Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

Acid adaptation and biocontrol efficacy of antagonistic marine yeast Rhodosporidium paludigenum

The objectives of this work were to assess the optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the biocontrol activity of non-adapted and acid-adapted yeasts in controlling apple blue mold caused by Penicillium expansum. R. paludigenum grown in malic and lactic acid treatments were stimulated after 12 h incubation. Moreover, medium modified with malic and lactic acid significantly enhanced the acid tolerance of R. paludigenum (p?<?0.05). In acid tolerance response test, the highest viability of R. paludigenum was obtained at initial pH of 5.5 in the NYDB medium modified with malic acid (91.6 %). In addition, all R. paludigenum treatments significantly reduced the disease incidences and lesion diameters of blue mold in apples. Furthermore, there was no significant difference between acid-adapted and unadapted yeasts in the apple wounds after 48 h dynamics. Acid stress improved R. paludigenum viability under acidic conditions. However, there was no significant difference between acid-adapted and unadapted yeasts in controlling P. expansum on apple fruit (p?<?0.05). These results indicate the potential for maintaining the survival level of biocontrol agents by physiological inducement strategy.     

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10⁸ CFU ml^?1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10⁵ spores ml^?1; While the blue mold decay was completely inhibited at concentrations of 5 × 10⁸ CFU ml^?1, at challenged with P. expansum spores suspension of 5 × 10⁴ spores ml^?1_. These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10⁵ CFU per wound to 2.24 × 10⁷ CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.     

Effect of light quality on Bacillus amyloliquefaciens JBC36 and its biocontrol efficacy

Light is one of the most important environmental signals regulating physiological processes of many microorganisms. However, very few studies have been reported on the qualitative or quantitative effects of light on control of postharvest spoilage using antagonistic bacteria. In this study, we investigated the effects of white, red, green, and blue light at photon flux densities of 40, 240, and 360 μmol m^?2 s^?1 on Bacillus amyloliquefaciens JBC36 (JBC36), which has been reported as a promising candidate for biocontrol of green and blue mold on mandarin fruit. With the exception of blue light at 240 and 360 μmol m^?2 s^?1, light generally stimulated growth of JBC36 compared to the controls grown in the dark. Red light increased swarming motility irrespective of intensity and significantly enhanced biofilm formation at 240 μmol m^?2 s^?1. Production of antifungal metabolites and antifungal activity on Penicillium digitatum was also affected by light quality. Interestingly, antifungal activity was significantly increased when JBC36 and P. digitatum was co-incubated under red and green light at an intensity of 240 μmol m^?2 s^?1. We also demonstrated that the quality of light resulted in changes in colonization of JBC36 on mandarin fruit and control of green mold. In particular, red light increased the population level on mandarin fruit and biocontrol efficacy against green mold. These results represent the first report on the effect of light quality on an antagonistic bacterium for the control of postharvest spoilage. We believe that an improved understanding of the JBC36 response to light quality may help in the development of strategies to increase biocontrol efficacy of postharvest spoilage.     

Salicylic Acid Enhances Biocontrol Efficacy of the Antagonist Cryptococcus laurentii in Apple Fruit

Biological control and induced resistance are two of the promising approaches to the control of postharvest diseases. This study was conducted to evaluate the efficacy of salicylic acid (SA) alone or in combination with an antagonistic yeast, Cryptococcus laurentii, in controlling the blue mold disease caused by Penicillium expansum on apple fruit wounds. SA alone significantly inhibited the spore germination of P. expansum in vitro when its concentration was increased to 1000 μg ml⁻¹, but it was not effective in controlling the disease in vivo. Simultaneous application of SA and C. laurentii to the wounds on the apple fruit surface showed that SA could improve the efficacy of C. laurentii against P. expansum in a concentration-dependent manner, being most effective at 10 μg ml⁻¹ but less effective at a higher or lower concentrations. Besides reducing the blue mold incidence in the local wound sites, the combination of C. laurentii with SA at 10 μg ml⁻¹ also had a synergistic effect on the induction of fruit resistance to the disease, which might be associated with a rapid increase in peroxidase, phenylalanineamonialyase and lipoxygenase activities. In addition, SA at 100 μg ml⁻¹ or above showed an adverse effect on the growth of C. laurentii in vitro and in vivo, whereas it had no effect when its concentration was decreased to 10 μg ml⁻¹ or lower. This suggested that SA could enhance the biological activity of C. laurentii in apple fruit by inducing resistance to pathogens based on the antagonistic activity of C. laurentii.     

Screening of antagonistic yeast strains for postharvest control of Penicillium expansum causing blue mold decay in table grape

Blue mold decay caused by Penicillium expansum is one of the most important postharvest diseases of grapes, leading to considerable economic losses. Regarding the increasing demand for pesticide-free foods, this study aimed to find potential yeast strains for biological control of blue mold on table grapes. A total of 50 yeast strains were screened for antagonistic activity against P. expansum using the dual culture method and six strains significantly inhibited the fungal growth. All six yeast strains (Coniochaeta euphorbiae, Auerobasidium mangrovei, Tranzscheliella sp., Geotrichum candidum, Basidioascus persicus, and Cryptococcus podzolicus) reduced the fungal growth (29.6–85.0%) and the decay degree of wounded grape berries inoculated with P. expansum while G. candidum was found to be the most efficient biocontrol agent. On the basis of antagonistic activity, the strains were further characterized by in vitro assays involving inhibition of conidial germination, production of volatile compounds, iron competition, production of hydrolytic enzymes, biofilm-forming capacity, and exhibited three or more putative mechanisms. To our knowledge, the yeasts are reported for the first time as potential biocontrol agents against the blue mold of grapes but more study is required to evaluate their efficiency related to field application.     

Biocontrol of apple blue mould by new yeast strains: Cryptococcus albidus KKUY0017 and Wickerhamomyces anomalus KKUY0051 and their mode of action

Seeking new yeast strains having the ability to protect apple fruits against blue mould for a long time under different storage conditions was the main goal of this work. Based on the in vitro test, yeast strains KKUY0017 and KKUY0051 were selected as the most effective antagonists against Penicillium expansum. Sequencing of 26S rDNA of both yeasts confirmed that the identity of KKUY0017 and KKUY0051 was Cryptococcus albidus and Wickerhamomyces anomalus, respectively. The two strains protected the apple fruits from the blue mould disease under a wide range of temperature (5–30°C); however, W. anomalus KKUY0051 was more effective. At 25°C, W. anomalus KKUY0051 involved in the reduction of disease severity and disease incidence of blue mould by 56.49% and 57.78%, respectively. When either of the two yeasts was applied in concentration of 10⁸ or 10⁹ cells/mL, the maximum reduction in disease severity and disease incidence was achieved. Under cold storage (5°C), both yeast strains succeeded to protect the apple fruits free from the infection up to 24 days. Electron micrograph showed a fit attachment between the cells of C. albidus KKUY0017 and the fungal hyphae leading to the degrading of the hyphae; however, W. anomalus killed the fungal hyphae without direct attachment to them. Gas chromatography–mass spectrometry analysis of the cell-free extract of W. anomalus KKUY0051 revealed the presence of toxic compounds such as the nitrophenol derivatives. The results support the assumption that the main mode of action of this yeast is by killer toxins. We conclude that application of these yeasts under cold storage condition could keep the apple fruits free from blue mould infection for a long time.     

Antagonistic action of siderophores from Rhodotorula glutinis upon the postharvest pathogen Penicillium expansum

Rhodotorulic acid produced by Rhodotorula glutinis strains improved the biological control of blue rot caused by Penicillium expansum in harvested apples. The production of the siderophore was closely associated with the iron concentration in the medium. Thus, very low additions of the metal reduced the siderophore production considerably. The antagonistic effect of R. glutinis and rhodotorulic acid was studied by using in vitro and in vivo assays. In the in vitro assays, rhodotorulic acid reduced the growth of P. expansum, whereas the chelate (rhodotorulic acid plus iron) did not. Siderophore antagonism was then related to competition for iron. In biocontrol assays on apple wounds, the blue mold was more effectively controlled by the antagonistic agent plus siderophore than by the antagonistic agent alone. The disease incidence (DI: percentage of treated wounds that developed rot) was 34% when apples were protected by R. glutinis alone, whereas it was 6% when the fruits were protected by R. glutinis plus rhodotorulic acid.     

Effects of yeast antagonist in combination with UV-C treatment on postharvest diseases of pear fruit

The yeast antagonist Candida guilliermondii and ultraviolet-C (UV-C) treatment were investigated for controlling infection following artificial inoculation with Penicillium expansum or Botrytis cinerea, or natural infection in pear fruit stored at 20°C. Applied separately, both C. guilliermondii and UV-C (5 kJ m⁻²) effectively inhibited decay caused by P. expansum or B. cinerea, and natural infection. The combination of C. guilliermondii and UV-C showed better control efficacy. Application of UV-C did not affect the growth of C. guilliermondii in pear fruit wounds, while UV-C induced a significant increase in the activities of chitinase, β-1,3-glucanase, catalase and peroxidase in pear fruit. The mechanism by which UV-C enhanced the biocontrol efficacy of C. guilliermondii may be related to the elicitation of defense responses in pear fruit. The combination of C. guilliermondii and UV-C radiation could be a promising method for the control of P. expansum and B. cinerea in pear fruit.     

Biological control of postharvest blue mold of oranges by <Emphasis Type="Italic">Cryptococcus laurentii </Emphasis>(Kufferath) Skinner

Cryptococcus laurentii (Kufferath) Skinner was evaluated for its activity in reducing postharvest blue mold decay of oranges caused by Penicillium italicum in vitro and in vivo. The results showed that washed cell suspensions of yeast provided control of blue mold decay better than yeast in culture broth. Autoclaved cell culture and cell-free culture filtrate failed to provide protection against the pathogen. The concentrations of antagonist had significant effects on biocontrol effectiveness. When the washed yeast cell suspension reached the concentration of 1 × 10⁹ CFU/ml, challenged with pathogen spore suspension at 1 × 10⁴ spores/ml, the blue mold decay was completely inhibited during 5 days of incubation at 20 °C. No complete control was obtained when oranges were stored at 4 °C for 30 days, but the decay was distinctly prevented. Efficacy of C. laurentii was maintained when applied simultaneously or prior to inoculation with P. italicum. Efficacy was reduced when C. laurentii was applied after inoculation. In drop-inoculated wounds of oranges, the populations of C. laurentii increased by approximately 50-fold during the first 24 h at 20 °C. The maximum yeast populations, approximately 250-fold over the initial populations, were reached 15 days after inoculation at 4 °C.     

A new improved strategy for the selection of cold-adapted antagonist yeasts to control postharvest pear diseases

Postharvest diseases cause considerable losses of harvested fruits during transportation and storage. Many yeast species have been reported as good antagonists against postharvest pear pathogens. In this work, we used a novel selection strategy that involves the isolation of yeasts from washing fluids, showing biocontrol activity against a regional Penicillium expansum strain (primary screening), originally obtained from fruit wounds after long time storage at ?1/0°C. About 26 isolates representative of the 11 yeast species identified in the 27 selected washing waters were chosen to be evaluated in a secondary screening against a regional Botrytis cinerea strain on pear wounds. Among yeasts tested, 38% showed complete control of P. expansum, but only 15% reduced the decay incidence of B. cinerea to 60–80% at ?1/0°C. These results reveal that some of the yeasts found can be biological alternatives to fungicides in the control of P. expansum and B. cinerea infections. Based on the data obtained, our strategy seems to be much more effective than the previously reported methods in obtaining successful biocontrol agents.     

Biocontrol of green and blue molds in postharvest satsuma mandarin using Bacillus amyloliquefaciens JBC36

In order to control postharvest rot of satsuma mandarin (mandarin) fruits, a new strain of Bacillus amyloliquefaciens JBC36 (JBC36) was isolated from rhizosphere of a mandarin orchard and tested for its suppression of decay due to green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, and its mode of action was investigated. In addition, carnauba wax-based and paraffin oil-based coating formulations were developed to increase the control efficacy of the antagonist. The strain JBC36 at 10⁸ CFU mL^?1 inhibited incidence of green and blue molds on wounded mandarin fruits with control efficacies of 88 and 80.2%, respectively. Mycelial growth and spore germination of P. digitatum and P. italicum were strongly inhibited in the presence of JBC36 or antagonistic metabolites. In order to determine antifungal activity, three kinds of antibiotics were isolated by reverse-phase high performance liquid chromatography (RP-HPLC) and identified as lipopeptide families, iturin A, fengycin and surfactin by RP-HPLC and thin-layer chromatography (TLC) analysis. Fengycin was further identified as C16 fengycin A by liquid chromatography/electrospray ionisation-mass spectrometry (LC/ESI-MS) and mass spectrometry/mass spectrometry (MS/MS) analysis. Volatile organic compounds from the antagonist also reduced the mycelial growth of P. digitatum and P. italicum. Carnauba wax-based and paraffin oil-based coating formulations containing 10⁸ CFU mL^?1 of JBC36 efficiently decreased the incidence of green mold with control efficacy of 91 and 80.9%, respectively. Overall, the antagonistic rhizobacterium JBC36 is a promising biocontrol agent for use in preventing postharvest spoilage of mandarin fruits by green and blue molds.     

Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit

Biocontrol potential of Bacillus subtilis strain CPA-8 was tested against the main postharvest diseases of orange, apple and stone fruit. Previously, CPA-8 growth was characterized and its antifungal activity in vitro determined against Botrytis cinerea, Monilinia laxa, M. fructicola, Penicillium digitatum, P. expansum, and P. italicum. In vivo activity against these pathogens was tested by treating fruits with cells, endospores or cell free supernatants. CPA-8 treatments cannot control decay caused by P. digitatum and P. italicum on oranges. The higher concentrations of CPA-8 studied were effective in controlling B. cinerea on apple, showing grey mold incidence from 70 to 12.5% in comparison with 100% in the control. However, in general, CPA-8 treatments were not effective in controlling P. expansum. The best results of CPA-8 treatments were obtained in stone fruit against M. laxa and M. fructicola where most treatments resulted in brown rot incidence of 0% compared with 70 and 90% in the control. Based on these results, cultures, cells and cell free supernatants at different concentrations were tested against M. laxa and M. fructicola on stone fruit. Most bacterial concentrations were effective in controlling M. laxa and M. fructicola as well as or better than Serenade® Max, in some treatments showing brown rot incidences of 0% in comparison with 100% of control. Bacterial populations of CPA-8 were maintained stable or increased up to 2-log inside wounds, showing the ability of the bacteria to colonize injured tissues. Experimental evidence suggests that B. subtilis CPA-8 has biocontrol potential for control of postharvest disease on several fruit types.     

Gamma irradiation‐induced disease resistance of pear (Pyrus pyrifolia “Niitaka”) against Penicillium expansum

In this study, the effects of gamma irradiation on the resistance of pear fruit against Penicillium expansum, the causal agent of blue mould disease, were investigated. A low dose of gamma irradiation for 14 days increased the disease resistance and firmness of pear fruits. Remarkably, exposure to 200 Gy of gamma irradiation significantly maintained fruit firmness, markedly reduced disease incidence and enhanced the activity of defence‐related enzymes (e.g., β‐1,3‐glucanase, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase) and expression of pathogenesis‐related (PR) genes (e.g., PR‐1, PR‐3 and PR‐4). Therefore, the gamma irradiation‐induced resistance against P. expansum involves both metabolic changes and the induction of expression of defence‐related genes. In addition, scanning electron microscopic analysis revealed that gamma irradiation significantly inhibits the growth of P. expansum. These results suggest that exposure of mature harvested pear fruits to artificial gamma irradiation confers fungal disease resistance; therefore, gamma irradiation represents an important strategy for controlling postharvest diseases in pear fruit.     

Inhibitory Effect of Selenium Against Penicillium expansum and Its Possible Mechanisms of Action

Some organic and inorganic salts could inhibit the growth of many pathogens. Selenium (Se), as an essential micronutrient, was effective in improving the plant resistance and antioxidant capacity at a low concentration. Penicillium expansum is one of the most important postharvest fungal pathogens, which can cause blue mold rot in various fruits and vegetables. In this study, the inhibitory effect of Se against P. expansum was evaluated. The result showed that Se strongly inhibited spore germination, germ tube elongation, and mycelial spread of P. expansum in the culture medium. The inhibitory effect was positively related to the concentration of Se used. Fluorescence microscopy observation of P. expansum conidia stained with propidium iodide (PI) indicated that the membrane integrity decreased to 37 % after the conidia were treated with Se (20 mg/l) for 9 h. With the use of an oxidant-sensitive probe 2,7-dichlorofluorescin (DCHF-DA), we found that Se at 15 mg/l could induce the generation of intracellular reactive oxygen species (ROS). Furthermore, methane dicarboxylic aldehyde (MDA) content, hydrogen peroxide (H₂O₂), and superoxide anion (O₂ ^?) production rate in P. expansum spores exposed to Se increased markedly. Compared with the control, the activities of superoxide dismutase (SOD) and the content of glutathione (GSH) were reduced, confirming that damage of Se to cellular oxygen-eliminating system is the main reason. These results suggest that Se might serve as a potential alternative to synthetic fungicides for the control of the postharvest disease of fruit and vegetables caused by P. expansum.     

Control of apple blue mould disease with Torulaspora delbrueckii in combination with Silicon

In this study, Torulaspora delbrueckii alone and in combination with silicon were evaluated for the control of apple blue mould disease caused by Penicillium expansum. In vitro, the antagonistic effects of T. delbrueckii in controlling mycelial growth of P. expansum on potato-dextrose-agar (PDA) in dual cultures, and the growth of P. expansum alone with cell-free metabolites and volatile components of T. delbrueckii were assayed. In vitro, to evaluate the direct effect of silicon on mycelial growth of pathogen, silicon at different concentrations (0.2, 0.4, 0.6, 1 and 2% (wt./vol.)) was added to PDA medium. Silicon at 0.6% (wt./vol.) and above concentrations completely inhibited the mycelial growth of P. expansum. However, it had no significant effect on population dynamics of yeast in vitro and in apple wounds. In vivo, silicon at 0.2 and 1% (wt./vol.) in combination with antagonistic yeast (1 × 10⁸ cell/ml) was a more effective approach to reduce the lesion diameter of blue mould decay of apples than the application of silicon or T. delbrueckii alone at 20 and 4°C, respectively.     

Biological control of postharvest diseases of apples,peaches and nectarines by Bacillus subtilis S16 isolated from halophytes rhizosphere

Penicillium expansum, Botrytis cinerea and Colletotrichum acutatum are the most common postharvest pathogens of apples, peaches and nectarines. In this study, 96 bacteria were isolated from halophytes rhizosphere and assayed for biocontrol activity under in vitro conditions. Among the 96 isolates tested, isolate S16 effectively inhibited the growth of P. expansum, B. cinerea and C. acutatum. The isolate S16 has reduced 78.33±1.53 to 82.98±2.13% of disease severity in apples, peaches and nectarines. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely surfactins, iturins and fengycins. Lipopeptides and hydrolytic enzymes produced by the isolate S16 play an important role in the antifungal activity. Polymerase chain reaction analysis using ituD, srfAD, fenD and fenE gene-specific primers showed that the isolate S16 carry sequences similar to ituD, srfAD, fenD and fenE genes. Based on the 16S rDNA sequencing, the effective isolate S16 was identified as Bacillus subtilis.