Effect of incubation temperature and relative humidity on lesion diameter of Botrytis cinerea Pers. and Penicillium expansum Link. on apple fruits.
Plant Pathology Unit, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des déportés 2, 5030 Gembloux, Belgium.
Previous studies carried out in our laboratory demonstrated that the growth of B. cinerea and P. expansum was highly influenced by water activity and temperature in 'in vitro' conditions. Regardless the temperature, a, minimal for growth was < or = 0.89 and equal to 0.89 for P. expansum and B. cinerea respectively. The effect of incubation temperature (5-25 degrees C) and relative humidity (RH 75-98%) on the lesion diameter of two common fungi B. cinerea and P. expansum was studied and modelled in the controlled laboratory conditions in order to validate previous findings. The obtained results showed that only temperature had a significant effect on fungal growth on wounded apples. The relative humidity of air had no direct influence on growth of fungi. The part of variation explained by both studied factors is 52 and 55% respectively for P. expansum and B. cinerea.'Lack of Fit' test was no significant for models, suggesting a greater difference between observed and predicted values. The difference between observed and predicted values was 14 and 29% respectively for P. expansum and B. cinerea. Theses results is in contradiction as compared to in vitro conditions and underlined that the humidity inside wounded apple sites is highly sufficient to start the growth of both postharvest pathogens of apples.
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A Box-Behnken design for predicting the combined effects of relative humidity and temperature on antagonistic yeast population density at the surface of apples.
The objective of this work was to develop models predicting the combined effects of relative humidity (RH, 75-98%), temperature (5-25 degrees C), and initial applied yeast concentration (10(4)-10(8) CFU/ml) on the apple-surface population densities of two biocontrol agents fused against postharvest diseases; the antagonistic yeasts Pichia anomala strain K and Candida oleophila strain O. Experiments were carried out according to a Box-Behnken matrix. Multiple regression analyses showed that both models yielded a good prediction of yeast density. The effect of relative humidity appeared greater than that of temperature. The number of yeast colony-forming units per square centimeter of apple fruit surface increased with increasing relative humidity, temperature, and initial applied yeast concentration. The models predict that under optimal growth conditions (25 degrees C, 98%), strains O and K should reach a density of 10(4) CFU/cm(2) when applied initially at 2x10(7)(strain O) or 10(7) CFU/ml (strain K). The model results suggest that rainfall was likely the principal cause of the variability of yeast efficacy reported for previous preharvest orchard trials spanning two successive years. Temperature may also contribute to this variation. The models developed here are important tools for predicting population densities of both strains on the apple surface within the experimental limits. The use of these results should contribute to achieving yeast densities of 10(4) CFU/cm(2) on apples by controlling yeast application and environmental factors such as relative humidity and temperature. The results of this study also confirm our previous in vitro findings that water activity has a greater effect than temperature on yeast population density.
Response surface methodology study of the combined effects of temperature, pH, and a(w) on the growth rate of Trichoderma asperellum.
Aims: To evaluate the influence of environmental parameters (water activity a(w), temperature, and pH) on the radial growth rate of Trichoderma asperellum (strains PR10, PR11, PR12, and 659-7), an antagonist of Phytophthora megakarya, the causal agent of cocoa black pod disease. Methods and Results: The radial growth of four strains of T. asperellum was monitored for 30 days on modified PDA medium. Six levels of a(w)(0.995, 0.980, 0.960, 0.930, 0.910, and 0.880) were combined with three values of pH (4.5, 6.5, and 8.5) and three incubation temperatures (20, 25, and 30 degrees C). Whatever the strain, mycelial growth rate was optimal at a(w) between 0.995 and 0.980, independently of the temperature and pH. Each strain appeared to be very sensitive to a(w) reduction. In addition, all four strains were able to grow at all temperatures and pH values (4.5-8.5) tested, highest growth rate being observed at 30 degrees C and at pH 4.5-6.5. The use of response surface methodology to model the combined effects of a(w), temperature, and pH on the radial growth rate of the T. asperellum strains confirmed the observed results. In our model, growth of the T. asperellum strains showed a greater dependence on a(w) than on temperature or pH under in vitro conditions. Conclusion: a(w) is a crucial environmental factor. Low a(w) can prevent growth of T. asperellum strains under some conditions. The observed and predicted radial growth rate of strain PR11 showed its greater capacity to support low a(w)(0.93) as compared with other tested strains at 20 degrees C. This is in agreement with its better protective level when applied in medium-scale trials on cocoa plantations. Significance and Impact of the Study: This study should contribute towards improving the biocontrol efficacy of T. asperellum strains used against P. megakarya. Integrated into a broader study of the impact of environmental factors on the biocontrol agent-pathogen system, this work should help to build a more rational control strategy, possibly involving the use of a compatible adjuvant protecting T. asperellum against desiccation.
Int J Food Microbiol. 2006 Dec 15;: 17175054
Predictive modelling of temperature and water activity (solutes) on the in vitro radial growth of Botrytis cinerea Pers.
Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés 2, 5030 Gembloux, Belgium.
The objective of this work was to develop validated models predicting the 'in vitro' effect of a(w) and temperature on the radial growth of Botrytis cinerea. The growth rate (g, mm d(-1)) of B. cinerea was calculated at three incubation temperatures (25 degrees C, 15 degrees C, 5 degrees C) and six water activities (ranging from 0.995 to 0.890). The water activity was adjusted with glucose, NaCl, glycerol, or sorbitol. Statistical analysis showed a significant effect of temperature, solute, a(w), and their two- and three-way interactions on the growth rate. No growth was observed at a(w)=0.93 in the presence of NaCl or at 0.89 in the presence of a non-ionic solute. The maximum colony growth rate decreased when the incubation temperature and water activity was lowered. Secondary models, relating the colony growth rate with a(w) or a(w) and temperature were developed. Optimum a(w) values for growth ranged from 0.981 to 0.987 in glycerol-, sorbitol-, or glucose-modified medium and were close to 1 in NaCl-modified medium. A quadratic polynomial equation was used to describe the combined effects of temperature and a(w) on g (mm d(-1)) in the presence of each solute. The highest and lowest radial growth rates were observed in models based on glucose and NaCl respectively, whatever the incubation temperature. All models prove to be good predictors of the growth rates of B. cinerea within the limits of experiments. The quadratic polynomial equation has bias factors of 0.957, 1.036, 0.950, and 0.860 and accuracy factors of 1.089, 1.070, 1.120 and 1.260 in media supplemented with glucose, NaCl, glycerol and sorbitol respectively. The results from modelling confirm the general finding that a(w) has a greater influence on fungal growth than temperature.
In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc.
Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés, Belgium.
To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.
Isolation and evaluation of bacteria and fungi as biological control agents against Rhizoctonia solani.
Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Belgium. email@example.com
Rhizoctonia solani is one of the most important limiting factors for potato production and storage in Belgium and worldwide. Its management is still strongly dependent on chemical treatments. The aim of this work was to evaluate the possibility of exploiting bacteria and fungi in order to control this pathogen. Among a collection of 220 bacterial strains isolated from different organs of healthy potato plants and rhizospheric soils, 25 isolates were selected using screening methods based on in vitro dual culture assays. The mycelial growth inhibition rate of the pathogen was ranged from 59.4 to 95.0%. Also seven fungal strains isolated from the rhizospheric soil and potato roots showed a highly mycelial growth inhibition of R. solani. The mycelial growth inhibition rate obtained with these fungi was included between 60.0 and 99.4%. From this preliminary study, the further investigations will be planned to determine the bacterial isolates systematic, species of fungal strains by using molecular tools and to assess their efficacy against R. solani in greenhouse trials.
Separate and combined disruptions of two exo-beta-1,3-glucanase genes decrease the efficiency of Pichia anomala (strain K) biocontrol against Botrytis cinerea on apple.
Plant Pathology Unit, University of Agricultural Sciences, 5030 Gembloux, Belgium.
The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71%(parental strain) to 8%(mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.
Plant Pathology Unit, Faculté Universitaire des Sciences Agronomiques de Gembloux Passage des Déportés 2, BE-5030 Gembloux, Belgium.
Candida oleophila strain O was previously selected for its high and reliable antagonistic activity against Botrytis cinerea and Penicillium expansum, two important wound pathogens on post-harvest apples. The application of these antagonistic strains on wound pathogens of Citrus was more recently undertaken. The efficacy of yeast (applied at several concentrations from 10(5) to 10(8) CFU/ml) was assessed against P. digitatum and P. italicum inoculated after one hours (at a concentration of 10(5), 106 and 10(7) spores/ml) on 'Clementine' and 'Valencia late' varieties. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. The antagonistic activity of this strain was also dependent on the incubation time before pathogen inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Finally, the efficacy of biomass of C. oleophila strain O (produced at an industrial scale), and two different formulations of that biomass was assessed in comparison with fungicidal treatment (Thiabendazole) under semi-practical conditions against P. digitatum. This efficacy of strain O (whatever its formulation) was statistically comparable to that for TBZ at commercial dose, indicating that both formulations could be used as an alternative for conventional fungicide in postharvest treatments.
Studying and modelling the combined effect of temperature and water activity on the growth rate of P. expansum.
The effect of solutes, water activity (a(w), 0.890--0.980) and temperature (5--25 degrees C) on the mycelial growth rate of Penicillium expansum was evaluated. The growth rate dropped as the temperature and a(w) of the medium decreased. NaCl was the solute causing the greatest growth rate reduction, followed by glucose, glycerol and sorbitol. Statistical analysis of the results showed a significant effect of solute, a(w), temperature and combinations of two or three of these factors (P<0.0001). Whatever the solutes and a(w) values, the initiation of colony growth required an additional day at 15 degrees C and 5 degrees C as compared to initiation at 25 degrees C. Growth models based on the results obtained with sorbitol and glycerol differed only slightly, with R(2) values of 97.00% and 97.95%, respectively. The response surfaces of both quadratic polynomial models showed that P. expansum should be able to grow at low a(w)(0.890) and that growth at 25 degrees C should be fastest at a(w) values ranging from 0.960 to 0.980. Both models presented a good fit between predicted and observed values.
Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco.
Faculté Universitaire des Sciences Agronomiques, Passage des Déportés, 2, 5030 Gembloux, Belgium.
Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.
Genetic characterization of the yeast Pichia anomala (strain K), an antagonist of postharvest diseases of apple.
Plant Pathology Unit, University of Agricultural Sciences, Gembloux, Belgium.
AIMS To obtain information about the genomic organization of Pichia anomala (strain K) and about its genomic diversity at species and intraspecies level. METHODS AND RESULTS The PFGE karyotype of strain K was composed of four bands ranging in size from 1.1 to 3.2 Mb. The number of chromosomes was estimated at six since bands 2 and 3 seemed to result from the comigration of two chromosomes with similar size. A comparison of strain K and Hansenulawingeii migration profiles led to the estimate of K strain genome size at 11.7 Mb. Comparison with isogenic strains, resulting from the sporulation of strain K, highlighted some major karyotypic differences. Two segregants (KH6 and KH7) showed supernumerary chromosomes and one (KH9) displayed chromosomal length polymorphism. This genomic instability was confirmed by molecular hybridization with four probes, consisting of URA3, LEU2, PAEXG1 and PAEXG2 genes of P. anomala. URA3 and LEU2 probes showed second hybridization signals on supernumerary chromosomes of strain KH7 and on chromosome 6 of strain K for LEU2 only. Karyotypic comparison of seven non-isogenic P. anomala strains revealed chromosomal length polymorphism, a sign of intraspecies variation. CONCLUSIONS This work has supplied information about genome size and chromosome number of strain K of P. anomala. The strain seems to be aneuploid because of the presence of supernumerary chromosomes and additional hybridization signals for URA3 and LEU2 probes in the chromosomal profile of some segregants. The work also highlighted genomic diversity within the P. anomala species. SIGNIFICANCE AND IMPACT OF THE STUDY Results obtained here increase information about the aneuploidy of P. anomala (strain K). Information about the genomic diversity of the segregants will be of great interest for further studies on strain K mode of action. The genome size and chromosomal profile of P. anomala presented here are different from the results obtained elsewhere for Hansenula anomala, while Hansenula is included as a synonym of Pichia. This warrants further studies to investigate this taxonomic relationship.
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Effect of storage conditions (relative humidity, duration, and temperature) on the germination time of Aspergillus carbonarius and Penicillium chrysogenum.
Laboratoire des Procédés Alimentaires et Microbiologiques, UMR Agro-Sup Dijon/Université de Bourgogne, France.
Fungal conidia are disseminated, often in the air, for a certain period of time, prior to contaminating food products. The objective of this study was to examine the effects of the relative humidity, RH (%), time (day), and temperature (°C) during this period of time, called "storage", on the germination time, τ (h), of Aspergillus carbonarius and Penicillium chrysogenum. A Doehlert design was used in the range, 20-100% RH, 2-28days, and 5-25°C. As compared to un-stored conidia, the germination time of conidia stored at 60% RH, 15days, 5°C was increased by 23 and 28%, for A. carbonarius and P. chrysogenum, respectively. Stored conidia exhibited a minimum τ value at 60% RH, and 100% RH for A. carbonarius and P. chrysogenum, respectively. For these species, τ was minimum for 2days of storage. The effect of temperature was RH dependent for A. carbonarius. The germination time of stored conidia was clearly greater than that of fresh conidia obtained in the laboratory. This result should be taken into account to determine the mould free shelf-life of food products.
Forecasting ARIMA models for atmospheric vineyard pathogens in Galicia and Northern Portugal:[i] Botrytis cinerea[/i] spores.
María Fernández-González, Francisco Javier Rodríguez-Rajo, Victoria Jato, María Jesús Aira, Helena Ribeiro, Manuela Oliveira, Iida Abreu
Department of Plant Biology and Soil Sciences. Sciences Faculty of Ourense. University of Vigo. Ourense Spain.
Botrytis cinerea is the cause of the most common disease in the Galician and Portuguese vineyards. Knowledge of the spore levels in the atmosphere of vineyards is a tool for forecasting models of the concentration of spores in order to adjust the phytosanitary treatments to real risk infection periods. The presented study was conducted in two vineyards, one located in Cenlle (Spain) and other in Amares (Portugal), from 2005-2007. A volumetric trap, model Lanzoni VPPS-2000, was used for the aerobiological study. Phenological observations were conducted on 20 vines of three grape varieties in Cenlle (Treixadura, Godello and Loureira) and in Amares (Trajadura, Loureiro and Pedernã), by using the BBCH scale. The highest total spore concentrations during the grapevine cycle were recorded in 2007 in both locations (Cenlle:16,145 spores; Amares:1,858 spores), and the lowest, in 2005 in Cenlle (1,700 spores) and in Amares (800 spores) in 2006. In Cenlle, the best adjusted model was an ARIMA (0,2,2), including the relative humidity four days earlier, while in Amares there was an ARIMA (1,2,3), considering the relative humidity three days earlier and rainfall two days earlier. The t-test showed no significant difference between observed and predicted data by the model.
Phytopathology. 2012 Jun 26;: 22734558
Institute of Biotechnology, Life Science, National Dong-Hwa University, Hualien, Taiwan; firstname.lastname@example.org.
Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative L-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves 2 steps, that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The 2-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an L-amino acid oxidase (Th-L-AAO) was identified from T. harzianum ETS 323. The secretion of Th-L-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-L-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-L-AAO also showed disease control against the development of B. cinerea on postharvest apple fruits and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-L-AAO, suggesting that Th-L-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.
Changes in peroxidase and phenols activity in apple fruit inoculated with antagonistic Pseudomonas fluorescens isolates and Botrytis mali.
Department of Plant Protection, Abourayhan Campus, University of Tehran, P.O. Box 11365/4117, Tehran, Iran.
The biocontrol activity of three isolates of Pseudomonas fluorescens against gray mold of apple fruit caused by Botrytis mali and their ability to induce biochemical defense response in apple tissue were investigated. Apple fruit (Malus domestica) wounds were inoculated with 20 microL bacterial suspension (10(8) CFU mL(-1)) of Pseudomonas fluorescens followed 24 h later by 20 microL of conidial suspension of B. mali (10(5) conidia mL(-1)). The apples were then incubated at 20 degrees C for 11 days. Lesion diameters were evaluated 6 and 10 days after pathogen inoculation. In addition to controlling gray mold, these three isolates of P. fluorescens caused increase in peroxidase activities that reached maximum levels 2-6 days after pathogen inoculation. Phenolic accumulation was increased in apple fruit treated with antagonists and inoculated with B. mali and exhibited the highest level 6-8 days after treatment. The ability of P. fluorescens to increase activities of peroxidase and levels of phenol compounds maybe one of mechanism responsible its biocontrol activity.
Effects of airflow and changing humidity on the aerosolization of respirable fungal fragments and conidia of Botrytis cinerea.
The National Research Centre for the Working Environment, Copenhagen, Denmark. email@example.com
The purpose of this study was to investigate the aerosolization of particles (micro- and macroconidia and fragments) from Botrytis cinerea cultures in relation to potential human inhalation in indoor environments. The influence of the following factors on the aerosolization of B. cinerea particles was studied: exposure to airflow, relative humidity (rh), changing rh, and plant or building materials. The aerodynamic diameter (d(a)) and the respirable fraction of the aerosolized particles were determined. Conidia and fragments of B. cinerea were not aerosolized as a response to a decrease in the rh. In contrast, both micro- and macroconidia and fungal fragments were aerosolized when exposed to an airflow of 1.5 m s(-1) or 0.5 m s(-1). Significantly more particles of microconidial size and fragment size were aerosolized at a low rh (18 to 40% rh) than at a higher rh (60 to 80% rh) when cultures were exposed to airflow. The size of the respirable fraction of the aerosolized particles was dependent on the rh but not on the growth material. At high rh, about 30% of the aerosolized particles were of respirable size, while at low rh, about 70% were of respirable size. During low rh, more fungal (1→3)-β-d-glucan and chitinase were aerosolized than during high rh. In conclusion, exposure to external physical forces such as airflow is necessary for the aerosolization of particles from B. cinerea. The amount and size distribution are highly affected by the rh, and more particles of respirable sizes were aerosolized at low rh than at high rh.
Increase in antioxidant gene transcripts, stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure.
Jia Liu, Michael Wisniewski, Samir Droby, John Norelli, Vera Hershkovitz, Shiping Tian, Robert Farrell
US Department of Agriculture - Agricultural Research Service (USDA-ARS), Kearneysville, WV, USA; Institute of Botany, Chinese Academy of Sciences, Beijing, China.
A pretreatment of the yeast, Candida oleophila, with 5 mM H(2) O(2) for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H(2) O(2)), high temperature (40 °C), and low pH (pH 4). Compared with non-stress-adapted yeast cells, stress-adapted cells exhibited better control of apple fruit infections by Penicillium expansum and Botrytis cinerea and had initially higher growth rates in apple wounds. Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semiquantitative reverse transcription-PCR. Seven antioxidant genes were upregulated. The elevated expression of these genes was associated with less accumulation of reactive oxygen species and a lower level of protein and lipid oxidation under subsequent stresses. These data support the premise that induction of abiotic stress tolerance in biocontrol yeast can improve biocontrol efficacy by upregulation of genes involved in the amelioration of oxidative stress.
Intraspecific variability of growth and patulin production of 79 Penicillium expansum isolates at two temperatures.
Food Technology Department, Lleida University, XaRTA-TPV, Rovira Roure 191, 25198 Lleida, Spain.
Penicillium expansum is the main species responsible for patulin production in apples and pears. Generally, fruit is stored at suboptimal conditions for mould growth and this situation could influence on the intra-species variability in both capability for growth and mycotoxin production. The aim of this research was to assess the impact of suboptimal environmental conditions on the intra-specific variability of P. expansum growth and patulin production using seventy nine isolates of this mould. Petri dishes with Apple Concentrate Agar Medium (ACAM) were inoculated centrally and incubated at two temperatures, one near optimal (20 °C) and the other representative of suboptimal cold storage (1 °C). For each condition, 10 Petri dishes were inoculated, and colony growth and patulin production was measured over time. The Kruskal-Wallis test revealed significant differences among growth rate (μ) and lag phase (λ) within the seventy nine assayed isolates. Coefficients of variation revealed a wider dispersion of μ (mm/day) and λ (days) at 1 °C compared with 20 °C. There were significant differences (p<0.05) among patulin levels (ng/mm²) for the different conditions, values being lower at the lower temperature. Coefficients of variation revealed a wider dispersion of mycotoxin production at 1 °C. In order to address the strain variability in growth initiation and prove the well-established notion of reducing patulin production in foods by preventing fungal growth, a greater number of strains should be included.
Laboratorio de Microbiología y Biotecnología, Instituto Multidisciplinario de Investigación y Desarrollo de la Patagonia Norte (IDEPA, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional del Comahue), Buenos Aires 1400,(8300) Neuquén, Argentina.
To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at -1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.
Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action.
College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, People's Republic of China.
The efficacy of Rhodotorula mucilaginosa against postharvest gray mold, blue mold and natural decay development of apples and the possible mechanisms involved were investigated. The decay incidence and lesion diameter of gray mold and blue mold of apples treated by R. mucilaginosa were significantly reduced compared with the control fruits, and the higher concentration of R. mucilaginosa, the better the efficacy of the biocontrol. R. mucilaginosa also significantly reduced the natural decay development of apples following storage at 20°C for 35 days or at 4°C for 45 days followed by 20°C for 15 days. Germination and survival of spores of Penicillium expansum and Botrytis cinerea were markedly inhibited by R. mucilaginosa in an in vitro test. Rapid colonization of the yeast in apple wounds was observed whether stored at 20°C or 4°C. In apples, the activities of peroxidase (POD) and polyphenoloxidase (PPO) were significantly induced and lipid peroxidation (malondialdehyde (MDA) content) was highly inhibited by R. mucilaginosa treatment compared with those of the control fruits. All these results indicated that R. mucilaginosa has great potential for development of commercial formulations to control postharvest pathogens on fruits. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, PPO and inhibition of lipid peroxidation (MDA content) of apples, so as to enhance the resistance and delay the ripening and senescence of apples.
Food Science Program, Division of Food Systems & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA.
Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l(-1) were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l(-1) can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.