Bioprospection of antagonistic yeasts for biocontrolling postharvest pathogenic fungi and physicochemical characterization of a yeast exopolysaccharide

Abstract

The leading cause of food loss or fruit and vegetable waste was associated with fungi-induced postharvest diseases. Since fruit is rich in water, sugar, and essential nutrients, phytopathogenic and/or mycotoxigenic fungi can easily spread and find an ideal habitat, especially in damaged fruits. At this point, synthetic fungicides appear to be the first way of protection. Although these fungicides are efficient and economical, they can develop resistance in fungi, and their toxic residues may pose a health and environmental threat. The European Food Safety Authority (EFSA) has set tight regulations for the maximum pesticide residue levels in crops. It also encourages using low-risk or non-chemical fungicides for health and the environment. Recently, climate change has reduced crop yields and triggered multi-mycotoxin formation. Therefore, there is a need for non-toxic, natural, and efficient bio-fungicides to be used in agroecosystems. Harnessing beneficial microorganisms, especially bacteria, yeasts, and moulds, has emerged as an alternative to synthetic fungicides. Among these microorganisms, antagonistic yeasts have a great potential for biopreservation with their GRAS (generally regarded as safe), biodegradable, non-toxic, genetically stable, and safety characteristics (non-pathogenic without producing antibiotics, mycotoxins, and allergenic spores). Additionally, antagonistic yeasts can use a variety of substances as carbon and energy sources, thereby limiting nutrients for spoilage fungi. They can also tightly attach to fruit and secrete hydrolytic enzymes, volatile organic compounds, mycocin, and biofilm. In line with the United Nations' Sustainable Development Goals of combating hunger and poverty, biocontrol yeasts have sparked much interest as a green and sustainable solution. The biocontrol yeasts can be a viable alternative to synthetic fungicides by bio-managing the notorious fungi and their mycotoxins, increasing agricultural productivity and food security. Regarding yeast metabolites, yeast-based exopolysaccharides (EPS) have received much attention due to their high yields, ease of extraction, and bioactive compounds. Considering the above, a research strategy has been developed to investigate the biocontrol potential of fruit-isolated yeasts against selected fungi on lemons, mandarins, and grapes, along with their exopolysaccharide production and characterization. The objectives of this PhD dissertation were (i) bioprospecting Metschnikowia sp. isolates as biocontrol agents against postharvest fungal decays on lemons with their potential modes of action; (ii) evaluating the combinatorial effect of antagonistic yeasts (Hanseniaspora uvarum, Meyerozyma guilliermondii, and Metschnikowia aff. pulcherrima P01A016) on the bio-management of green mould disease on mandarin fruits; (iii) recruiting grape-isolated Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hansenispora uvarum yeasts on the biopreservation of Botrytized grapes; (iv) characterization of a novel exopolysaccharide produced by a cold-adapted yeast Rhodotorula glutinis. Four research concepts (Chapters 3-6) were carried out to accomplish these objectives. Firstly, various fruits (grape, rosehip, hawthorn, blackberry, and cornelian cherry) and leaves were employed for yeast isolation. These yeasts were screened for their in vitro antifungal activity against various fungi. Since the highest antagonism was observed on Penicillium spp., lemons were selected as a substrate for further experiments. Then, pectinase-free antagonistic yeasts were applied to wounded lemon fruits to test their biocontrolling efficacy (Chapter 3). The compatible combinations of three antagonistic yeasts were employed to manage green mould decay on wounded and intact mandarin fruits. Furthermore, the findings were compared to the effectiveness of the synthetic fungicide, imazalil (Chapter 4). Grape-isolated yeasts Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hanseniaspora uvarum were used in the biopreservation of grapes against Botrytis cinerea, along with elucidating antifungal mechanisms of action (Chapter 5). Finally, yeast isolates were screened for their exopolysaccharide production capabilities. A blackberry isolate, R. glutinis, was the most promising one with its high yield, characterized by its structural, rheological, antioxidant, and antibiofilm properties (Chapter 6). The research framework and objectives of this PhD thesis are introduced in Chapter 1. Following that, Chapter 2 provided a comprehensive review of the exploitation of antagonistic yeasts for sustainable bio-management of postharvest phytopathogenic and mycotoxigenic fungi in fruits. The potential use of antagonistic yeasts for biocontrol purposes was mentioned, along with explaining their antifungal mechanisms of action. The current literature was screened to determine how antagonistic yeasts could be used with other agents or processes for improved biocontrolling activity. Subsequently, biocontrol yeasts' principles, advantages, disadvantages, and applications for commercial use were discussed. In Chapter 3, eleven distinct yeast cultures of Metschnikowia sp. belonging to six different species were identified using polymerase chain reaction (PCR) with sequence-based analysis of the D1/D2 domain of 26S rDNA. Yeast antagonism (1 × 108 cells/mL) against various fungi (Fusarium oxysporum, Botrytis cinerea, Penicillium digitatum, Penicillium expansum, and Alternaria alternata) was determined through a dual culture method on plates. The highest antagonism was obtained against Penicillium digitatum NRRL 1202 and Penicillium expansum DSM6284 (83.63-100 %). Chitinase activity was present in all of the examined yeasts, while others also exhibited protease, pectinase, cellulase, β-1-3 glucanase, and gelatinase activities. Due to the high pectin content in lemons, three pectinase-free cultures with strong in vitro antagonistic effects on Penicillium were chosen. The interaction of lytic enzyme secretion, biofilm formation, iron depletion, and volatile organic compound (VOC) formation determined the antifungal mechanism of action. Pectinase-free Metschnikowia sp. yeasts significantly reduced the disease incidences and lesion diameters on lemons. M. aff. fructicola had the best biocontrol efficacy against Penicillium on lemons. In Chapter 4, the antagonistic yeasts belonging to genera Hanseniaspora uvarum, Meyerozyma guilliermondii, and Metschnikowia aff. pulcherrima P01A016 were employed to inhibit P. digitatum in wounded and whole mandarins. All yeast cultures (73.85% - 80.64%) and their combinations (1:1, v/v; 1:1:1, v/v/v, 1 × 108 cells/mL) (78.40% - 83.18%) reduced the mycelial growth of green mould in vitro. M. aff. pulcherrima reduced the disease incidence and lesion diameters by 75.5% and 91.3%, respectively, demonstrating the highest biocontrolling activity alone. M. guilliermondii exhibited the highest biofilm formation (OD 0.93 ± 0.01) and antifungal activity (71.13%) via volatile organic compounds (VOCs), whereas H. uvarum exhibited cell-wall degrading β-1,3 glucanase activity in addition to mycocinogenic and VOCs activity. M. guilliermondii and M. aff. pulcherrima (M-1) were the most compatible, while M. aff. pulcherrima and H. uvarum (M-3) had the least compatibility. Combining these three yeasts resulted in synergistic cooperation, demonstrating the highest biocontrolling efficacy in vitro and in vivo. In Chapter 5, grape-isolated yeasts (Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hansenispora uvarum) were proposed to control B. cinerea-caused grey mould disease in grapes, while also elucidating their potential inhibitory mechanisms. The antifungal mechanisms included iron depletion, secretion of hydrolytic enzymes, diffusible compounds, inhibition of conidial germination, biofilm formation, wound-site colonisation, and VOC emission. Metschnikowia yeasts yielded comparable in vitro results but in in vivo experiments M. aff. fructicola outperformed all other yeasts. Interestingly, VOCs from H. uvarum held considerable potential as an antifungal biofumigant. All yeasts are adapted well to their ecological niche to protect the grapes against grey mould. In Chapter 6, a novel EPS from R. glutinis was characterized by its monosaccharide composition by high-performance liquid chromatography (HPLC), molecular characterization by Fourier transform infrared (FT-IR) spectroscopy, thermal stability by differential scanning calorimetry (DSC), morphological characterization by scanning electron microscopy (SEM), crystallographic characteristics by X-ray diffraction (XRD) analysis, structural characterization by nuclear magnetic resonance (NMR) spectroscopy, antioxidant activity by •OH, DPPH•, CUPRAC, and ABTS radical scavenging activities, and rheological characterization by a rheometer. The findings revealed that EPS was a heteropolysaccharide composed of glucose and galactose. NMR and FT-IR analyses confirmed that the EPS-BMD26 structure had glucose and galactose without mannose. Differential scanning calorimetry (DSC) analysis revealed its thermal stability up to 326.16 °C. Scanning electron microscopy (SEM) revealed the porous microstructure with fissures. X-ray diffraction (XRD) analysis revealed that it was semi-crystalline. It demonstrated moderate and concentration-dependent antioxidant potential through •OH, DPPH•, CUPRAC, and ABTS radical scavenging methods. EPS-BMD26 exhibited high water holding capacity of 190 ± 0.22% with a water solubility index of 60.6 ± 8.3%. The EPS-BMD26 also inhibited biofilm formation by Staphylococcus aureus ATCC 25923 (79.5% inhibition at 1250 ppm). Rheological analysis revealed its shear thinning and pseudoplastic behaviour. Finally, in Chapter 7, based on the main outcomes of the previous chapters, the general discussion, conclusion, and future prospects in the biocontrol yeasts and yeast-derived exopolysaccharides are presented. From a biocontrol perspective, further research is required to examine the relationships between yeast, fungal pathogens, fruit, and microbiome. Integrating antagonistic yeasts with different microorganisms, natural agents, and physical processes can open new opportunities for designing effective microbial consortia for a tailored biofungicide. Further research is required to develop novel yeast-based biofungicides and understand their precise mode of action in large-scale applications.