Reduction In Fungal Growth Research Articles

Antifungal potential of punicalagin against Cryptococcus neoformans species complex.

ABSTRACTThis study evaluated the antifungal activity and cytotoxicity profile of the ellagitannin punicalagin, a compound extracted from the L. pacari A. St.-Hil (Lythraceae) leaf, against Cryptococcus neoformans species complex. Minimum inhibitory concentrations (MIC) were checked using the broth microdilution method. Minimum fungicidal concentrations (MFC) and time of death were used to confirm the antifungal activity of the compound. The in vitro cytotoxicity of punicalagin was tested in BALB/c3T3 fibroblasts and A549 human lung cancer cell line, while the hemolytic potential was tested on sheep erythrocytes. The morphological changes induced in yeast strains by the presence of punicalagin were also analyzed. Tested on eight isolates of the C. neoformans complex punicalagin showed MIC of 0.5 to 4.0 μg/mL and MFC> 256 μg/mL. Punicalagin also demonstrated a good growth inhibitory activity in time-kill curves, but it was not able to achieve a statistically significant reduction of fungal growth suggesting a fungistatic effect of the compound. In vitro cytotoxicity studies using the two cell lines showed that punicalagin has low activity on these cells and no activity on sheep erythrocytes. Morphological changes were seen in the yeasts strains studied when treated with punicalagin. Therefore, punicalagin is a potential antifungal for important pathogenic yeasts and presents a low cytotoxicity profile associated with no hemolytic effects.

Efficacious fungicidal potential of composite derived from nano-aggregates of Cu-Diclofenac complexes and ZnO nanoparticles

The composite-based formulations have appeared as prominent “antimicrobial materials” because it provide superior antimicrobial efficacy than single component based therapeutic formulations. Herein, we report the synergistically enhanced antifungal activity of the composite developed from self-assembled nano-aggregates of copper complexes of non-steroidal anti-inflammatory drug, diclofenac (Cu-Dc) in combination with zinc oxide (ZnO) nanoparticles. The composites (ZnO-Cu-Dc) were prepared at different weight ratios (Cu-Dc/ZnO; 1:0.125–1:2) and characterized thoroughly by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), fourier-transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS) measurements which revealed them to be nanostructured aggregates. The composite mediated treatments showed substantially increased zones of inhibition in agar well-diffusion method against a human pathogenic fungus (Candida albicans) and three plant pathogenic fungi (Penicillium funiculosum, Aspergillus niger and Fusarium oxysporum). Notably, the individual treatments using the respective concentrations of ZnO nanoparticles did not show any inhibitory activity against all the fungal strains. Optical density measurements profiled maximum reduction in fungal growth for composite treatment when compared to that of ZnO nanoparticles and Cu-Dc complexes alone. The morphological analysis of fungal cell growth by SEM and optical microscopic studies evidenced maximum deformation of biofilm mass for composite mediated treatment. Concisely, the ZnO-Cu-Dc, a composite system has been developed which can be utilized as an effective formulation against fungal infections.

Involvement of MaSom1, a downstream transcriptional factor of cAMP/PKA pathway, in conidial yield, stress tolerances, and virulence in Metarhizium acridum.

Flo8/Som1, which functions downstream from the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway, plays important roles in hyphal development, spore formation, and virulence in yeast and several filamentous fungi. However, the functions of Som1 in entomopathogenic fungi are still a mystery. In this study, MaSom1, a Flo8/Som1 homolog, was identified and functionally characterized in a model entomopathogenic fungus Metarhizium acridum. Similar to Flo8/Som1 in other fungi, MaSom1 mainly localized to the nucleus in M. acridum. Disruption of MaSom1 reduced conidial yield, delayed conidial germination, and impaired the fungal tolerances to heat and UV-B. The expression levels of some genes involved in defenses of heat shock and UV-B radiation were significantly reduced in ΔMaSom1. MaSom1 is also important for cell wall integrity and conidial surface structures in M. acridum. Some genes related to fungal cell wall synthesis were downregulated in ΔMaSom1. Bioassays showed that ΔMaSom1 had a dramatically decreased virulence after both topical inoculation and intrahemocoel injection of the fungus in locusts. Moreover, inactivation of MaSom1 reduced appressorium formation, diminished fungal growth in locust hemolymph in vitro, and enhanced insect immune responses. Taken together, these results indicate that disruption of MaSom1 leads to a decline of fungal virulence because of impairments in conidial germination and appressorium formation, reduction of fungal growth in host hemolymph, and enhancement of insect immune responses owing to the changes in conidial surface structures.

Feasibility of 3D UV-C treatment to reduce fungal growth and mycotoxin loads on maize and wheat kernels.

Fungal disease of grain crops is a concern for the agricultural industry, resulting in economic losses. Aside from severe yield losses, mycotoxigenic fungi such as Penicillium and Fusarium can produce harmful mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and ochratoxin A (OTA). This proof-of-concept study explored the feasibility and effects of ultraviolet (UV) C light at 253.7nm to reduce fungal and mycotoxin loads on model surfaces as well as on maize and wheat kernels using benchtop 2D and 3D illumination strategies. Reduction of Penicillium verrucosum (98.6%) and Fusarium graminearum (88.8%) on agar was achieved using a UV-C dose of 100mJcm-2. Naturally occurring fungal growth resembling P. verrucosum on maize was reduced by 79% after exposure to 5000mJcm-2. Similarly, fungal growth resembling F. graminearum on maize was reduced by 60% with 1000mJcm-2. On wheat, significant reduction of fungal growth was not observed. Maximal reduction of DON (97.3%), ZEN (75.4%), and OTA (91.2%) on filter paper was obtained using 15,000mJcm-2. The overall reduction of DON (30%; 14%), ZEN (52%; 42%), and OTA (17%; 6%) on maize and wheat, respectively, was lower than on filter paper. Moisture and crude protein content as well as percent germination of maize kernels were not affected by UV-C treatment up to 5000mJcm-2. This study has shown that 3D UV-C treatment is a feasible option for reducing Fusarium and Penicillium growth on maize kernels and, at higher doses, decreasing ZEN by ~ 50%.

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus.

RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.

The Aspergillus flavus Spermidine Synthase (spds) Gene, Is Required for Normal Development, Aflatoxin Production, and Pathogenesis During Infection of Maize Kernels.

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76–98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Antifungal exploitation of fungicides against Fusarium oxysporum f. sp. capsici causing Fusarium wilt of chilli pepper in Pakistan.

The research was conducted to evaluate in-vitro efficacy of numerous fungicides against Fusarium oxysporum f. sp. capsici. In present research, six treatments (T) viz. Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete along with control, various concentrations (C), days (D), and their interactions, i.e., (T × C), (T × D), (C × D), and (T × C × D) were exploited in a laboratory through food poison technique. Alliete expressed maximum colony growth (1.93cm) as compared to all other fungicides with respect to control. Interaction between treatments and concentration (T × C) exhibited maximum colony growth of all treatments (Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete), i.e., 0.87, 1.23, 1.73, 2.20, 2.53, and 2.93cm at 300ppm as compared to 500 and 700ppm concentrations, respectively. Similar trend was also observed concerning interaction between (fungicides × days) and (tested concentrations × days). Results of the present study revealed that among tested fungicides, Carbendazim at 700ppm expressed significant reduction in fungal growth.

Cryphonectria hypovirus 1-Induced Epigenetic Changes in Infected Phytopathogenic Fungus Cryphonectria parasitica.

Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. As a model pathosystem for mycovirus-host interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards.

Antifungal activity of native yeasts from different microenvironments against Colletotrichum gloeosporioides on ripe olive fruits

Anthracnose is a fungal disease caused by Colletotrichum species, which mainly affects olive fruits during ripening. The use of yeasts from related and unrelated microenvironments in the control of plant pathogens has become an alternative to the use of synthetic fungicides. The objectives of the present study were to evaluate in vitro biocontrol strategies against C. gloeosporioides, in order to select native antagonistic yeasts isolated from different microenvironments, and to verify the in vivo biocontrol activity on ripe olive fruits. The potential human pathogenicity of the antagonistic yeasts was also considered. A total of 241 wild yeasts from diverse olive cultures and vitivinicultural microenvironments were assayed in vitro and in vivo for their antagonistic effect against the pathogen C. gloeosporioides. Ninety-two yeasts with in vitro characteristics of competitiveness such as niche overlap, dominance against the pathogen, reduction of fungal growth and production of antifungal metabolites were examined on ripe olive fruits. Nine biocontrol yeasts, three from fermenting grape must and six from olive fruits, leaves and branches in vivo reduced the incidence and severity of C. gloeosporioides. According to the 42°C test, which assays for pseudohyphal formation and phospholipase production, these biocontrol yeasts did not present a health risk to humans. Microenvironments with selective stress factors can be relevant sources of microorganisms with biocontrol purposes.

Antifungal activity of indigenous Bacillus spp. isolated from soil

Biocontrol using plant growth-promoting rhizobacteria (PGPR) represents an alternative approach to disease management, since PGPR are known to promote growth and reduce diseases in various crops. Among the different PGPR, members of the genus Bacillus are prefered for most biotechnological uses due to their capability to form extremely resistant spores and produce a wide variety of metabolites with antimicrobial activity. The objective of this research was to identify antagonistic bacteria for management of the plant diseases. Eleven isolates of Bacillus spp. were obtained from the soil samples collected from different localities in the Province of Vojvodina. The antifungal activity of bacterial isolates against five fungal species was examined using a dual plate assay. Bacillus isolates exhibited the highest antifungal activity against Fusarium proliferatum, Fusarium oxysporum f. sp. cepae and Alternaria padwickii, while they had the least antagonistic effect on Fusarium verticillioides and Fusarium graminearum. Molecular identification showed that effective bacterial isolates were identified as Bacillus safensis (B2), Bacillus pumilus (B3, B11), Bacillus subtilis (B5, B7) and Bacillus megaterium (B8, B9). The highest antagonistic activity was exhibited by isolates B5 (from 39% to 62% reduction in fungal growth) and B7 (from 40% to 71% reduction in fungal growth). These isolates of B. subtilis could be used as potential biocontrol agents of plant diseases.

The Pmt2p-Mediated Protein O-Mannosylation Is Required for Morphogenesis, Adhesive Properties, Cell Wall Integrity and Full Virulence of Magnaporthe oryzae.

Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT–PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae.

Anthracnose of lucky bamboo Dracaena sanderiana caused by the fungus Colletotrichum dracaenophilum in Egypt

Dracaena sanderiana, of the family Liliaceae, is among the ornamental plants most frequently imported into Egypt. Typical anthracnose symptoms were observed on the stems of imported D. sanderiana samples. The pathogen was isolated, demonstrated to be pathogenic based on Koch’s rule and identified as Colletotrichum dracaenophilum. The optimum temperature for its growth ranges from 25 to 30°C, maintained for 8days. Kemazed 50% wettable powder (WP) was the most effective fungicide against the pathogen, as no fungal growth was observed over 100ppm. The biocontrol agents Trichoderma harzianum and Trichoderma viride followed by Bacillus subtilis and Bacillus pumilus caused the highest reduction in fungal growth. To the best of our knowledge, this report describes the first time that this pathogen was observed on D. sanderiana in Egypt.

Bacterial and Fungal Counts of Dried and Semi-Dried Foods Collected from Dhaka, Bangladesh, and Their Reduction Methods.

Food is a basic necessity for human survival, but it is still the vehicle for the transmission of food borne disease. Various studies have examined the roles of spices, herbs, nuts, and semi-dried fruits, making the need for safe and convenient methods of decontamination a necessity. The current study determined the bacterial and fungal loads of 26 spices and herbs, 5 nuts, 10 semi-dried fruits and 5 other foods. Spices, herbs and semi-dried foods demonstrated the highest bacterial and fungal loads with the majority showing over 104 CFU/mL. Nuts and other foods showed growths ranging from 102 to 106 CFU/mL. The current study also attempted to determine the effects of heat and plasma treatment. The log reduction of bacterial growth after heat treatment (maximum: 120 min for 60℃) was between 0.08 to 4.47, and the log reduction after plasma treatment (maximum: 40 min) ranged from 2.37 to 5.75. Spices showed the lowest rates of reduction, whereas the semi-dried and other foods showed moderate to high levels of decrease after heat treatment. The log reduction of fungal growth after heat treatment ranged from 0.27 to 4.40, and log reduction after plasma treatment ranged from 2.15 to 5.91．Furthermore, we validated the sterilization effect of plasma treatment against Bacillus spp. and Staphylococcus spp. by using scanning electron microscopy. Both treatment methods could prove to be advantageous in the agriculture related fields, enhancing the quality of the foods.

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection.

Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

Control of citrus molds using bioactive coatings incorporated with fungal chitosan/plant extracts composite.

The ongoing postharvest loss in citrus fruits, due to fungal infection, is a chronic economic and agricultural problem. Most of citrus damage is caused by Penicillium spp., e.g. green mold (P. digitatum) and blue mold (P. italicum). Fungal chitosan, from Mucor rouxii, and plant extracts from cress seeds, olive leaves, pomegranate peels and senna pods, were evaluated as antifungal agents against the phytopathogenic fungi, P. digitatum and P. italicum, using in vitro qualitative and quantitative assays. All natural agents tested exhibited potent antifungal activity; the most powerful agent was cress (Lepidium sativum) seed extract, followed by pomegranate (Punica granatum) peel extract. Fungal chitosan also had a remarkable fungicidal potentiality using both evaluation assays. Penicillium digitatum was generally more resistant than P. italicum toward all examined agents. The incorporation of each individual natural agent in coating material resulted in a great reduction in fungal growth and viability. The addition of chitosan combined with cress and pomegranate extracts, to the coating materials, prevented coated citrus fruit from decay by green and blue mold for a 2-week storage period. Natural derivatives could be recommended as powerful antifungal alternatives to protect citrus fruits from postharvest fungal decay.

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55mg/mL) and savory (0.32mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40mg/mL for thyme oil and 0.21mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).

The Penicillium digitatum protein O-mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26.

The activity of protein O-mannosyltransferases (Pmts) affects the morphogenesis and virulence of fungal pathogens. Recently, PMT genes have been shown to determine the sensitivity of Saccharomyces cerevisiae to the antifungal peptide PAF26. This study reports the identification and characterization of the three Pdpmt genes in the citrus post-harvest pathogen Penicillium digitatum. The Pdpmt genes are expressed during fungal growth and fruit infection, with the highest induction for Pdpmt2. Pdpmt2 complemented the growth defect of the S. cerevisiae Δpmt2 strain. The Pdpmt2 gene mutation in P. digitatum caused pleiotropic effects, including a reduction in fungal growth and virulence, whereas its constitutive expression had no phenotypic effect. The Pdpmt2 null mutants also showed a distinctive colourless phenotype with a strong reduction in the number of conidia, which was associated with severe alterations in the development of conidiophores. Additional effects of the Pdpmt2 mutation were hyphal morphological alterations, increased sensitivity to cell wall-interfering compounds and a blockage of invasive growth. In contrast, the Pdpmt2 mutation increased tolerance to oxidative stress and to the antifungal activity of PAF26. These data confirm the role of protein O-glycosylation in the PAF26-mediated antifungal mechanism present in distantly related fungal species. Important to future crop protection strategies, this study demonstrates that a mutation rendering fungi more resistant to an antifungal peptide results in severe deleterious effects on fungal growth and virulence.

Control of Penicillium sp. on the Surface of Italian Salami Using Essential Oils.

The goal of this study is to evaluate the in vitro effects of rosemary, salvia, oregano and clove oils at volume fractions of 1000, 750, 500, 250, 100, 50, 26, 10 and 5 µL/mL (100, 75, 50, 25, 10, 5, 3, 1 and 0.5%) on the growth of contaminating fungi in salami. The in vitro effect of the oils against fungal growth was indicated by zones of inhibition. Rosemary oil showed an inhibition zone of 9.6 mm only at the maximal volume fraction (1000 µL/mL). Salvia oil showed inhibition zones of 12.2, 11.2 and 10.5 mm only at the three highest fractions tested. Based on the inhibition zones, clove oil at 125 and 250 µL/mL, oregano oil at 250 and 500 µL/mL and a mixture (1:1 by volume) of the two oils at 100 µL/mL were selected to be applied to the surface of salamis. A significant reduction of fungal growth in all of the oil-treated samples was confirmed by visual inspection. A sensory analysis revealed that the samples treated with 125 µL/mL of clove oil or 100 µL/mL of a mixture of oregano and clove oil showed no significant flavour differences compared with the control. Carvacrol and eugenol were the principal compounds in oregano and clove oils, respectively, and were most likely responsible for the antifungal activity.

Development and Evaluation of a New Packaging System for Fresh Produce: a Case Study on Fresh Cherries Under Global Supply Chain Conditions

A new packaging system to extend the shelf life of fresh produce in a cold chain with changing temperatures has been developed. This new packaging system allows rapid increase of the CO2 concentration (while reducing the O2 concentration) when stored at low temperature, and an immediate adjustment to temperature rises in the cold chain in order to maintain optimal gas compositions. The storage stability of fresh cherries, both stem-on and stem-free, was monitored under domestic (road transport) and international (ocean transport) supply chain conditions. The predominant retail package for fresh cherries, flexible, macroperforated polyethylene bags, was used as controls. Package headspace gas composition, fungal growth, weight loss, color, texture, pH, and titratable acidity were monitored for up to 17 and 27 days for domestic and international supply chain conditions, respectively. Consumer acceptance sensory tests were conducted after 14 and 27 days of storage (N > 70). The beneficial effects of the use of this new packaging system included the reduction of fungal growth (P ≤ 0.05) and the maintenance of firmness and red color (P ≤ 0.05). The specific package design also allowed minimization of weight loss due to transpiration. Consumers demonstrated a significant purchasing preference for the new package versus the control (P ≤ 0.05) and rated cherries stored in these packages significantly higher than those stored in the controls using a 9-point hedonic scale. Significantly higher consumer acceptance was found for appearance under simulated domestic supply chain conditions and aroma and flavor under simulated international supply chain conditions.