Using combinations of biocontrol agents to control Botrytis cinerea on strawberry leaves under fluctuating temperatures

Experiments were conducted with Botrytis cinerea on strawberry leaves to investigate where combinations of commercially available biological control agents (BCAs) might control B. cinerea more effectively than individual BCAs. Specifically, we studied the persistence of biocontrol activities, spread of BCAs among leaves, and biocontrol efficacy in relation to application regimes: mixed versus single BCA, pre-versus post-inoculation application, and sequential versus simultaneous application. Three BCA products (Sentinel, Serenade and Trianum) were used for this study. Overall, Serenade did not significantly reduce sporulation of B. cinerea on strawberry leaf discs whereas Sentinel and Trianum gave a similar and significant biocontrol efficacy. Biocontrol efficacy remained almost unchanged 10 days after application at 20/20°C (day/night) or 24/16°C temperature regimes. In contrast, reduced biocontrol efficacy at 26/14°C suggests BCA survival was reduced under these conditions. Incidence of B. cinerea sporulation on leaf discs was ca. 60% higher on leaves that emerged after the BCA application than on leaves directly exposed to BCA, indicating insufficient amount of the BCA had managed to spread to new leaves. Combinations of BCAs, whether applied simultaneously or sequentially (48 h apart), did not improve disease control over the most effective BCA within the combination applied alone. This indicated possible antagonism or interference between the BCAs. Results suggested that there was significant antagonism for most combinations of the three BCAs tested and the degree of antagonism increased as the time from BCA application to pathogen introduction lengthened.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Physiological manipulation and formulation of the biocontrol yeast Pichia anomala for control of Penicillium verrucosum and ochratoxin A contamination of moist grain

The major hurdle in the production of commercial biocontrol agents (BCAs) has been the lack of production of appropriate formulations. Of particular importance is the conservation of viability and ecological competence after application. With this in mind studies were conducted to develop formulations of P. anomala which would have these attributes. Cells were grown in molasses-based medium modified with proline to different water availability levels (0.98 and 0.96) which significantly increased (up to 50%) the content of trehalose and arabitol in the yeast cells during liquid broth fermentation. The use of isotonic solutions for harvesting the yeast cells further increased the endogenous content of these compatible solutes as well as glycerol. Fluidised bed drying of cells at 30–80°C was carried out for 10 and 20 min and showed that viability was significantly decreased at 70–80°C. A temperature of 50°C for 20 min was found to be best for viability (70%) and moisture content of <10%. Several additives for conservation of viability showed that cotton seed flour+skimmed milk was the best treatment when dried at 50°C. The biocontrol efficacy of formulated P. anomala cells was tested in laboratory scale studies and this showed that they inhibited growth of Penicillium verrucosum and reduce ochratoxin A production in moist wheat grain under some combinations of water availability. Physiologically modified formulated yeast cells with increased levels of trehalose and arabitol gave similar efficacy as fresh cells. This suggests that ecophysiological manipulation of such BCAs can result in improved ecological competence of such formulations and effective biocontrol.     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

Biocontrol of strawberry fruit infected by Botrytis cinerea: Effects on the microbial communities on fruit assessed by next‐generation sequencing

Fruit grey mould, caused by the fungus Botrytis cinerea, is known to be a harmful disease of strawberry at postharvest stage. However, effects of an application of biological control agents (BCAs) on strawberry fruit in terms of shift in the microbial community are still unknown. The present research aimed to investigate the effects of an application of BCAs on postharvest microbial populations present on strawberry fruits. Strawberry plants were sprayed with three kinds of BCA, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum‐P (Trichoderma harzianum T22) and Naturalis (Beauveria bassiana ATCC 74040), targeting Botrytis cinerea fungus. Control plots were composed of water and fungicide treatments. Microbial communities (bacteria and fungi) were analysed via next‐generation sequencing on an Illumina MiSeq. Analysis of 16S RNA and ITS rRNA sequences indicated that the BCAs application modified both bacterial and fungal community compositions and diversity. An application of two BCAs together had more effects on microbial community composition than a single application. These results suggest that BCAs can modify bacterial and fungal community composition and diversity on strawberry fruits, which may consequently improve the efficiency and establishment of these products on control of postharvest diseases of fruits, such as grey mould.     

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying

Aims: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA‐1 cells by mild heat treatments to enhanced survival of formulations using spray‐drying. The possible role of heat‐shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. Methods and Results: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33°C during mid‐ (16 h), late‐exponential (24 h), early‐ (30 h) and mid‐stationary (36 h) growth phases. The effect on viability was determined both before and after spray‐drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat‐adapted cells at 33°C in the early‐ or mid‐stationary phases had survival values after spray‐drying significantly higher (P ≤ 0·05) than nonadapted cells. However, viabilities were not high enough to be considered for commercial use with values up to 17%. HSPs were not implicated in thermotolerance acquired by mild heat‐adapted cells as similar viabilities were obtained in the presence of protein inhibitors. Little change was observed in sugar and sugar alcohols with an increase in glucose and arabitol in some treatments. Conclusions: This study suggests that it is possible to induce thermotolerance in biocontrol yeasts such as C. sake. However, this does not improve survival of cells exposed to spray‐drying sufficiently to consider this a suitable formulation method for this biocontrol agent. HSPs, sugars and sugar polyols were not directly responsible for induced thermotolerance in yeast cells. Significance and Impact of the Study: This type of information can be effectively applied to improve the viability of cells in the process of formulation.     

Assessing the viability of three Lactobacillus bacterial species protected in the cryoprotectants containing whey and maltodextrin during freeze-drying process

Freeze-drying of bacteria associates with different stresses such as osmotic pressure, temperature and oxidation, and decreases bacterial viability, which seem to reduce by applying cryoprotectants. The present study evaluated the effect of four cryoprotectants on decreasing the stress caused by freeze-drying process among three Lactobacillus species. Additionally, it highlighted the use of whey and maltodextrin as a substitute for peptone and sucrose in cryoprotectants respectively. The viability of lactobacilli was measured after freeze-drying, 1 month of storage at 25 and 4°C. Based on the results, the viability rate of bacteria in protectants during freeze-drying stage was dependent on their strains. The best viability of Lacticaseibacillus rhamnosus GG and Ligilactobacillus salivarius 20687 was, respectively, observed in the protectants containing sucrose and whey, while Lactiplantibacillus plantarum NRRL B-14768 viability was equal in all protectants. The number of live bacteria reduced significantly by storing bacteria for 1 month at 25°C compared to the 4°C storage. During the storage period, the viability of L. salivarius improved by adding sucrose in protectant. Due to the positive effect of whey and sucrose in the drying and storage stage, on bacterial viability, the protectant consisting of whey and sucrose is suggested for all of the species under study.     

Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans

The use of insects for evaluating the virulence of microbial pathogens and for determining the efficacy of antimicrobial drugs is increasing. When larvae of the greater wax moth Galleria mellonella were incubated at 4 or 37°C for 24 h. prior to infection, they manifested increased resistance to infection by the yeast Candida albicans compared to larvae that had been pre-incubated for 24 h at 30°C. Incubation at 4 or 37°C led to an increase in haemocyte density and the expression of genes coding for gallerimycin, transferrin, an inducible metalloproteinase inhibitor (IMPI) and galiomicin. Peak expression of these genes was recorded at approximately 24 h after the commencement of the 4 or 37°C incubation. These results indicate that exposure of larvae to mild thermal shock conditions induces a protective cellular and humoral immune response mediated by increased numbers of haemocytes and elevated expression of antimicrobial peptides.     

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics–calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p?>?0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.     

Shelf-life of a Biocontrol Pseudomonas putida Applied to Sugar Beet Seeds Using Commercial Coatings

Pseudomonas putida 40RNF is a putative biological control agent (BCA) of Pythium damping-off of sugar beet. The survival of 40RNF during commercial seed treatment and its subsequent shelf-life (i.e. long-term viability and biocontrol activity) were assessed. Two methods were used to apply 40RNF to sugar beet seeds: incorporation into film-coats sprayed on to pre-pelleted seeds and incorporation into the pellet material prior to pelleting. Only 7.1% of applied 40RNF survived film-coating, but an initial concentration of 7 × 10⁸ ensured that 83.3% of a pre-determined target rate of 6 × 10⁷ |pellet was achieved. After 52 weeks of storage at 4°C,the numbers of 40RNF had declined by one to two orders of magnitude, with a decrease of approximately 50% in disease control. After 52 weeks at 18-20°C, 40RNF was below detectable limits (< 100|pellet), yet the biocontrol activity of the seed treatments was not reduced. The survival of 40RNF during incorporation into the pellet material was poor (< 0.2% of those applied, i.e. 5 × 10⁵ pellet). However, bacterial viability and biocontrol efficacy were maintained at 100% of the control value for 24 weeks when stored at 18-20°C. The results indicate that commercial seed treatments and the storage of pellets at ambient temperatures has potential for the introduction of bacterial BCAs into the spermosphere.     

Screening tomato-associated bacteria for biological control of grey mold on tomato

Aiming at discovering effective biocontrol agents (BCAs) against grey mold on tomato caused by Botrytis cinerea Pers., we selected 819 bacterial isolates from the surface as well as the interior of the roots, stems, and leaves of tomato plants grown in B. cinerea-infested fields. In a dual-culture assay, 116 isolates (14.16%) showed antagonism against B. cinerea and fewer ones against five additional tomato-associated fungal pathogens – Pythium ultimum, Phytophthora capsici, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Ralstonia solanacearum. Thirty-one isolates with antagonism to B. cinerea and at least one of the five additional pathogens were assessed for their efficacy in controlling grey mold on tomato in a greenhouse test. Thirteen of them attained the efficacy over 50% and were subjected to the second greenhouse test, in which 12 isolates consistently accomplished the biocontrol efficacy over 50%, with isolates ABc28 and ABc22 achieving the efficacy of 66.71% and 64.90%, respectively. Under greenhouse conditions, the above two as well as isolates ABc2, ABc11 and ABc17 increased tomato biomass by more than 20% in comparison with the control. The 12 antagonistic isolates accomplishing the biocontrol efficacy over 50% in both greenhouse tests were considered potential BCAs against grey mold, which were identified as Pseudomonas spp., Pantoea spp., Bacillus spp. and Chryseobacterium spp. Ten of them were found to produce at least one of the three hydrolytic enzymes (protease, cellulase and chitinase) and/or siderophore, which might be involved in their mechanisms of suppressing the disease. Based on the origin of these 12 strains, the leaf tissue, especially the leaf interior, of tomato plants grown in a B. cinerea-infested field appears to be a good source of potential BCAs against grey mold.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Environmental Effects on Agonistic Interactions between Males of the Cockroach Nauphoeta cinerea

The effects of different environments on the agonistic behavior of males of the cockroach, Nauphoeta cinerea were investigated. We compared the social interactions between pairs of males that had been reared during the period of sexual maturation, when social behavior develops, under one of four environmental treatments: (1) control (28°C with ad libitum food and water) (2) heat stress (35°C, ad libitum food and water) (3) water deprivation (28°C), or (4) food and water deprivation (28°C). Different environments affected the structure of the interactions between males and the behavior of both dominant and subordinate individuals. The mean number of agonistic acts per minute was similar for all treatment groups except the water-deprived group, which was significantly lower. Water-deprived, food- and water-deprived, and heat-stress rearing conditions reduced the stability of agonistic interactions relative to the control group. When reared under stressful conditions, dominant-scored males decreased the number of aggressive acts and increased the number of submissive acts, and subordinate-scored males decreased the number of submissive acts and increased the number of aggressive acts. Thus, stressful environmental conditions can disrupt agonistic interactions and cause both dominant and subordinate male N. cinerea to adopt different behavioral strategies during male-male competition.     

Evaluation of biocontrol agents and potassium silicate for the management of powdery mildew of zucchini

Investigations were conducted under greenhouse and field conditions to evaluate the effects of potential biocontrol agents (BCAs) and soluble silicon (Si) on powdery mildew of zucchini caused by Podosphaera xanthii. Five BCAs were applied as foliar sprays to zucchini leaves and Si was drenched weekly into the rhizosphere of these plants.In the greenhouse, all BCAs provided significant control of powdery mildew with fungal isolates, reducing disease levels by up to 90%. Si alone reduced powdery mildew by as much as 35% and improved the efficacy of most of the biocontrol agents. Higher disease pressure reduced the efficacy of Si on powdery mildew but did not affect the performance of the BCAs. In the field, a disease reduction of 10–70% was achieved by BCAs and Si. Lower temperatures and high humidity ranges were suitable for optimal performances. The efficacy of the bacterial BCA, Serratia marcescens – B15 and silicon diminished at temperatures above 25 °C. The fungal BCAs (Clonostachys rosea – EH and Trichothecium roseum – H20) were better suited to higher temperatures (25–30 °C) and were tolerant of low RH values. Application of K₂SiO₂ to zucchini roots increased the level of Si in the leaves, which was responsible for suppression of the disease.     

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims: The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments. Methods and Results: Cronobacter sakazakii stationary growth‐phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l^?1 H₂O₂, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm^?1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV‐C light (88·55 mW cm^?2, 25°C) treatments. The inactivation kinetics of C. sakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV‐C treatments, the osmotic shock to heat treatments and the oxidative shock to UV‐C treatments. The maximum adaptive response was observed when heat‐shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times. Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions. Significance and Impact of the Study: The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.     

Field applications of improved formulations of Candida sake CPA-1 for control of Botrytis cinerea in grapes

There is increasing interest in the use of biological control agents (BCAs) to replace synthetic fungicides. The aim of this study was to evaluate the effectiveness of combining different formulation strategies on the establishment, survival, persistence and efficacy of Candida sake CPA-1 applied in the field in order to control Botrytis cinerea in grapes. Different formulations of C. sake cells were applied at flowering, pea sized berries, veraison and before harvest. In 2005, heat adapted or non-adapted fresh cells of C. sake were tested. In 2006, these same treatments were tested but, in this case, the C. sake cells were also formulated in an isotonic solution. The compound Fungicover® (FC) an edible coating was evaluated as a potential additive for C. sake treatments. Spray applications of different formulations of C. sake resulted in colonisation of bunches under field conditions and when combined with FC, C. sake had significantly higher survival rates (up to 50% higher) compared with C. sake without additive FC. Formulation of C. sake cells in an isotonic solution combined with FC resulted in Botrytis spp. control similarly than fungicide treatment. The potential of C. sake for biocontrol of Botrytis bunch rot of grapevine has been demonstrated. FC has a beneficial effect on the BCA, improving the persistence of C. sake cells on the host and its efficacy to levels comparable to fungicide treatment. It is possible to broaden the spectrum of use of BCAs using different formulation strategies and to thereby develop practical uses under field conditions.     

The restriction of Cladosporium fulvum and Botrytis cinerea, attacking glasshouse tomatoes, by automatic humidity control

Decreasing periods of atmospheric humidity in excess of 90 and 75 % r.h. by automatic control decreased the incidence of C. fulvum and B. cinerea and sometimes increased tomato yields. The desired value of humidity was not always achieved but nevertheless environments which were both physically and biologically different were obtained with humidistats set at 90 and 75 % r.h. in glasshouses maintaining two temperature régimes–20 °C day and night, or 20 °C by day and 13 °C at night. Less B. cinerea and C. fulvum occurred on tomatoes grown constantly at 20 °C than on those grown in conditions with lower night temperatures. In the latter regime the end-of-season incidence of C.fulvumvas decreased from 25.0% where humidity was not controlled to 2.8% and 0.0% where humidistats were set at 90 and 75 % r.h. In the same conditions the proportion of blemished fruit damaged by B. cinerea decreased from 2.6% to 1.6% and 0.2%.     

Life history and coldhardiness ofAthrycia cinerea (Dipt.: Tachinidae) in western Canada

Athrycia cinerea Coq. is a univoltine parasitoid of the bertha armyworm,Mamestra configurata Walker (Lepidoptera: Noctuidae), in western Canada. This parasitoid overwinters as a pupa in the soil. These pupae are less sensitive than their host to increased cold stress caused by lower temperatures or danger durations of exposure. Exposure to −7.5°C for 140 days did not significantly reduce survival. Survival decreased with exposure to temperatures from −10 to −20°C, but survival was 48% even after 40 days exposure to −20°C. The frequency distribution ofA. cinerea puparia per host is highly contagious. Other aspects of the life history are described. Contribution No. 1403 Winnipeg Research Station.