCC. laurentii), and evaluated the effects of CMCC. laurentii on phenylpropane and reactive oxygen metabolism in grapefruit fruit. Transcriptome analysis revealed that the upregulation of gene expression associated with yeast growth and antagonistic ability occurred in CMCC. laurentii after 72 h cultivation. Meanwhile, CMCC. laurentii reduced lesion diameter and disease incidence in fruit. This treatment promoted phenylpropane metabolism by activating PAL, C4H, 4CL, POD, and PPO and increasing the secondary metabolites. CMCC. laurentii also activated the AsA-GSH cycle, enhanced the activities of SOD and CAT, and reduced the accumulation of H2O2 and O2•-. The results suggested that CMCC. laurentii maintained high postharvest fruit quality in grapefruit fruit by elevating the phenylpropane and reactive oxygen metabolism.

Effective control of citrus green mold using plasma-activated water with hydrogen peroxide vapor: A non-thermal approach

In this study, a novel approach for controlling Citrus sinensis Salustiana green mold disease in oranges was developed using plasma-activated water (PAW) generated by a Surface Dielectric Barrier Discharge (SDBD) system reinforced with H2O2 cold vapor. Penicillium digitatum, the primary cause of orange mold, was the target of the treatment. The efficiency of the setup was investigated in three stages. First, a suspension of P. digitatum spores was directly treated with Ar/H2O2 plasma to estimate the optimal plasma exposure time required for maximum fungal inactivation. Second, the impact of Argon/H2O2 plasma-treated water (PTW), generated from various plasma exposure times, on mold growth on orange peels was examined. For this, orange peels were placed in Ar/H2O2-PTW for different durations, and mold occurrence was monitored. Finally, the effect of PTW, generated from various Ar/H2O2 plasma exposure times, on mold growth on whole oranges was investigated. Oranges were treated with Ar/H2O2-PTW for different durations, and mold occurrence was analyzed. The results demonstrated that a 20-min treatment of oranges with PTW produced from a 420-s Ar/H2O2 plasma exposure time led to complete inactivation of P. digitatum. This study showed that this hybrid scenario can be effectively used for fungal decontamination in the food industry.

Harnessing Nature's Potential: Innovating Antibacterial Cream through Garlic and Clove Extracts

The onset of the antibiotics period was characterised by the clinical implementation of penicillin, the inaugural antibiotic, unearthed in 1928 by Alexander Fleming in the green mould Penicillium notatum. Garlic and clove have long been used as traditional treatments for different diseases, including infections. Their antibacterial qualities have been attributed to their sulfur-containing components, such as allicin and ajoene in garlic, and eugenol and isoeugenol in clove. In this study, we wanted to produce a novel antibacterial cream leveraging the synergistic properties of garlic and clove extracts. The cream was manufactured utilizing a simple and scalable technique, and its antimicrobial activity was assessed against a panel of microbes present in our surrounding. The results indicated that the cream had considerable antibacterial action against all tested microorganisms, with minimum inhibitory concentrations (MICs) comparable to conventional antibiotics. This study establishes a groundbreaking, ecologically-oriented method for safeguarding and healing the skin. The tropical antimicrobial cream provides a secure and efficient substitute for traditional antimicrobials that may have harsh effects, while also including the nourishing and rejuvenating qualities of tropical botanicals. This groundbreaking research marks a significant advancement in the realm of natural, holistic skin health solutions. The tropical antimicrobial cream stands as a potent alternative to conventional, potentially harsh antimicrobials, offering a safe and effective way to combat skin infections and irritations. Furthermore, its incorporation of nourishing and restorative tropical botanicals transcends mere protection, promoting overall skin health and well-being. Beyond the individual benefits, this innovation holds immense potential for promoting sustainable practices within tropical ecosystems by leveraging resources responsibly and ethically. This paves the way for a future where nature's wisdom empowers us to safeguard our skin with gentle effectiveness, while simultaneously cherishing and preserving the biodiversity that holds the key to our well-being.

Citrus bliss: potassium, sodium, and calcium silicates secrets for post-harvest diseases of fruit defense

Biotic stress significantly challenges the global citrus industry. Major post-harvest issues include diseases caused by Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, Alternaria alternata, and Phytophthora citrophthora. The negative impact of chemical fungicides on the environment and health necessitates eco-friendly alternatives. This study examines the effectiveness of sodium, potassium, and calcium silicates against common citrus diseases. In vitro tests evaluated mycelial growth inhibition using silicate concentrations from 0 to 10,000 ppm after 7 days at 25°C. Sodium silicate showed the highest efficacy, completely inhibiting P. digitatum and P. italicum at 2000 ppm. Potassium and calcium silicates achieved 100% inhibition against Penicillium spp. at a concentration of 1%. In vivo tests on Sidi Aissa clementines assessed the preventive and curative effects of 1, 2, and 6% silicate salt solutions. Sodium silicate prevented 41% of brown rot, 72% of sour rot, and 100% of green mold at 6%. Calcium silicate at 6% significantly reduced blue mold and black rot by 32% and 74%, respectively. Sodium silicate was most effective in curative treatments, suggesting its potential as a pre- or post-harvest spray to control P. digitatum, P. italicum, and G. citri-aurantii.

In Vitro Antagonistic Activity of Plant Growth Promoting Rhizobacteria Against Aggressive Biotypes of the Green Mold.

During the cultivation of button mushrooms, the green mold epidemic, which causes a decrease in productivity, is a very important problem. The environmental harm of chemicals used in the control of such epidemics and the demand of consumers for organic products without chemicals have brought environmentally friendly biological control to the fore. Biological control can be achieved by the use of antagonistic microorganisms and their metabolites. In this study, the effectiveness of Bacillus spp. and Pseudomonas spp. for the biological control of the aggressive biotypes of the green mold disease agent Trichoderma aggressivum strains was examined in vitro. For this purpose, the antifungal effects of Bacillus spp. and Pseudomonas spp. against T. aggressivum strains were examined by in vitro dual culture test. Afterward, the antifungal activity of Bacillus spp. metabolites was assessed further using the agar well diffusion method. Then, it was determined whether the bacterial strains showing antifungal activity showed antagonistic activity against A. bisporus. Although none of the Pseudomonas spp. showed antifungal activity against T. aggressivum strains, most of the Bacillus spp. were found to have high activity. It has been concluded that Bacillus sp. Ö-4-82 may be potential biological control agent for button mushroom cultivation.

Effect of Neem Leaf Powder Supplementation in Casing Mixture on the Growth and Yield Parameters of white Button Mushroom [Agaricus bisporus (Lange) Imbach

Green mould caused by Trichoderma harzianum is a severe threat of white button mushroom [Agaricus bisporus (Lange) Imbach] cultivation causing heavy crop losses. The present experiment was conducted to manage the green mould fungus Trichoderma harzianum using neem leaf extracts. In vitro, results showed maximum mycelial growth inhibition of 64.59 % in T. harzianum using neem leaf extract at 4.5% concentration. Neem leaf extracts at 4.5% concentration showed maximum efficacy against pathogens. Further, an In vivo experiment was conducted to evaluate neem leaf powder at different concentrations @0.5%, 1.5%, 2.5%, 3.5% and 4.5% supplemented in the casing mixture combination viz., Farm yard manure (FYM) + Cocopeat + Sawdust (1:1:1) to monitor the growth and yield aspects of white button mushroom. Among all treatments used in the study, the results revealed that minimum average time taken for completion of spawn run (15.14 days) and pinhead initiation (18.28 days) as well as maximum number of fruiting bodies (14.71), fruiting bodies weight (22.54g), stalk length (2.81 cm) stalk diameter (1.85 cm) and pileus diameter (5.64 cm), and were recorded in T2 [FYM + Cocopeat + Sawdust (1:1:1) + neem leaf powder @ 1.5%]. Results have shown that T2 [FYM + Cocopeat + Sawdust (1:1:1) + neem leaf powder @ 1.5%] had the highest yield (470.09 g) and biological efficiency (27.11%).

Exploration of antimicrobial activities and mechanisms of biocontrol agent Serratia nematodiphila BC-SKRU-1 against Penicillium digitatum in tangerine fruit

The isolate BC-SKRU-1, identified as Serratia nematodiphila from fungal culture contamination, was investigated for its biocontrol potential against Penicillium digitatum in vitro and on tangerine fruit, with a focus on its mechanisms of action. In vitro tests on PDA plates revealed BC-SKRU-1's broad-spectrum antifungal activity, achieving up to 74.68% inhibition against seven plant pathogenic fungi, including P. digitatum NKP4321. Bacterial culture filtrates from BC-SKRU-1 (BCF BC-SKRU-1) at 5–10% concentrations inhibited NKP4321 mycelial growth in PDB medium (78.41–83.68%) more effectively than in PDA medium (46.20–72.61%), with complete suppression at 15% (v/v) in both media. BCF BC-SKRU-1's efficacy was comparable to chemical fungicides such as propiconazole®, prochloraz®, and mancozeb®. Notably, BCF BC-SKRU-1 retained its antifungal activity after dilution (1/1000), autoclaving (at 121 °C), and storage (at −20 °C). In vivo evaluations on tangerine fruit showed significant reduction in the severity of postharvest green mold disease with BC-SKRU-1 treatments, especially at a concentration of 108 CFU mL−1. Both preventive and curative applications were effective, with curative treatments being more successful. Mechanistic studies indicated that BCF BC-SKRU-1 reduces intracellular ergosterol content, compromises plasma membrane integrity, and attenuates antioxidant defense activities (SOD, CAT, GSH, GSSG, and GSH/GSSG ratio). These findings highlight BC-SKRU-1 and its metabolites as promising biocontrol agents against green mold in tangerine fruits and provide insights into their antifungal mechanisms.

Green mould contamination of Pleurotus pulmonarius cultivation in Malaysia: Unravelling causal agents and water source as critical factors

Green mould contamination causes a significant challenge to mushroom growers in Malaysia leading to reduced yields and economic losses in the widely cultivated and marketed edible grey oyster mushroom, Pleurotus pulmanorius. This study aimed to identify the causal agents of green mould contaminants and determine the critical points in the cultivation process in the farm that contribute to green mould contamination. Samples of mushroom substrate (sawdust), spawn substrate (corn), environmental sources and tools were collected at different stages of mushroom cultivation. As results, the causal agents of green mould contamination were identified as Trichoderma pleuroti, T. harzianum and T. ghanese. Prior to steam pasteurisation and after steam pasteurisation, the spawn substrate and mushroom substrate were found to be free of Trichoderma. However, Trichoderma was detected in water, air within the production house and on cleaning tools. This findings suggests that water could serve as the source of green mould introduction in mushroom farms, while cultivation practices such as watering and scratching during the harvesting cycle may contribute to adverse green mould. Understanding these critical points and causal agents provides information to mitigate the green mould contamination throughout the grey oyster mushroom cultivation process.

Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit.

To develop novel fungicides for controlling postharvest fungal diseases in citrus fruits, 12 essential oil (EO)-based thiosemicarbazones compounds, termed hydrazine-carbothioamide, were prepared according to the condensation method. In vitro assays showed that compound 13j exhibited the strongest antifungal activity (minimum inhibitory concentration [MIC] = minimum fungicidal concentration [MFC] = 0.0125 mg/mL) against Penicillium digitatum. An in vivo study revealed that 5 × MFC of compound 13j can effectively mitigate the green mold incidence of citrus fruit inoculated with P. digitatum, as well as fruit rot during natural storage, at a level comparable to that of the chemical fungicide prochloraz. Throughout this process, fruit quality was maintained. The hemolysis assay showed that these thiosemicarbazone compounds have good biocompatibility and that their safety is comparable to that of prochloraz. The antifungal activity of compound 13j was attributed to membrane damage, as confirmed using scanning electron microscopy (SEM), Calcofluor white (CFW) staining, propidium iodide (PI) staining, Fourier transform-infrared (FT-IR) spectroscopy, optical density (OD)260, and relative conductivity assays. Collectively, our results indicate that compound 13j can be used as an antifungal agent to control the postharvest decay of citrus fruits.

Characterizations of the pullulan/sodium alginate coatings incorporated with sodium silicate and EDTA-2Na and the antifungal effect on citrus green mold

Citrus has suffered great losses from postharvest fungal diseases. The strategy of polysaccharide-based coating combined with antimicrobial salts was introduced to preserve citrus and control green mold. Sodium silicate and ethylene diamine tetraacetic acid-2Na (EDTA-2Na) (SE) were incorporated with pullulan (PUL)/sodium alginate (SA) solutions to fabricate PSA-SE systems. The results indicated that the PSA-SE films had good biocompatibility based on hydrogen bonds and showed uniform and dense. The molecular of PUL and SA were assembled through entanglement to form a network structure and the surface roughness of the PSA-SE films increased with the addition of SA. The P3SA2-SE film indicated appropriate tensile strength and elongation at break, as well as the lowest water vapor permeability. The carbon dioxide and oxygen permeability and dissolution of P3SA2-SE film were at low values. The P3SA2-SE film had the highest water contact angle (66°). With the addition of SA, the absorbance of the PSA-SE films increased, which was conducive to light blocking. The antifungal experiments revealed that the P3SA2-SE coating showed a significant effect on controlling citrus green mold, especially in Citrus sinensis Osbeck ‘Lane Late’ almost completely inhibited Penicillium digitatum (P44). This study will promote the development of citrus postharvest disease control.

Green Silver Nanoparticles and Their Activity as Antifungal Agents Against Penicillium digitatum Causing Green Mould of Citrus

AbstractThe green synthesis of silver nanoparticles (SNPs) using aqueous extracts of five plants viz., Ocimum americanum, Azadirachta indica, Allium sativum, Eucalyptus hybrida, Zingiber officinale were evaluated for their antifungal property against Penicillium digitatum, a green mould pathogen of citrus fruits. The five plant species were selected from a screening of aqueous extracts from 16 plant species. The efficacy of aqueous extracts, at 50 % concentration, ranged from 14.44–68.52 % inhibition in mycelia growth of P. digitatum. Among SNPs, O. americanum was found most efficacious, at 1000 ppm concentration, with 80.7 % mycelial inhibition followed by A. indica with 77.4 % mycelial growth inhibition of fungal pathogen. Further, physical and structural characterization of SNPs of O. americanum and A. indica was carried out. The characterization analysis executed via transmission and scanning electron microscope revealed size distribution and nanoparticles were spherical to irregularly shaped ranging from 22.8–250 nm in O. americanum and 26.5–500 nm in A. indica. In UV‐visible spectrophotometer, the absolute absorption peak was recorded at 448 nm and 442 nm for O. americanum and A. indica, respectively. FTIR analysis of SNPs of A. indica and O. americanum confirmed the presence of carboxyl, amide group and secondary amines which were the main constituent of proteins. The novelty of the present study is in the green synthesis of silver nanoparticles with potential in the management of postharvest pathogens of fruit crops. The synthesis of SNPs used in this study is simple, environmentally benign and cost‐effective using aqueous extract of native plants.

Pullulan-based coatings carrying biocontrol yeast mixed with NaCl to control citrus postharvest disease decays

The decline in postharvest citrus quality due to fungal infections necessitates innovative packaging solutions. This study presents a pullulan-based edible film (PBYFs) with biocontrol yeasts, designed to dissolve in both liquid and soil. This film is capable of enveloping entire citrus fruits, effectively managing postharvest diseases, and extending their shelf life. The formulation of PBYFs includes NaCl 0.1 M, Kloeckera apiculata 34–9 at 1.0 × 107 CFU mL−1, pullulan 3 % w/v, SA 0.5 % w/v, and glycerin 1 % w/v. Our experiments, conducted on eight citrus varieties, demonstrated that PBYFs significantly reduced the occurrence of green and blue molds, sour rot, and anthracnose in vivo. Moreover, PBYFs-coated fruits exhibited an extended shelf life without compromising the quality parameters such as weight loss, TSS (total soluble solids), TA (titratable acidity), VC (vitamin C), or the accumulation of off-flavor volatiles. This research presents a promising approach for creating scalable, cost-effective, and environmentally sustainable biodegradable antifungal packaging systems for citrus fruits.

Correction to: Improvement of post-harvest quality of Balady lime fruit with Aloe vera gel and tea tree oil against green mold disease caused by Penicillium digitatum

Correction to: Improvement of post-harvest quality of Balady lime fruit with Aloe vera gel and tea tree oil against green mold disease caused by Penicillium digitatum

Combination of Cinnamaldehyde/β-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit

The essential oil and β-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-β-cyclodextrin inclusion complex (β-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of β-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of β-CD to form the inclusion complex. β-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that β-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC β-CDCA treated hyphae. This implied that β-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that β-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, β-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Altering Microbial Communities in Substrate to Stimulate the Growth of Healthy Button Mushrooms

Green mould, caused by Trichoderma aggressivum, is one of the major fungal diseases of button mushrooms. The main problems in chemical disease control include a lack of effective agents, the occurrence of pathogen resistance to pesticides, and the harmful impact on the environment. In an attempt to find a solution, the interaction between two beneficial microorganisms, Bacillus amyloliquefaciens B-241 (an antifungal agent) and Streptomyces flavovirens A06 (a yield stimulant), was investigated in vivo. The synergy factor (SF) was calculated as a ratio between the observed and expected impact on the yield or efficacy of disease suppression after artificial inoculation with T. aggressivum. The highest control of T. aggressivum was achieved by joint application of the two beneficial microorganisms. The additive interaction between microorganisms in efficacy against the pathogen was revealed. The largest yield was obtained in mushroom beds sprayed with the two beneficial microorganisms combined (B-241 80% and A06 20%). Regarding the impact on the yield, synergistic interaction between the two microorganisms was confirmed (SFs were 1.62 or 1.52). The introduction of optimized microbial combinations could create new possibilities for biorational edible mushroom protection, with improved yield and quality and reduced risks to human health and the environment.

Postharvest biocontrol ability and involved mechanism of volatile organic compounds from Serratia nematodiphila BC-SKRU-1 against Penicillium digitatum tangerine fruit

Volatile organic compounds (VOCs) emitted by microorganisms offer a promising strategy for managing green mold disease in citrus fruits caused by Penicillium digitatum. This study evaluated the biocontrol efficacy of VOCs produced by Serratia nematodiphila BC-SKRU-1 (BC-SKRU-1 VOCs) against green mold in postharvest tangerine fruits, targeting P. digitatum NKP4321. Our findings demonstrated that BC-SKRU-1 VOCs exhibited a broad spectrum of activity against seven plant pathogens, with the highest efficacy observed against NKP4321 (68.21%). A concentration of 106 CFU mL−1 for VOC production by BC-SKRU-1 completely inhibited the mycelial growth of NKP4321, whereas a concentration of 107 CFU mL−1 was required to fully inhibit spore germination. The application of four plates of BC-SKRU-1 VOCs at 108 CFU mL−1 significantly reduced the severity of green mold disease, with optimal biocontrol achieved through continuous fumigation during storage. Notably, 2-nonanol, identified as the predominant component among the 20 VOCs using HS-SPME/GC-MS, exhibited superior antifungal activity both in vitro and in vivo. It effectively decreased ergosterol production and disrupted plasma membrane integrity in NKP4321 cells. Furthermore, 2-nonanol altered the cellular content of non-enzymatic antioxidants (e.g., GSH, GSSG) and enzymatic antioxidants (e.g., CAT, SOD), elucidating its comprehensive antifungal mechanism. These findings highlight the potential of BC-SKRU-1 VOCs, particularly 2-nonanol, as effective biocontrol agents for enhancing postharvest fruit preservation by combating fungal pathogens.

Increasing the efficiency of CRISPR/Cas9-mediated genome editing in the citrus postharvest pathogen Penicillium digitatum

BackgroundPenicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use.ResultsIncreasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54–83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol.ConclusionGenome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.

Bioactive molecules isolated from Cymbopogon flexuosus and Monarda citriodora essential oils: Chemical profiling, antimycotic potential, and molecular docking studies against green and blue molds of Kinnow

Among the most alarming postharvest fungal pathogens affecting the shelf life of citrus fruits are green (Penicillium digitatum) and blue (Penicillium italicum) molds. This in vitro study was aimed to evaluate the essential oils (EOs) of Cymbopogon flexuosus (CF) and Monarda citriodora (MC) and their principal components against these fungal pathogens. The composition of selected EOs was analyzed using Gas Chromatography- Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID). Citral (83.81%) and thymol (62.51%) were identified as major components and were isolated by column chromatography from CF and MC EO respectively followed by characterization using spectral techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR) and Carbon Nuclear Magnetic Resonance Spectroscopy (13C-NMR). Among EOs and their major components, thymol displayed promising potential against both Penicillium species with MIC values of 50 and 80 mg L-1 respectively. Molecular docking studies also confirmed the stable binding interaction of both components, particularly thymol with the active sites of target proteins i.e. 14-α-demethylase and chitin synthase of P. digitatum and P. italicum with the docking scores of -5.07, -5.41 kcal/mol and -5.78, -5.23 kcal/mol respectively. Optical microscopy studies revealed hyphal thinning and fragmentation at frequent sites with contraction of cytoplasmic content indicating incipient plasmolysis. The tested principal components, citral and thymol were predicted to exhibit similar mechanism of action to that of synthetic recommended molecules as revealed by molecular docking. Therefore, these findings provide empirical support to explore thymol as a promising eco-friendly antifungal agent for postharvest application in Kinnow.