Rhodosporidium paludigenum induces resistance and defense-related responses against Penicillium digitatum in citrus fruit

Induced disease resistance against plant pathogens is a promising non-fungicidal decay control strategy. In this study, a potential biocontrol yeast, Rhodosporidium paludigenum, was investigated for its induction of disease resistance against Penicillium digitatum in citrus fruit. The results showed that R. paludigenum is the most effective yeast among three selected yeasts in stimulating the resistance of citrus fruit to green mold. When R. paludigenum was applied 48–72 h before inoculation with P. digitatum, disease incidence and disease severity in citrus fruit significantly decreased. Application of R. paludigenum at concentrations of 1 × 10⁸ and 1 × 10⁹ cells mL⁻¹ respectively resulted in 49.6% and 52.5% reductions in the percentage of infections. Induction of resistance to P. digitatum by R. paludigenum treatment significantly enhanced the activities of defense-related enzymes, including β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase, and polyphenoloxidase, which may be an important mechanism by which the biocontrol yeast reduces the fungal disease of citrus fruit caused by P. digitatum.     

Synergistic effect of Lentinula edodes and Pichia membranefaciens on inhibition of Penicillium expansum infections

The feasibility of using a lyophilized culture filtrate (LCF) from Lentinula edodes at different concentrations to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mold decay in apple fruit was investigated. Application of LCF alone was effective in inhibiting the blue mold rot in apple fruit wounds in a dose-dependent manner. Moreover, the biocontrol activity of P. membranefaciens against blue mold caused by Penicillium expansum in apple fruit could be enhanced by addition of LCF. The combination of P. membranefaciens and LCF resulted in a more effective control than individual treatment of P. membranefaciens or LCF alone. The combined treatment induced higher phenolic accumulation and the upregulation of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities in fruit than LCF or yeast alone, and resulted in a lower lesion diameter and disease incidence. The use of LCF may be an effective method to improve the biological activity of P. membranefaciens, and induced host defenses appear to contribute to the control mechanism of P. membranefaciens and LCF.     

Control of blue mold decay of apple during commercial controlled atmosphere storage with yeast antagonists and sodium bicarbonate

A mixture of two yeast antagonists, Metschnikowia pulcherrima and Cryptococcus laurentii, originally isolated from apples and exhibiting greater biocontrol activity against blue mold of apple than either yeast applied alone, were used in combination with sodium bicarbonate (SBC) in a pilot test in which treated fruit were stored under commercial controlled atmosphere (CA) storage conditions. Conidia of Penicillium expansum, antagonists cells and SBC were added to the drench solution. The treatments were applied to apples by drenching entire bins filled with apples containing 100 wounded fruit evenly distributed among five positions in each bin. The treated fruit were stored in commercial CA storages for approximately six months in the 2005–2006 and 2006–2007 storage seasons and then evaluated for incidence of decay. In both years, the treatments with the antagonist alone or in combination with SBC were equally effective and reduced blue mold incidence by 84–97% in 2005–2006 and 73–82% in 2006–2007. SBC alone significantly reduced blue mold incidence compared to the non-treated control but was less effective than the antagonist alone or in combination with SBC. This pilot test showed that the combination of these two antagonists and SBC can be an effective decay control method under commercial CA conditions, confirming results from our earlier laboratory studies using similarly treated fruit stored under CA conditions.     

Rhodosporidium paludigenum induced resistance in Ponkan mandarin against Penicillium digitatum requires ethylene-dependent signaling pathway

This work determined if the ethylene dependent signal pathway was required for antagonist-mediated fruit defense mechanisms through investigation of disease resistance against Penicillium digitatum in Ponkan mandarin induced by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, and Rhodosporidium paludigenum. Blocking ethylene perception with 1-MCP resulted in an increase in ACS1, ACS2 and ACO expression, and consequently an increase in ethylene production during mechanical wounding and resistance induction. The expression of the ethylene receptors ETR1, ETR2 and ETR4 as well as ethylene response factor (ERF) were observed with similar responses to yeast and 1-MCP stimuli, with ETR3 mRNA accumulation being the most sensitive to yeast application while ERS1 was the least sensitive. When applied at concentrations greater than 500 nL L⁻¹, 1-MCP pre-fumigation significantly reduced the fruit's natural protection and R. paludigenum induced disease resistance to Penicillium decay, indicating that ethylene perception was involved in inducting disease resistance. Moreover, expression of the defensive genes CHI, β-1,3-glucanase, PAL and CIN up-regulated by yeast was inhibited to different degrees by the 1-MCP pre-treatment. This study provides evidence that the biocontrol yeast R. paludigenum increased disease resistance in Ponkan mandarin against P. digitatum infection due to ethylene and signaling pathway dependent mechanisms.     

The potential role of PR-8 gene of apple fruit in the mode of action of the yeast antagonist,Candida oleophila,in postharvest biocontrol of Botrytis cinerea

Several pathogenesis-related (PR) genes of apple have been cloned and their response to different pathogens has been studied. Different PR genes, however, may have a variable response depending on the specific organ or tissue as well as microbe. The objective of the current study was to characterize the expression of specific apple PR genes in fruit tissues in response to the antagonistic yeast, Candida oleophila (a common postharvest biocontrol agent), and the fungal pathogen, Botrytis cinerea (a major postharvest pathogen). Apple PR-5 and PR-8 gene expression was characterized in fruit in response to C. oleophila and B. cinerea. Results indicated that PR-8 expression was significantly elevated in response to both fungi. In contrast, neither C. oleophila nor B. cinerea treatment markedly affected PR-5 expression. The PR-8 gene was then synthesized and cloned into a Pichia pastoris expression system to study the antifungal activity of the PR-8 protein against B. cinerea both in vitro and in vivo. Collectively, the PR-8 gene of apple is associated with the response to B. cinerea infection, and may play a role in the mechanism by which C. oleophila effectively inhibits B. cinerea disease in apple fruit, namely by the induction of this specific host PR gene.     

Induction of H2O2-metabolizing enzymes and total protein synthesis by antagonistic yeast and salicylic acid in harvested sweet cherry fruit

The immersion of sweet cherry fruit in Pichia membranefaciens at a concentration of 5 × 10⁷ cells ml⁻¹ or in salicyclic acid (SA) at 0.5 mM for 10 min reduced the incidence of decay and lesion size caused by Penicillium expansum. Without pathogen inoculation, peroxidase (POD) activity was enhanced in yeast-treated fruit, but activities of catalase (CAT) and superoxide dismutase (SOD) showed a decrease in the same fruit. SA-treatment significantly inhibited CAT activity, but stimulated SOD and POD activities. After inoculation with P. expansum, CAT activity decreased and SOD activity increased in both yeast- and SA-treated fruit. No obvious difference was found in POD activity between treatments and water control. Treatments with yeast and SA changed the expression of POD isozymes. In addition, yeast and SA treatment increased total protein content of sweet cherry and up-regulated 33 and 47 kDa protein bands shown by SDS-PAGE. These results indicated that yeast- and SA-treatments induced synthesis of anti-oxidant enzymes and specific proteins, which may play a role in the resistance against postharvest blue mold.     

Superoxide anion and hydrogen peroxide in the yeast antagonist–fruit interaction: A new role for reactive oxygen species in postharvest biocontrol?

The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that the yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate greater levels of super oxide anion (O₂⁻) on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though yeast antagonists show a high level of O₂⁻ on nutrient-rich media, when applied on fruits around wounds (areas abundant in nutrients) accumulation of O₂⁻, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H₂O₂ accumulation in host tissue. In citrus fruit, the level of H₂O₂ around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water) as early as 18 h after inoculation. Yeast continued to stimulate H₂O₂ production in citrus fruit up to 66 h after inoculation and H₂O₂ levels were still 3-fold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. Similar increase in H₂O₂ accumulation around yeast-inoculated wounds was observed in apple fruit exocarp. The present data, together with our earlier discovery of the importance of H₂O₂ production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases.     

Inhibition of green mold disease in mandarins by preventive applications of methyl jasmonate and antagonistic yeast Cryptococcus laurentii

The objective of this study was to evaluate the preventive activity of methyl jasmonate (MeJA) alone and in combination with antagonistic yeast in suppressing green mold decay in citrus fruit, and to explore the mechanisms involved. At 100 μmol/L, MeJA inhibited disease incidence and lesion diameter of mold decay compared with the control (P < 0.05) The preventive application of Cryptococcus laurentii at 1 × 10⁸ cells/mL combined with 100 μmol/L MeJA reduced green mold incidence compared to the control and the other treatment groups (P < 0.05) when tested in wounded citrus fruit inoculated with Penicillium digitatum. MeJA and C. laurentii induced higher activity of polyphenol oxidase, peroxidase and catalase than control. Moreover, treatment with MeJA and C. laurentii induced a rise in the mRNA expression level of PR5 (pathogenesis-related protein family 5), which was stronger than in the single-treatment groups and the control. In addition, 100 μmol/L MeJA improved the rapid proliferation of C. laurentii in citrus fruit wounds. This combined treatment can induce natural resistance and stimulate the proliferation of antagonistic yeast on the fruit surface.     

Metschnikowia andauensis as a new biocontrol agent of fruit postharvest diseases

Potential antagonists were isolated from the epiphytic flora associated with oranges and pome fruit. A total of 1465 microorganisms were tested in a preliminary screening against blue and green moulds on pome and citrus fruit, respectively. Among them, approximately 3% reduced incidence and severity by more than 50% and 4 microorganisms fulfilled the selection criteria of reduction in severity and incidence by 75%. The most effective was a yeast identified as Metschnikowia andauensis, strain NCYC 3728 (PBC-2), isolated from the surface of ‘Bravo de Esmolfe’ apple fruit cultivated in North Portugal. The biocontrol activity of M. andauensis PBC-2 was dependent on its applied concentration. At 5 × 10⁶ cfu/mL incidence (% of infected wounds) and severity (lesion diameter) were reduced by 62 and 70%, respectively and at 1 × 10⁷ cfu/mL, the greatest reduction was achieved, 90% of incidence and 95% of severity. The broad spectrum of action of M. andauensis PBC-2 was evaluated with effective control being achieved against Rhizopus stolonifer, Penicillium expansum and Botritys cinerea, on ‘Rocha’ pears and on different apple cultivars and against Penicillium digitatum and Penicillium italicum on mandarins and oranges. In semi-commercial trials in cold storage, the reduction of blue mould was 90%. Rapid colonization of fresh apple fruit wounds was observed during the first 24 h of cold storage, followed by a significant population increase during the first 15 days of storage and then the population remained stable until the end of storage.     

Biocontrol of Botrytis cinerea in table grapes by non-pathogenic indigenous Saccharomyces cerevisiae yeasts isolated from viticultural environments in Argentina

Botrytis cinerea, the causal agent of gray mold, is an important disease of grapes. Yeasts are members of the epiphytic microbial community on surfaces of fruits and vegetables and because some yeasts inhibit fungi they are used as biocontrol agents. The major objective of the present work was to isolate yeasts from grapes, vineyard soil, and grape must and select them for their ability to prevent gray mold onset after harvest. Yeasts that were found effective against the fungus were also assayed for their possible pathogenicity in humans. Two antagonism experiments were performed to study the effect of yeasts on B. cinerea, an in vitro study with Czapeck Yeast Extract Agar and an in vivo study with grape berries at 2 °C and 25 °C; both experiments were conducted at different yeast concentrations (10⁵, 10⁶ and 10⁷ cfu/mL). Antagonists were subsequently assayed for their ability to colonize and grow in fruit wounds. The biocontrol yeasts were also examined for their possible pathogenicity in humans: phospholipase and proteolytic activity, growth at 37 °C and 42 °C, pseudohyphal formation and invasive growth. A total of 225 yeasts belonging to 41 species were isolated from must and grape berries and 65 of them, representing 15 species, exhibited in vitro inhibition of B. cinerea at 25 °C. These 65 biocontrol yeasts were subsequently assayed in vivo and 16 of them (15 Saccharomyces cerevisiae and 1 Schizosaccharomyces pombe) showed antagonistic properties against B. cinerea at 25 °C. Only one isolate (S. cerevisiae BSc68) was able to inhibit mycelial growth of B. cinerea on grape berries at both 2 °C and 25 °C. The biomass of this strain in grape wounds increased 221.5-fold at 25 °C after 3 d and 325.5-fold at 2 °C after 10 d of incubation. An increase in the concentration of certain yeasts significantly enhanced their antagonistic activity. All yeast isolates determined as biocontrol agents under in vivo conditions were isolated from fermenting musts. Twelve biocontrol agents (S. cerevisiae) revealed one or more phenotypical characteristics associated with pathogenicity in humans but none of them showed all characteristics together. The fact that there exist few reports on S. cerevisiae and none on Sch. pombe as biocontrol agents against B. cinerea makes our results even more relevant.     

A new strain of Metschnikowia fructicola for postharvest control of Penicillium expansum and patulin accumulation on four cultivars of apple

The efficacy of three antagonistic yeasts, Metschnikowia pulcherrima strain MACH1, M. pulcherrima strain GS9, and Metschnikowia fructicola strain AL27, against Penicillium expansum and patulin accumulation was evaluated on apples stored at room (22 ± 1 °C for 7 days) and cold temperatures (1 ± 1 °C for 56 days). To increase the potential range of application of the biocontrol agents (BCAs), their efficacy was evaluated on four cultivars of apple, i.e. ‘Golden Delicious’, ‘Granny Smith’, ‘Red Chief’ and ‘Royal Gala’. AL27 was more effective than MACH1 and GS9 in the control of blue mold rot and in the reduction of patulin accumulation. The efficacy of AL27 was in most cases similar to the chemical control used, making the antagonist as competitive as chemical fungicides. In vitro experiments showed that AL27 reduced the conidial germination and germ tube length of P. expansum more than the other strains. The three BCAs were more effective in the control of blue mold rot on ‘Golden Delicious’ apples than on the other tested cultivars.     

Real time polymerase chain reaction for rapid and quantitative determination of Cystofilobasidium infirmominiatum on the surfaces of apple,pear, and sweet cherry fruit

The objectives of this study were to develop primers and a real time PCR protocol for the postharvest biocontrol yeast Cystofilobasidium infirmominiatum (Cim). The application of this technology was developed to quantify Cim on the surfaces of apple, two pear cultivars, and sweet cherry fruit treated over a range of concentrations. Statistically significant relationships were observed between Cim DNA on fruit surfaces, expressed as μg/m², and CFU/L of dip suspensions for apple, pear, and sweet cherry. In addition, the relationship for each fruit was significantly different from the other three fruits. Threshold values of concentrations of Cim DNA on the fruit surface were calculated based on regression equations and a dose of 2.0 × 10¹¹ CFU/L of dip suspension, the dose for optimum decay control, and were 4.8, 7.0, 16.5, and 25.2 μg/m² for Bosc pear, Lapins sweet cherry, d’Anjou pear, and Golden Delicious apple, respectively. Monitoring Cim DNA concentration on fruit surfaces will assure that Cim is being properly applied to fruit and that a sufficient number of cells are present for optimum decay control.     

Control of citrus green mold with Aspire is impacted by the type of injury

Aspire, a formulation of the yeast Candida oleophila registered for postharvest application to citrus for the control of green mold (Penicillium digitatum), competes with the pathogen for nutrients at injuries to prevent infection. A major factor affecting efficacy is how quickly and well the yeast colonizes injuries to the fruit surface, including minor injuries involving only oil vesicles. Colonization of puncture-related injuries that either encompassed oil glands or individually ruptured glands was achieved within 1–2 days at 21°C. Colonization of puncture injuries by C. oleophila was comparable after 2 days at 21 and 30°C, but no colonization occurred at 13°C. Ruptured oil glands were colonized more effectively if treated 7 h after injury rather than immediately. Peel oil was toxic to cells of C. oleophila but not to spores of P. digitatum. Candida oleophila colonized punctures more uniformly than individually damaged oil glands, and provided more effective control of green mold originating at punctures than at oil gland injuries. Incubating treated fruit at 30°C for 2 days before storage at 21°C enhanced the control of green mold, and control was significantly improved by the addition of Aspire in one of two trials.     

Control of postharvest diseases of fruit with an invert emulsion formulation of Trichoderma harzianum Rifai

Control of primary postharvest diseases caused by Rhizopus stolonifer, Botrytis cinerea, and Penicillium expansum on a variety of fresh fruit was evaluated with an invert emulsion formulation of Trichoderma harzianum. Diseases evaluated were quantified by the period of protection conferred by the antagonist and the diameter of decay lesions. Treatment of the various fruit species with formulated T. harzianum conidia in an invert emulsion significantly (P ≤ 0.05) reduced the mean lesion diameters of R. stolonifer on apple, pear, peach and strawberry, B. cinerea on grape, pear, strawberry, and kiwifruit, and P. expansum on grape, pear, and kiwifruit in comparison with the control treatment. Significant differences (P ≤ 0.05) were obtained in the mean percent reduction in lesion diameter caused by the same postharvest pathogens on the same fruit species due to the treatment with the formulated T. harzianum conidia relative to control treatment. The greatest mean percent reduction (86.7%) was obtained on apple fruit for the infection with R. stolonifer. Significant differences (P ≤ 0.05) were also obtained in the mean durations of the minimum protection period due to treatment with the formulated T. harzianum against the infection with the same postharvest pathogens on the same fruit species. The longest mean duration of the minimum protection period (up to 59 days) was obtained for unwounded apple fruit against the infection with R. stolonifer. Overall, the results indicate that the treatment with the invert emulsion formulation of T. harzianum protected fruit from infection by the primary postharvest pathogens of the fruit tested for up to 2 months and reduced the diameters of decay lesion up to 86% and is a promising treatment to prolong the postharvest shelf-life of fresh fruit.     

Sensitivity of Penicillium expansum to diphenylamine and thiabendazole and postharvest control of blue mold with fludioxonil in ‘McIntosh’ apples

Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples. Due to the development of resistance to the postharvest fungicide, thiabendazole (TBZ), an increase in blue mold has been observed in apple storages. The sensitivity of three TBZ-sensitive and three TBZ-resistant isolates of P. expansum to diphenylamine (DPA), scald inhibitor, was tested in vitro. Of the 94 isolates, collected later in the storage season, 41% were found resistant to both DPA and TBZ. To manage the fungicide resistance, a reduced-risk fungicide, fludioxonil, was tested against blue mold caused by TBZ-sensitive and -resistant P. expansum on ‘McIntosh’ apples treated with or without 1000 μg ml⁻¹ of diphenylamine. Fruit were assessed for disease and scald incidence during storage. Diphenylamine controlled scald in treated fruit. Higher disease incidence of blue mold was observed in apples treated with diphenylamine and low concentrations of fludioxonil (3, 5, and 75 μg ml⁻¹). DPA neither positively nor negatively affected the control of blue mold when DPA was applied together with 150, 300 and 600 μg ml⁻¹ of fludioxonil during 12 weeks of storage at 4 °C.     

Assessment of blue light treatments on citrus postharvest diseases

Exposure of mature ‘Fallglo’ tangerine fruit to blue light with a photon fluence rate 40 μmol m⁻² s⁻¹ reduced symptom development of blue mold (Penicillium italicum), green mold (Penicillium digitatum), and stem end rot (Phomopsis citri) postharvest decays. Direct exposure to blue light was required to reduce decay caused by Penicillium. Blue light (40 μmol m⁻² s⁻¹) reduced in vitro fungal growth of P. italicum and P. citri. The growth of P. digitatum was more tolerant to blue light, however, the activity of fungal polygalacturonase was reduced by blue light at the intensity of 40 μmol m⁻² s⁻¹. Gas chromatography–mass spectrometry analysis identified 29 chemical constituents in flavedo oil; blue light induced only octanal accumulation. Application of octanal suppressed growth of P. italicum, P. digitatum, and P. citri in vitro. Treatment of fruit with octanal at 5 mM or 50 mM suppressed symptom development caused by Penicillium and P. citri, but discolored the peel. Inhibition of postharvest decays by blue light may be due to a combination of inhibition of fungal growth and induction of defensive responses in the host.     

Effect of rhamnolipids on Rhodotorula glutinis biocontrol of Alternaria alternata infection in cherry tomato fruit

This study was conducted to evaluate the efficacy of the biocontrol yeast, Rhodotorula glutinis with a surfactant produced by Pseudomonas aeruginosa, rhamnolipids, on the inhibition of Alternaria alternata on cherry tomato fruit. A combination of R. glutinis with rhamnolipids at 500 μg/mL was more effective in suppressing A. alternata infection than application of R. glutinis or rhamnolipids alone. Moreover, integration of R. glutinis with rhamnolipids significantly stimulated peroxidase, polyphenoloxidase and phenylalanine ammonialyase activities of cherry tomato fruit, which were stronger than the single treatments. Rhamnolipids with reduced concentrations did not affect the growth of R. glutinis in vitro, but were utilized as carbon sources by yeasts under conditions of limited carbon sources. Moreover, rhamnolipids themselves reduced decay incidence of A. alternata on cherry tomato fruit and promoted the population growth of R. glutinis in vivo. This combined treatment could induce natural resistance as well as accelerate colonization of yeasts on the fruit surface, providing an effective and safe strategy to control postharvest diseases.     

拮抗酵母添加方式对哈密瓜保鲜效果的研究

研究了拮抗酵母以不同载体和不同时间的添加方式对哈密瓜保鲜效果影响。研究表明,1.0%壳聚糖配制的1×10~8 CFU/mLWJ-1酵母悬液,处理哈密瓜能够显著(p<0.05)抑制哈密瓜果实发病率和病斑直径(6.8 mm)的扩展,显著提高WJ-1对M1的生防效力,显示了良好的应用前景;病原菌早于拮抗菌接种且间隔时间越长,拮抗菌对病害抑制效果就越差,甚至出现果实发病率高于对照,说明拮抗菌WJ-1对已经侵染哈密瓜果实软腐病致病菌M1基本没有杀死作用,初步推测空间竞争可能是拮抗菌WJ-1的主要抑菌机理之一。