Thymol Encapsulated into HP-ß-Cyclodextrin as an Alternative to Synthetic Fungicides to Induce Lemon Resistance against Sour Rot Decay.

Consumers demand the use of eco-friendly fungicides to treat fruit and vegetables and governmental authorities have unauthorized the application of chemical antifungals for the efficient control of sour rot. In the present research, the microwave irradiation (MW) method was used to encapsulate thymol into 2-hydroxylpropyl-beta-cyclodextrin (HP-ß-CD) and the effect of these HP-ß-CD on controlling sour rot in citrus fruit, caused by Geotrichum citri-aurantii, was evaluated. Amounts of 25 and 50 mM of HP-ß-CD-thymol were used, and compared with propiconazole, to control the decay of inoculated lemon fruit. The treatments were performed in curative and preventive experiments. The incidence and severity of Geotrichum citri-aurantii in 25 and 50 mM HP-ß-CD-thymol-treated fruit were reduced in both experiments. The preventive 50 mM HP-ß-CD-thymol treatment showed the best effect, reducing the sour rot, respiration rate and fruit weight loss during storage at 20 °C. HP-ß-CD-thymol increased polyphenol concentration and the activity of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) in lemon peel, and the highest effects were found with the 50-mM dose. In conclusion, the results show that the use of thymol encapsulated by MW into HP-ß-CD could be an effective and sustainable tool, a substitute to the synthetic fungicides, for G. citri-auriantii control in citrus fruit.

Dietary supplementation of Geotrichum candidum improves growth, gut microbiota, immune-related gene expression and disease resistance in gibel carp CAS â ¢ (Carassius auratus gibelio).

Geotrichum candidum is a filamentous fungus mostly used in the dairy industry for cheese ripening and flavoring. The current study was designed to evaluate the potential probiotics effect of dietary supplementation of G. candidum on growth, digestive enzymes activities, gut microbiota and immune-related gene expression and disease resistance in Gibel carp CAS Ⅲ (Carassius auratus gibelio) against A. hydrophila infections. For this purpose, 420 healthy fish (initial body weight: 7.09 ± 0.02g) were randomly divided into 7 feeding groups in triplicates of 20 fish in each tank. Seven isocaloric and isonitrogenous diets were prepared, supplemented with 6 different doses of G. candidum viz; C: 0, T1:106, T2:107, T3:108, T4:109, T5:1010, T6:1011 CFU/kg diet and fed to fish twice a day for 60 days. Results showed significantly higher weight gain (WG) and feed efficiency (FE) in groups fed 106 and 108 CFU/kg diet compared to the control (P < 0.05). All G. candidum fed groups showed higher intestinal α-amylase activity (P < 0.05) while no difference in lipase and trypsin activity compared to the control group. Before challenge test, gut microbial diversity analysis revealed relatively more abundance of bacteria belonging to phylum Proteobacteria, Actinobacteria, Firmicutes and Bacteriodetes in group fed G. candidum supplemented diet and higher percentage of pathogenic bacteria belonging to order Aeromonadales and Vibrionales in control post-challenge. After challenge test immune response expressed numerically higher survival rate (P > 0.05) and significantly higher (P < 0.05) Respiratory Burst Activity(RBA), Immunoglobulin M level (IgM), Aspartate Transaminase activity (AST) and improved (P > 0.05) lysozyme activity, and Alanine Transaminase activity (ALT) in groups fed 106 and 108 CFU/kg G. candidum supplemented diet. The expression of immune related gene (IL-I ß, TNF- α, HSP70 and TLR-2) in liver of the fish were significantly affected (P < 0.05) by supplementation of G. candidum post bacterial challenge. The results showed that G. candidum is a potential probiotic as it could improve feed utilization, immunity and cause no harmful effects on growth of gibel carp at higher supplementation levels. The productive results can be achieved by using the lower dose as 106-108 CFU/kg diet.

Biocontrol ability and action mechanism of Metschnikowia citriensis against Geotrichum citri-aurantii causing sour rot of postharvest citrus fruit.

This study investigated the biocontrol efficiency of Metschnikowia citriensis strain FL01 against Geotrichum citri-aurantii, and evaluated possible mechanisms. The results showed that M. citriensis could effectively control the development of sour rot, and significantly inhibit the mycelial growth and spore germination of G. citri-aurantii. The population dynamics results and Scanning electron microscopy (SEM) analysis indicated that M. citriensis could rapidly colonize wounds and tightly adhere to the surface of the wounds to compete with G. citri-aurantii for nutrition and space. M. citriensis also showed the biofilm formation action in vitro. The response of G. citri-aurantii to different components of M. citriensis culture showed that only the yeast cells but not the extracellular metabolites and the volatile organic compounds (VOCs) exhibited inhibitory effect on the growth of G. citri-aurantii. M. citriensis adhered to the hyphae of G. citri-aurantii loosely and sparsely, and the production of lytic enzymes ß-1, 3-glucanase (GLU) and Chitinase (CHI) could not be induced by G. citri-auranti. Iron affected the pulcherrimin pigment production and antagonism of M. citriensis indicating iron depletion as the most important antagonistic mechanism. Besides, M. citriensis also induced resistance of fruit against sour rot. These results suggested that M. citriensis could be used as the potential alternative of fungicides to control postharvest pathogens on citrus fruit.

Regulation of resveratrol O-methyltransferase gene in pterostilbene defensing the sour rot of wine grape.

Sour rot, caused by Geotrichum citriaurantii (G. citri-aurantii.), is one of the most serious grapevine secondary infection diseases in China. We have determined that pterostilbene, the most important phytoalexin, effectively inhibits the activity of G. citri-aurantii. To study the synthesis mechanism of pterostilbene against G. citri-aurantii in grape, we initially detected the content of pterostilbene present in grapes infected by G. citri-aurantii with the use of UHPLC-QQQ-MS2 . Pterostilbene levels are controlled by the resveratrol O-methyltransferase (ROMT) gene, and within grape samples is positively related to the accumulation of resveratrol. The pPIC9k-ROMT vector and plasmid pCAMBIA2300-GFP-ROMT were constructed for expression purposes. The pPIC9k-ROMT vector was expressed in Pichia pastoris GS115 and plasmid pCAMBIA2300-GFP-ROMT was expressed in onion. Analysis of qRT-PCR amplification samples revealed that gene expression was induced rapidly in grape as a defense against G. citri-aurantii. Western blot analysis verified that the recombinant protein was successfully expressed. PRACTICAL APPLICATIONS: Pterostilbene is a trace and efficient phytoalexins produced by plant' secondary metabolism, which exhibits good pharmacological activity. As an important protective agent in plants, it can improve the antioxidant capacity and resistance to adversity of plants. However, the method which could be used for mass production of pterostilbene has not been reported currently. The key gene of pterostilbene biosynthesis was investigated and verified in this paper, which provides the theoretical basis for the industrial production of pterostilbene. The study of pterostilbene is significant for the prevention and treatment of G. citri-aurantii disease, and has important practical applications for the development and production of pesticides.

Fungal Infection Caused by Geotrichum capitatum in a Severe Aplastic Anemia Patient: a Case Report and Review of the Literature.

BACKGROUND: Invasive fungal infections often occur in immunocompromised patients. Here, we report an infection case caused by Geotrichum capitatum in a severe aplastic anemia patient. METHODS: Identification of the pathogenic bacteria was done by sequencing and mass spectrometric analysis. RESULTS: The fungal infection was isolated from blood cultures. The pathogenic bacteria were identified as Geotrichum capitatum. The infection was primarily cured by voriconazole and caspofungin monotherapy. However, the effect was not obvious. Then a combination of liposomal amphotericin B and caspofungin was used. Body temperature of the patient decreased, and clinical symptoms improved. CONCLUSIONS: Sequencing and mass spectrometric analysis could have a role for Geotrichum capitatum diagnosis. Curative effect of using a single antifungal drug was unsatisfactory. Using liposome amphotericin B combined with caspofungin might obtain certain curative effect. Early diagnosis and appropriate combined therapy were necessary to improve the outcome of patients with Geotrichum capitatum infection.

Biofilm production by Aureobasidium pullulans improves biocontrol against sour rot in citrus.

The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 against Geotrichum citri-aurantii, causal agent of sour rot in citrus, and their interactions were evaluated. For this, were evaluated the incorporation of nutrients in optimizing the antagonistic activity of the yeast, the competition for nutrients between microorganisms, the effect of nutrients on yeast cell and biofilm production and their correlation in the biocontrol of disease, the survival of yeast in citrus fruits and the interaction between microorganisms using scanning electron microscopy. Micronutrients (boric acid, cobalt chloride and ammonium molybdate) favoured the antagonistic action of A. pullulans. Ammonium sulfate 1% and sucrose 0.5% favoured the yeast during the competition between the microorganisms. The addition of ammonium sulfate (1%) in the yeast culture stimulated biofilm production and increased the antagonistic activity against the disease, as also allowed the better survival of yeast in wounded sites of citrus fruit. The yeast was found to be able to form biofilms on citrus, deforming the pathogen hyphae. These results showed the importance of the addition of nutrients in A. pullulans based-formulations when aiming for their use on a commercial scale. This is the first report of a positive correlation between the increase in the quantity of biofilm produced by A. pullulans, with increased antagonistic activity.

Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than <1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability.

Invasive Geotrichum clavatum fungal infection in an acute myeloid leukaemia patient: a case report and review.

Invasive Geotrichum clavatum fungal infections are extremely rare and unusual, occurring nearly exclusively in patients experiencing prolonged neutropenia during the treatment for acute myeloid leukaemia. Several groups of cases of fatal G. clavatum infection were reported in France between 2011 and 2012, but the ecological niche has not yet been identified. We report a case of a 32-year-old patient with acute myeloid leukaemia who developed G. clavatum sepsis with primary peritonitis, hepatic nodular lesions, and multivisceral failure during aplasia after induction followed by salvage chemotherapy. He was treated with voriconazole and is still alive 1 year after with controlled disease. We then discuss the epidemiological, clinical, and therapeutic features of these serious fungal infections compared to the published data.

Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2d-dige technology and samespots software analysis.

Geotrichum candidum is a micro-fungus widely used as a ripening starter in cheese making. In anthropogenic environments such as dairy industries, this microorganism is subjected to many environmental and technological stresses including low temperature exposure. Our aim was to study the proteomic response of G. candidum to cold stress using a comparative proteomic approach by two-dimensional Differential In Gel Electrophoresis (2D DIGE). This technique consists on the labeling of proteins by specific fluorescent dyes (CyDyes). The results, obtained with G. candidum cells subjected to cold temperature, show significant proteomic patterns differences compared with the standard conditions. Furthermore, this biochemical response seems strain specific. 2D DIGE technology combined with SameSpots™ software analysis support these results through an important statistical validity. The comparative studies in a single gel, using two different fluorescent CyDyes (Cy3 and Cy5), lead to proteins differentiation. Selected spots were treated and analyzed by mass spectrometry.

The use of FAME analyses to discriminate between different strains of Geotrichum klebahnii with different viabilities.

A considerable decline in viability of spray dried cells of Geotrichum klebahnii was observed and was attributed to an undefined alteration of the used strain. As common techniques were not able to distinguish the altered from the still viable strains, we used the fatty acid methyl ester (FAME) analysis. On the basis of FAME data we were able to discriminate the three strains under investigation. Especially the ratios of cis/trans fatty acid ratios and of saturated/unsaturated fatty acid were significantly reduced in the less viable strain, pointing to an increased stress level in this strain. These findings clearly show the applicability of the FAME analysis to detect strain alterations and that this method is therefore a suitable, fast and feasible tool for quality assurance.

Relationship between growth behaviour, micro and macroscopic morphologies and freezing sensitivity of the ripening starter Geotrichum candidum is strain specific and mostly related to the morphotypes: the arthrospores/hyphae parameter.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml-1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes "yeast", "mould" or "intermediate". The two "mould" strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the "yeast" strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the "mould" morphotype respond similarly to freezing stress.

Case of fatal Blastoschizomyces capitatus infection occurring in a patient receiving empiric micafungin therapy.

We report the first case, to our knowledge, of Blastoschizomyces capitatus infection occurring in a patient receiving empirical echinocandin therapy for neutropenic fevers. Clinicians should consider B. capitatus infection in those neutropenic patients who remain febrile despite echinocandin therapy or who develop yeast bloodstream infections while receiving an echinocandin.

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species.

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.

Improvement of the cryopreservation of the fungal starter Geotrichum candidum by artificial nucleation and temperature downshift control.

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.

Nematicidal metabolites produced by the endophytic fungus Geotrichum sp. AL4.

From the endophytic fungal strain Geotrichum sp. AL4, cultivated from the leaves of the neem tree (Azadirachta indica), four compounds, 1-4, were isolated from the AcOEt extract, including two new, chlorinated, epimeric 1,3-oxazinane derivatives. All compounds were assessed for their nematicidal activities against the nematodes Bursaphelenchus xylophilus and Panagrellus redivivus, and three out of the four isolates showed noticeable bioactivities.

Three new asexual arthroconidial yeasts, Geotrichum carabidarum sp. nov., Geotrichum histeridarum sp. nov., and Geotrichum cucujoidarum sp. nov., isolated from the gut of insects.

Twenty arthroconidial yeasts were isolated from the digestive tract of basidiome-feeding beetles and lepidopteran larvae. All of the yeasts reproduced only asexually by arthroconidia and some by endo- or blastoconidia as well. Based on the comparisons of sequences in ribosomal RNA genes and other taxonomic characteristics, the yeasts were identified as three unknown Geotrichum species. The three new species are described as Geotrichum carabidarum (NRRL Y-27727T), G. histeridarum (NRRL Y-27729T), and G. cucujoidarum (NRRL Y-27731T). Phylogenetic analyses from ribosomal DNA sequences showed that members of the genus Geotrichum and related arthroconidial yeast taxa were divided into two major clades: (1) Dipodascus and Galactomyces with Geotrichum anamorphs including all the new species; and (2) Magnusiomyces with Saprochaete anamorphs. G. cucujoidarum formed a subclade with G. fermentans and Geotrichum sp. Y-5419, while the two closely related species, G. carabidarum and G. histeridarum, represent a new basal subclade in the clade of Geotrichum and its teleomorphs.

The inhibitory effect of Penicillium camemberti and Geotruchum candidum on the associated funga of white mould cheese.

The diameter of pinpoint inoculated cheese contaminates (Cladosporium herbarum, Penicillium roqueforti, Penicillium caseifulvum and Penicillium commune), isolated from either the dairy environment or directly from cheese, were inoculated 24 h after inoculation of the secondary starters to simulate contamination at the critical point of the salt brine. Pure P. camemberti had the largest inhibitory effect on the C. herbarum contaminant. Adding G. candidum in mixed cultures weakened the inhibitory effect of P. camemberti on C. herbarum. Low levels of G. candidum (10(3) spore/ml) promoted visible growth effects of C. herbarum, and this was most pronounced in the early stages of growth. The interaction mechanism of C. herbarum was not affected by the choice of the strain of P. camemberti whereas the Penicillium contaminants were very sensitive to the choice of the P. camemberti strain. The presence of G. candidum in the mixed cultures seems to decrease the suppressing effect of pour-plated P. camemberti. No correlation of any kind was found in the pour-plated spore concentration totals by the inhibition of the C. herbarum and P. roqueforti contaminants whereas P. caseifulvum and P. commune were sensitive to this.

Resin straw as an alternative system to securely store frozen microorganisms.

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Production of sulfur flavors by ten strains of Geotrichum candidum.

Ten strains of Geotrichum candidum were studied on a liquid cheese model medium for the production of sulfur compounds which contribute to the aroma of cheeses. The volatile components produced by each cultured strain were extracted by dynamic headspace extractions, separated and quantified by gas chromatography (GC), and identified by GC-mass spectrometry. It was shown that four strains of this microorganism produced significant quantities of S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, S-methyl thioisobutanoate, S-methyl thioisovalerate, and S-methyl thiohexanoate. This is the first example of the production of these compounds by a fungus. In addition, dimethyldisulfide, dimethyltrisulfide, dimethylsulfide, and methanethiol, which are more commonly associated with the development of cheese flavor in bacterial cultures, were also produced by G. candidum in various yields, depending on the strain selected. The potential application of these strains in cultured microbial associations to produce modified cheeses with more desirable organoleptic properties is discussed.

Protection by fungal starters against growth and secondary metabolite production of fungal spoilers of cheese.

The influence of fungal starter cultures on growth and secondary metabolite production of fungal contaminants associated with cheese was studied on laboratory media and Camembert cheese. Isolates of the species Penicillium nalgiovense, P. camemberti, P. roqueforti and Geotrichum candidum were used as fungal starters. The species P. commune, P. caseifulvum, P. verrucosum, P. discolor, P. solitum, P. coprophilum and Aspergillus versicolor were selected as contaminants. The fungal starters showed different competitive ability on laboratory media and Camembert cheese. The presence of the Penicillium species, especially P. nalgiovense, showed an inhibitory effect on the growth of the fungal contaminants on laboratory media. G. candidum caused a significant inhibition of the fungal contaminants on Camembert cheese. The results indicate that G. candidum plays an important role in competition with undesirable microorganisms in mould fermented cheeses. Among the starters, P. nalgiovense caused the largest reduction in secondary metabolite production of the fungal contaminants on the laboratory medium. On Camembert cheese no significant changes in metabolite production of the fungal contaminants was observed in the presence of the starters.