Effectiveness of fungal, bacterial and yeast antagonists for management of mango anthracnose (Colletotrichum gloeosporioides)

Background Mango anthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases of mango crop. It mainly attacks leaves, flowers, young fruits and twigs and also appears as a post-harvest disease of ripened fruits. Application of bio-control agents has huge potential in plant disease management. The goal of the present research was to establish the potential of individual and combined bio-control agents for the management of mango anthracnose under in vitro and under field conditions. Results The antagonistic reaction of six fungi, six bacteria and nine yeasts against C. gloeosporioides on potato dextrose agar medium and malt extract agar medium was observed among which Trichoderma harzianum was found to be the most efficient with 89.26% mycelial growth inhibition. Evaluation of bio-control agents against anthracnose disease development on mango fruit revealed that dip treatment of mango fruits in spore suspension (1.2 × 104 cfu/ml) of T. harzianum for 5 min was the most effective and provided disease control to the tune of 81.67%. Combined application of effective bio-control agents as a post-harvest fruit dip treatment was also evaluated against the mango anthracnose on mango fruits, where the treatment of T. harzianum + Pichia anomala was very effective with 93.39% disease control. Under field conditions, three consecutive sprays of T. harzianum, starting with the initiation of disease on leaves, followed by other two sprays at an interval of 15 days during 2015 and 2016 were found the best for the management of mango anthracnose disease both on leaves and on fruits at two locations. Conclusions The combined and individual applications of bio-control agents, viz.T. harzianum, Bacillus subtilis and P. anomala, through foliar spray or by fruit dip had the potential to control mango anthracnose. The bio-formulations of these bio-control agents had the potential to replace chemical fungicides and also protect the natural environment, thus playing a significant role in integrated disease management.

Pichia anomala Induced With Chitosan Triggers Defense Response of Table Grapes Against Post-harvest Blue Mold Disease

To study the mechanism by which Pichia anomala induced with chitosan (1% w/v) controls blue mold disease in table grapes caused by Penicillium expansum, this study evaluated alterations in three yeast enzymatic activities. The changes in the five primary disease defense-related enzymes and two non-enzyme activities of table grapes were assayed. The results of the study showed that chitosan (1% w/v) significantly increased the yeast β-1,3-glucanase, catalase (CAT), and malondialdehyde (MDA) activities. Furthermore, P. anomala alone or induced with chitosan (1% w/v) significantly increased the table grapes enzymatic activities of Polyphenol oxidase (PPO), phenylalanine (PAL), peroxidase (POD), and catalase (CAT) compared to the control. The RT-qPCR results also confirmed that the genes of these major disease defense enzymes were up-regulated when the table grapes were treated with P. anomala. The highest results were recorded when the fruit was treated by yeast induced with chitosan (1% w/v). The phenolic compounds, in addition to their nutritional value, can also increase the antimicrobial properties of table grapes. The current experiment determined that the total phenol and flavonoid contents of table grapes showed the highest results for fruits treated by P. anomala induced with chitosan compared with the control. Generally, the increment of these fruit enzymatic and non-enzymatic activities shows improved table grape defense against the pathogenic fungus. The induction of the yeast with chitosan also increases its bio-control efficacy against the pathogen. This study will enable future detailed investigation in the yeast pathogen control mechanisms and the use of yeasts as bio-pesticides.

In Vitro Activity of Selected Phenolic Compounds against Planktonic and Biofilm Cells of Food-Contaminating Yeasts

Phenolic compounds are natural substances that can be obtained from plants. Many of them are potent growth inhibitors of foodborne pathogenic microorganisms, however, phenolic activities against spoilage yeasts are rarely studied. In this study, planktonic and biofilm growth, and the adhesion capacity of Pichia anomala, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Debaryomyces hansenii spoilage yeasts were investigated in the presence of hydroxybenzoic acid, hydroxycinnamic acid, stilbene, flavonoid and phenolic aldehyde compounds. The results showed significant anti-yeast properties for many phenolics. Among the tested molecules, cinnamic acid and vanillin exhibited the highest antimicrobial activity with minimum inhibitory concentration (MIC) values from 500 µg/mL to 2 mg/mL. Quercetin, (−)-epicatechin, resveratrol, 4-hydroxybenzaldehyde, p-coumaric acid and ferulic acid were also efficient growth inhibitors for certain yeasts with a MIC of 2 mg/mL. The D. hansenii, P. anomala and S. pombe biofilms were the most sensitive to the phenolics, while the S. cerevisiae biofilm was quite resistant against the activity of the compounds. Fluorescence microscopy revealed disrupted biofilm matrix on glass surfaces in the presence of certain phenolics. Highest antiadhesion activity was registered for cinnamic acid with inhibition effects between 48% and 91%. The active phenolics can be natural interventions against food-contaminating yeasts in future preservative developments.

Transcriptome Characterization and Expression Profiles of Disease Defense-Related Genes of Table Grapes in Response to Pichia anomala Induced with Chitosan

Transcriptome analysis (TA) was conducted to characterize the transcriptome changes in postharvest disease-related genes of table grapes following treatment with Pichia anomala induced with chitosan (1% w/v). In the current study, the difference in the gene expression of table grapes after treatment with P. anomala induced with chitosan and that of a control group was compared 72 h post-inoculation. The study revealed that postharvest treatment of table grapes with P. anomala induced with chitosan could up-regulate genes that have a pivotal role in the fruit’s disease defense. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results also confirmed that GO terms and the KEGG pathways, which have pivotal roles in plant disease resistance, were significantly enriched. The up-regulated genes of the treatment group have a unique function in the fruit’s disease resistance compared to the control group. Generally, most genes in the plant–pathogen interaction pathway; the plant Mitogen-activated protein kinase (MAPK) signaling pathway; the plant hormone signal transduction pathway; the pathway of glutathione metabolism; the pathway of phenylalanine, tyrosine, and tryptophan biosynthesis; and the pathway of flavonoid biosynthesis were all up-regulated. These up-regulations help the fruit to synthesize disease-resistant substances, regulate the reactive oxygen species (ROS), enhance the fruit cell wall, and enrich hormone signal transduction during the pathogen’s attack. This study is useful to overcome the lags in applying transcriptomics technology in postharvest pathology, and will provide insight towards developing other alternative methods to using bio-pesticides to control postharvest diseases of perishables.

Isolation, Identification and Function of Pichia anomala AR2016 and Its Effects on the Growth and Health of Weaned Pigs

This study isolates and identifies Pichia anomala (P. anomala) AR2016, and studies its effect on the growth and health of weaned pigs. A P. anomala strain from solid wine koji is isolated and identified using 26S rDNA analysis, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, artificial gastric, and intestinal juice tolerance are evaluated. In our methodology, thirty 28 d Large White × Landrace × Rongchang weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days. A P. anomala strain was identified and named P. anomala AR2016. P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our results show that compared with the CK group, orally administered P. anomala AR2016 increases average daily gain, the ileal villus height, the ileal mucosal concentrations of occludin and zonula occluens-1, the serum glucose and total protein concentration, total superoxide dismutase, glutathione peroxidase, and total antioxidative capacity activity, the trypsin and lipase activity in jejunal and ileal contents, the jejunal and ileal mucosa mRNA levels of ALP, TNF-α, and TLR-2, and the relative abundance of Bacteroidetes, Actinobacteria, Succinivibrionaceae, Lachnospiraceae, and Prevotellaceae (p < 0.05). Compared with the CK group, oral administration of P. anomala AR2016 decreased the incidence of diarrhea, aspartate aminotransferase activity, alanine amino-transferase-activity, malondialdehyde, D-lactic acid and endotoxin content in serum, the mRNA level of aminopeptidase N of ileum mucosa, and the relative abundance of Proteobacteria, Clostridiaceae, Campylobacteraceae, Vibrionaceae, Bacillus, and Pseudon (p < 0.05). Collectively, the study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. Oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.

Sustainable Second-Generation Bioethanol Production from Enzymatically Hydrolyzed Domestic Food Waste Using Pichia anomala as Biocatalyst

In the current study, a domestic food waste containing more than 50% of carbohydrates was assessed as feedstock to produce second-generation bioethanol. Aiming to the maximum exploitation of the carbohydrate fraction of the waste, its hydrolysis via cellulolytic and amylolytic enzymatic blends was investigated and the saccharification efficiency was assessed in each case. Fermentation experiments were performed using the non-conventional yeast Pichia anomala (Wickerhamomyces anomalus) under both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) modes to evaluate the conversion efficiencies and ethanol yields for different enzymatic loadings. It was shown that the fermentation efficiency of the yeast was not affected by the fermentation mode and was high for all handlings, reaching 83%, whereas the enzymatic blend containing the highest amount of both cellulolytic and amylolytic enzymes led to almost complete liquefaction of the waste, resulting also in ethanol yields reaching 141.06 ± 6.81 g ethanol/kg waste (0.40 ± 0.03 g ethanol/g consumed carbohydrates). In the sequel, a scale-up fermentation experiment was performed with the highest loading of enzymes in SHF mode, from which the maximum specific growth rate, μmax, and the biomass yield, Yx/s, of the yeast from the hydrolyzed waste were estimated. The ethanol yields that were achieved were similar to those of the respective small scale experiments reaching 138.67 ± 5.69 g ethanol/kg waste (0.40 ± 0.01 g ethanol/g consumed carbohydrates).

Wickerhamomyces anomalous: A Rare Cause of Fungemia Causing Febrile Neutropenia in Acute Lymphoblastic Leukemia

Candida bloodstream infection is the major cause of increased morbidity and mortality (20–49%) in hospitalized patients in both paediatric and adult age groups. Due to the increase in the number of immunocompromised patients, other important species such as Trichosporon asahii and Debaryomyces hansenii are emerging. One such organism, Wickerhamomyces anomalous, previously known as Pichia anomala (teleomorph stages of several Candida species), is increasingly being reported as a cause of fungemia in neonatal intensive care units and is now increasingly being reported in a lot of immunosuppressive conditions such as interstitial lung disease, endocarditis, enteritis, corticosteroids, and chemotherapy uptake. Though this yeast is ubiquitous in nature, systemic infections from isolated cases and sporadic outbreaks with high mortality have been reported in ICUs, which emphasize the importance to consider this fungus within the diagnostic possibilities. Here, we report a case of catheter-related bloodstream infection (CRBSI) caused by W. anomalus in a leukemic immunosuppressed patient who was successfully treated by early detection and treatment of this emerging fungus.

Assaying the effect of yeasts on growth of fungi associated with disease

Background Pathogenic fungi often cause serious infections mainly in immunocompromised persons. The number of infections caused by the non-albicans Candida or other species has significantly increased over the last years. These infections present a major challenge in the health sector because these pathogenic fungi have strong virulence and often show resistance to the commonly used antifungal treatments. To solve the problems caused by the drug resistant pathogenic fungi, it is necessary to find new antifungal agents and their sources. The aim of this study was to give evidence that yeasts can effectively fight against strains which belong to pathogenic fungi and reveal those yeasts which are able to inhibit growth of Kodamaea ohmeri, Pichia kudriavzevii, Naganishia albida or Candida tropicalis. Furthermore, we wanted to determine the effects of certain culturing factors on the growth inhibition. Results Our screening revealed that although the strains belonging to pathogenic species were much more tolerant to the yeast-produced bioactive agents than the non-disease-associated yeasts, growth of Kodamaea ohmeri and Candida tropicalis could be inhibited by Metschnikowia andauensis, while Naganishia albida could be controlled by Pichia anomala or Candida tropicalis. Our data proved that the experimental circumstances could have a serious impact on the inhibitory capacity of the yeasts. Appearance of inhibition strongly depended on media, pH and temperature. Our data also shed some light on the fact that Pichia kudriavzevii must have high natural resistance to the yeast-produced agents, while other species, such as Saccharomycopsis crataegensis belonged to the easily inhibitable species. Conclusions Our study suggests that yeast-produced bioactive agents could be potential growth inhibitory agents against the disease-associated fungi and yeasts can also contribute to alternative approaches to combat against pathogenic fungi. Our data revealed an important role of the culturing factors in inhibition and pointed to the complex nature of antagonism.

Seven New Cytotoxic and Antimicrobial Xanthoquinodins from Jugulospora vestita

During the course of a screening for novel biologically active secondary metabolites produced by the Sordariomycetes (Ascomycota, Fungi), the ex-type strain of Jugulospora vestita was found to produce seven novel xanthone-anthraquinone heterodimers, xanthoquinodin A11 (1) and xanthoquinodins B10–15 (2–7), together with the already known compound xanthoquinodin B4 (8). The structures of the xanthoquinodins were determined by analysis of the nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric data. Moreover, the absolute configurations of these metabolites were established by analysis of the 1H−1H coupling constants, nuclear Overhauser effect spectroscopy (NOESY) correlations, and Electronic Circular Dichroism (ECD) spectroscopic data. Antifungal and antibacterial activities as well as cytotoxicity of all compounds were tested. Xanthoquinodin B11 showed fungicidal activities against Mucor hiemalis [minimum inhibitory concentration (MIC) 2.1 µg/mL], Rhodotorula glutinis (MIC 2.1 µg/mL), and Pichia anomala (MIC 8.3 µg/mL). All the compounds 1–8 displayed anti-Gram-positive bacteria activity (MIC 0.2–8.3 µg/mL). In addition, all these eight compounds showed cytotoxicity against KB 3.1, L929, A549, SK-OV-3, PC-3, A431, and MCF-7 mammalian cell lines. The six novel compounds (1–3, 5–7), together with xanthoquinodin B4, were also found in the screening of other strains belonging to Jugulospora rotula, revealing the potential chemotaxonomic significance of the compound class for the genus.