Efficacy of Wickerhamomyces anomalus yeast in the biocontrol of blue mold decay in apples and investigation of the mechanisms involved

Blue mold decay is the one of most important postharvest disease of apples caused by the fungus, Penicillium expansum. This study aimed to investigate the biocontrol efficacy of the yeast, Wickerhamomyces anomalus, on postharvest blue mold decay of apples and the relative defense mechanisms. The results indicated that W. anomalus could significantly reduce the blue mold decay of apples, and the maximum inhibition was obtained when the concentration of W. anomalus was 1?×?10⁸ cells ml^?1. Furthermore, W. anomalus significantly reduced the fruit decay under ambient conditions, without generating any change in fruit quality. In vitro experiments showed that W. anomalus greatly inhibited the spore germination and germ tube elongation of P. expansum. Besides, its ease of adaptation, stable growth and potential colonization of in apple wounds or surfaces indicated that W. anomalus could compete with P. expansum for nutrients and space, leading to considerable inhibition blue mold decay. W. anomalus significantly induced the activities of polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and ascorbate peroxidase (APX) in apples. Moreover, W. anomalus increased the contents of flavonoid and total phenols. All these results suggested that W. anomalus has potential biocontrol efficacy to control the postharvest blue mold decay of apples

     

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.     

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.     

Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition

Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa.     

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.     

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.     

Isolation and Characterization of Antagonistic Bacillus Strains Capable to Degrade Ethylenethiourea

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.     

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.     

Biocontrol of aerial plant diseases in agriculture and horticulture: current approaches and future prospects

Until recently, the majority of research on the biological control of aerial plant diseases was focused on control of bacterial pathogens. Such research led to the commercialization of the biocontrol agent Pseudomonas fluorescens A506, as BlightBan A506™, for control of fire blight of pear. In contrast, chemical fungicides typically have provided adequate control of most foliar fungal pathogens. However, fungicide resistance problems, concerns regarding pesticide residues and revocation of registration of certain widely used fungicides have led to increased activity in the development of biocontrol agents of foliar fungal pathogens. Much of this activity has centered around the use of Trichoderma spp and Gliocladium spp to control Botrytis cinerea on grape and strawberry. The biocontrol agent Trichoderma harzianum T39 is commercially available in Israel, as Trichodex ™, for control of grey mold in grapes and may soon be registered for use in the US. Also targeted primarily against a foliar disease of grapes, in this case powdery mildew caused by Uncinula necator, is the biocontrol agent Ampelomyces quisqualis AQ10, marketed as AQ^{10  TM} biofungicide. Another promising development in the area of foliar disease control, though one which is not yet commercialized, is the use of rhizobacteria as seed treatments to induce systemic resistance in the host plant, a strategy which can protect the plant against a range of bacterial and fungal pathogens. Received 06 February 1997/ Accepted in revised form 05 June 1997     

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.     

Durability of rubberwood (Hevea brasiliensis) treated with peppermint oil,eucalyptus oil,and their main components

Anti-fungal activities of two essential oils (peppermint oil and eucalyptus oil) and their main components (menthol and eucalyptol, respectively) against molds (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) and a white-rot decay fungus (Trametes versicolor) identified from rubberwood surfaces were investigated. The broth dilution method and the agar diffusion technique were employed to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) using the concentration of substances between 100 and 800 μl ml^?1. Inhibitory effects of essential oils and their main components at the MICs against mold growth, fungal decay, and termite attack on rubberwood were further examined by means of the dip treatment method. It was found that MFC values against molds for all treatments examined were about 50–100 μl ml^?1 higher than MIC values. Peppermint oil and menthol exhibited high fungistatic and fungicidal activities, with MICs of 300 μl ml^?1 and 350 μl ml^?1, respectively, against the test molds and the decay fungus. Eucalyptus oil and eucalyptol were also effective against these microbes but at higher concentrations of 600 μl ml^?1 and 500 μl ml^?1, respectively. Only peppermint oil at the MIC was capable of providing a complete protection from mold growth on rubberwood for up to 12 weeks at storage conditions of 25 °C and 100% RH. Both peppermint oil and eucalyptus oil at the MICs showed moderate resistance to fungal decay and high resistance to termite attack.     

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.     

Evaluation of the biological control by the yeast <Emphasis Type="Italic">Torulaspora globosa</Emphasis> against <Emphasis Type="Italic">Colletotrichum sublineolum</Emphasis> in sorghum

The yeasts are microorganisms with great potential for biotechnological applications in diverse areas. The biological control of phytopathogens by yeasts has showed satisfactory results under laboratory conditions, and it has already produced commercial formulations. With this as focus, this work aims to perform in vitro and in vivo evaluations of the action of a Torulaspora globosa yeast strain (1S112), isolated from sugarcane rhizosphere, against the phytopathogenic mold Colletotrichum sublineolum, the causative agent of anthracnose in sorghum. In vitro experiments included the antagonism test in Petri dishes with morphological hyphal evaluation; yeast killer activity; siderophore, volatile compound and hydrolytic enzyme production. In vivo experiments were conducted in greenhouse conditions with a sorghum variety susceptible to C. sublineolum by evaluating the anthracnose disease for 6 weeks. The results indicated that the yeast strain significantly controlled the fungal growth, either in vitro or in vivo. The strain of T. globosa exhibited killer activity against two sensitive strains, which is a novel capacity for this species. The yeast did not produce siderophores, volatile compounds or hydrolytic enzymes, although it has reduced the mycelial growth, resulting in hyphal deformities but not cell death. The yeast controlled the anthracnose disease in sorghum, either inoculated before or after the fungal spores, suggesting that the competition for space and nutrients to dominate the mold and killer toxin production, altering the hyphal morphology, are mechanisms utilized by the yeast in the biocontrol.     

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.     

Characterization of new bacterial biocontrol agents Acinetobacter,Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea

A collection of 282 bacterial isolates from the rhizosphere and different organs of healthy field-grown grapevine plants was obtained and screened for their ability to protect grapevine leaves against Botrytis cinerea, the causal agent of gray mold. Twenty-six strains effectively controlled B. cinerea infections on leaves. After phenotypic and molecular analysis, seven strains were identified as Pseudomonas fluorescens PTA-268 and PTA-CT2, Bacillus subtilis PTA-271, Pantoea agglomerans PTA-AF1 and PTA-AF2, and Acinetobacter lwoffii PTA-113 and PTA-152. In vitro antifungal experiments showed that from these seven strains, only PTA-AF1 and PTA-CT2 exhibited a direct antagonism against B. cinerea. Furthermore, the biocontrol activity of the seven bacteria was associated with differential induction of defense-related responses lipoxygenase, phenylalanine ammonia-lyase and chitinase in grapevine leaves. Our results show that the selected bacteria can efficiently protect grapevine leaves against gray mold disease through an induction of plant resistance and in some cases by an additional antagonistic activity.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

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.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.