Improvement of Candida sake biocontrol activity against post-harvest decay by the addition of ammonium molybdate

AIM: To improve the efficacy of Candida sake by adding ammonium molybdate to control post-harvest decay in Golden Delicious apples. METHODS AND RESULTS: In laboratory trials, C. sake 2 x 10(6) cfu ml(-)1 plus 5 mmol l(-1) ammonium molybdate greatly reduced Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer in apples stored at 20 degrees C for 7 days, and reduced by more than 90% blue and grey mould in apples stored at 1 degrees C for 60 days. The consistency of these results was maintained in semi-commercial trials at 1 degrees C in air and in a low oxygen atmosphere for 120 days. The pre-harvest application of C. sake 2 x 10(7) cfu ml(-1) plus 1 mmol l(-1) ammonium molybdate did not improve post-harvest biocontrol of blue mould. The population of C. sake significantly decreased in the presence of ammonium molybdate in apple wounds. CONCLUSION: The addition of ammonium molybdate at 5 mmol l(-1) to C. sake enhanced the efficacy of the antagonist to control post-harvest diseases on apples. SIGNIFICANCE AND IMPACT OF THE STUDY: Ammonium molybdate significantly reduces the amount of C. sake biomass required to achieve post-harvest disease control, with a consequent reduction in costs. This may be useful in the industrial production of C. sake.     

Effect of package and storage conditions on viability and efficacy of the freeze-dried biocontrol agent Pantoea agglomerans strain CPA-2

AIMS: To reduce concentrations of protective and rehydrating media and to evaluate the effect of storage temperature, packaging and atmosphere conditions on the stability of freeze-dried Pantoea agglomerans cells. Efficacy against Penicillium digitatum of freeze-dried cells in orange fruits was also evaluated. METHODS AND RESULTS: Several concentrations of protective and rehydration media were tested to reduce processing costs. Freeze-dried cells were packed in glass vials or plastic bags under vacuum or nitrogen conditions at 4 and 25 degrees C. After 1 and 3 months, efficacy of freeze-dried P. agglomerans against P. digitatum was tested. CONCLUSIONS: The results indicate that it is possible to reduce the concentration of non-fat skimmed milk as a rehydration medium from 10% to 1%, maintaining viabilities of 100%. Moreover, freeze-dried cells could be stored in glass vials or in high barrier plastic bags at 4 degrees C for 3 months while maintaining high viabilities and efficacy against P. digitatum. SIGNIFICANCE AND IMPACT OF THE STUDY: The major obstacle in the commercialization of biocontrol products is the development of a shelf-stable formulated product that retains biocontrol activity at a level similar to that of fresh cells. This study suggests that it is possible to maintain viability and efficacy of freeze-dried P. agglomerans cells for at least 3 months.     

Water activity,temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

The effect of nitrogen and carbon sources on growth of the biocontrol agent Pantoea agglomerans strain CPA-2

AIM: The effect of several nitrogen and carbon sources on the growth of Pantoea agglomerans (strain CPA-2) was studied for the first time. METHODS AND RESULTS: Synthetic nitrogen and carbon sources were tested to obtain a suitable medium. Synthetic yeast extract provided maximum growth and disaccharides such as sucrose, lactose and trehalose improved this growth significantly from 3.2 x 10(9) to 5.5 x 10(9) cfu ml-1. CONCLUSION: Pantoea agglomerans can be produced in a combination of nitrogen sources such as yeast extract with carbohydrates such as sucrose in shake flask and a laboratory fermenter (5 l). SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest good production of this biocontrol agent on a laboratory scale and the potential of scaling up the process.     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments

AIMS: To study the improvement of tolerance to low water activity (aw) and desiccation during spray drying in Pantoea agglomerans cells subjected to mild osmotic stress during growth. METHODS AND RESULTS: The micro-organism was cultured in an unmodified liquid (control) or in aw-modified media, and viability of these cells was evaluated on unstressed (0.995) and 0.96 aw stressed solid media, in order to check total viability and aw stress tolerance respectively. Significant improvements in viability on unmodified medium were observed with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48 h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water stress tolerance were achieved with low aw media. Cells grown for 24 h in NaCl 0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glycerol 0.97 aw showed improved aw stress tolerance in comparison with control cells. The best results were obtained with NaCl treatments (0.98 aw and 0.97 aw) which also exhibited better survival rates than control cells during spray-drying process and maintained their efficacy against postharvest fungal pathogens in apples and oranges. CONCLUSIONS: NaCl treatments are very appropriate for improving P. agglomerans low aw tolerance obtaining high production levels and maintaining biocontrol efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Improving stress tolerance of biocontrol agents could be an efficient way to obtain consistency and maintain efficacy of biological control under practical conditions.     

Evaluation of antifungal activity of carbonate and bicarbonate salts alone or in combination with biocontrol agents in control of citrus green mold

The aim of this research was to determine if the attacks of green mold on orange could be reduced by edible salts alone or in combination with biocontrol agent. For this purpose toxicity to Pantoea digitatum and practical use of sodium carbonate (SC), sodium bicarbonate (SBC) and potassium carbonate, and potassium bicarbonate alone or in combination with antagonistic bacteria (Pseudomonas fluorescens isolate PN, Bacillus subtilis isolate VHN, Pantoea agglomerans isolate CA) to control green mold were determined. All were fungistatic. SC and SBC were equal and superior to the other salts for control of green mold on oranges inoculated 6h before treatment and were chosen for subsequent trails under cold storage conditions. The biocontrol agents were found completely tolerant to 3% sodium bicarbonate and sodium carbonate at room temperature; although their culturability was reduced by > 1000-fold after 60 min in 1% other salt solutions. Satisfactory results were also obtained with the combined treatment for control of green mold. A significant increase in biocontrol activity of all isolate was observed when combined with sodium carbonate and sodium bicarbonate. The treatments comprising CA combined with SB was as effective as fungicide treatment. Thus, use of sodium bicarbonate treatment at 3% followed by the antagonist P. agglomerans CA could be an alternative to chemical fungicides for control of green mold on oranges.     

Role of Pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (Gossypium hirsutum) grown in the field

AIMS: To investigate the aetiology of seed and boll rot of cotton grown in South Carolina (SC). METHODS AND RESULTS: Bacteria were isolated from diseased locules of cotton bolls collected in a field in SC, USA and tested for the ability to cause comparable disease symptoms in greenhouse grown cotton fruit. Spontaneously generated rifampicin-resistant (Rif(r)) mutants of the isolates were used in confirmatory pathogenicity tests. Resistance to the antibiotic was both stable and effective in differentiating between an inoculated Rif(r) strain, rifampicin-sensitive contaminants and/or endophytes. A series of inoculation methods was tested at various boll developmental stages and at different fruiting nodes on the plant. Field disease symptoms were reproduced by inoculating bolls at 2 weeks postanthesis with bacterial suspensions ranging from 10(3) to 10(6) CFU ml(-1). Pathogenic isolates were categorized as Pantoea agglomerans on the basis of phenotype testing, fatty acid profiling (similarity index = 0.94), and 16s ribosomal DNA sequence analysis (99% nucleotide identity). CONCLUSIONS: Pantoea agglomerans isolates from field-collected immature, diseased cotton caused comparable infection symptoms in greenhouse produced cotton fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: In 1999, significant yield losses in SC cotton resulted from a previously unobserved seed and boll rot that has since been reported in other southeastern states. This study demonstrated a role of P. agglomerans in producing opportunistic bacterial seed and boll rot of cotton.     

Relative importance of amino acids, glycine-betaine and ectoine synthesis in the biocontrol agent Pantoea agglomerans CPA-2 in response to osmotic, acidic and heat stress

AIMS: The objective of this work was to determine the role of different compatible solutes in adaptation of Pantoea agglomerans CPA-2 at different stages of growth to solute (0.98, 0.97, 0.96 aw), heat (35 and 40 degrees C) and acidic (pH 4.0, 5.0, 6.0) stress. METHODS AND RESULTS: Solute stress was imposed by using NaCl, glucose or glycerol, and pH was imposed with malic and citric acids. The accumulation of glycine-betaine, ectoine and amino acids in bacterial cells was quantified using high performance liquid chromathography (HPLC). There was a significant (P<0.05) accumulation of glycine-betaine (NaCl modified, 100-150 micromol g(-1) dry weight of cells) and ectoine (glucose modified media, >340 micromol g(-1) dry weight of cells) in the cells over a 48 h incubation period when compared with controls (<10 micromol g(-1) dry weight of cells). Chromatographic profile of amino acids was different with respect to control when NaCl or glucose was used as osmolyte. CONCLUSIONS: Pantoea agglomerans CPA-2 cells synthesised significant amounts of glycine-betaine and ectoine in response to imposed solute stress. However, these compounds and tested amino acids were not involved in cellular adaptation to either heat or pH stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This type of information can be effectively applied to improve ecophysiological quality of cells of bacterial biocontrol agents for better survival and biocontrol efficacy in the phyllosphere of plants.     

Activity of trans-2-hexenal against Penicillium expansum in 'Conference' pears

AIMS: To investigate the effects of trans-2-hexenal on blue mould disease, patulin content and fruit quality in 'Conference' pears. METHODS AND RESULTS: Fruits, wounded and inoculated with Penicillium expansum or non-inoculated, were exposed to trans-2-hexenal vapour treatment (12.5 microl l(-1)) at 20 degrees C. A greater reduction of decay was obtained by treatment application 24 or 48 h after inoculation, in contrast trans-2-hexenal application 2 h after inoculation was ineffective. Fruit storage temperature (-1 degrees C) after treatment did not affect the antifungal activity. Although 2-h exposure to trans-2-hexenal was effective in reducing blue mould, an exposure of at least 8 h was required to reduce fruit patulin content. Treatments did not affect fruit physical-chemical characteristics. After 6 days at 20 degrees C following exposure, trans-2-hexenal residue in treated fruits was less than the natural content of the compound in unripe fruits. CONCLUSIONS: trans-2-Hexenal treatment is effective in the reduction of blue mould infections and patulin content in Conference pears when applied 24-48 h after pathogen inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: trans-2-Hexenal could be a natural alternative to fungicides in the control of P. expansum infections. Further work is needed to study the methods and conditions avoiding the persistence of off-odours and off-flavours in pears after their exposure to trans-2-hexenal vapours.     

Spatial organization of dual-species bacterial aggregates on leaf surfaces

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.     

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.     

Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22 degrees C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30 degrees C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.     

Malt sprout extract medium for cultivation of Lactobacillus plantarum protective cultures

AIMS: The aim was to develop a cheap cereal-based alternative medium for the large-scale production of biopreservative Lactobacillus plantarum VTT E-79098. We examined the effect of growth medium and pH control on the cell yield of Lact. plantarum E-79098 and the antimicrobial activity of the cell-free extracts. METHODS: Fermentations using a novel Malt Sprout Extract Medium (MSE) were performed with different pH regimes. The antimicrobial activity of the cell-free extracts against Pantoea agglomerans VTT E-90396 and Fusarium avenaceum VTT D-80147 was assessed with automated turbidometry. SIGNIFICANCE AND IMPACT OF THE STUDY: When compared with MRS, the MSE medium cultures produced equal growth yields of Lact. plantarum VTT E-79098 and enhanced antimicrobial potential against the Gram-negative bacterium P. agglomerans and a Fusarium fungus. The MSE medium can be used as a low-cost alternative to MRS for producing high cell yields and good antimicrobial activity of Lact. plantarum.     

Genome sequence of the biocontrol agent Pantoea vagans strain C9-1

Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.     

Transmission of cotton seed and boll rotting bacteria by the southern green stink bug (Nezara viridula L.)

AIMS: To determine the ability of the southern green stink bug (SGSB) (Nezara viridula L.) to transmit Pantoea agglomerans into cotton (Gossypium hirsutum) bolls. METHODS AND RESULTS: An SGSB laboratory colony was kept on fresh green beans. A P. agglomerans variant resistant to rifampicin (Rif) (strain Sc 1-R) was used as the opportunistic cotton pathogen. Adult insects were individually provided green beans that were sterilized and then soaked in either sterile water or in a suspension of strain Sc 1-R. Insects were individually caged with an unopened greenhouse-grown cotton boll. After 2 days, live SGSB were collected, surfaced sterilized, ground, serially diluted, and then plated on nonselective media and media amended with Rif. Exterior and interior evidence of feeding on bolls was recorded 2 weeks after exposure to insects. Seed and lint tissue were harvested, ground, serially diluted, and then plated on media with and without Rif. Bacteria were recovered on nonselective media from all insects, and from seed and lint with signs of insect feeding at concentrations ranging from 10(2) to 10(9) CFU g(-1) tissue. The Sc 1-R strain was isolated only from insects exposed to the marked strain and from seed and lint of respective bolls showing signs of insect feeding. Evidence of insect feeding on the exterior wall of the carpel was not always apparent (47%), whereas feeding was always observed (100%) on the interior wall in association with bacterial infections of seed and lint. CONCLUSIONS: Nezara viridula readily ingested the opportunistic P. agglomerans strain Sc 1-R and transmitted it into unopened cotton bolls. Infections by the transmitted Sc 1-R strain caused rotting of the entire locule that masked internal carpel wounds incurred by insect feeding. Bacteria were recovered from penetration points by insects not exposed to the pathogen, but locule damage was limited to the area surrounding the feeding site (c. 3 mm). SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that demonstrates the ability of SGSB to acquire and transmit plant pathogenic bacteria into cotton bolls.     

Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere

To develop environment-friendly biofertilizer solubilizing insoluble phosphates, salt- and pH-tolerant, insoluble inorganic phosphate-solubilizing bacterium was isolated from soybean rhizosphere. On the basis of its physiological characteristics and Vitek analysis, this bacterium was identified as Pantoea agglomerans. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. agglomerans R-42 were 3% (w/v) of glucose, 0.1% (w/v) of NH4NO3, 0.02% (w/v) of MgSO4 x 7H2O, and 0.06% (w/v) of CaCl2 x 2H2O along with initial pH 7.5 at 30 degree C. The soluble phosphate production under optimal condition was around 900 mg/l, which was approximately 4.6-fold higher than the yield in the MPVK medium. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium. P. agglomerans R-42 showed resistance against different environmental stresses like 5-45 degrees C temperature, 1-5% salt concentration and 3-11 pH range. Insoluble phosphate solubilization was highest from CaHPO4 (1367 mg/l), hydroxyapatite (1357 mg/l) and Ca3(PO4)2 (1312 mg/l). However, the strain produced soluble phosphate to the culture broth with the concentrations of 28 mg/l against FePO4, and 19 mg/l against AlPO4, respectively.     

Patulin accumulation in apples by Penicillium expansum during postharvest stages

AIMS: The aim of this study was to assess the opportunities of Penicillium expansum to develop and produce patulin in apples during cold storage and in the steps prior to processing of apple products. METHODS AND RESULTS: Two lots of apples var. Golden with different ripeness degree were used. Half of each lot was fungicide treated. Apples were inoculated with P. expansum and stored at 1 degrees C for 6 weeks. The extent of lesions and patulin accumulation both at the end of cold storage and after 3 days at 20 degrees C were assessed. Short storage at 20 degrees C aimed to simulate the transport and storage steps at room temperature before processing. Lesion size significantly increased during the storage at 20 degrees C. An interaction between fungicide treatment and ripeness degree was found; efficiency of fungicide treatment was higher for ripe apples. Although lesions were evident after cold storage, no patulin was detected. Patulin was detected only when fruits were further stored at 20 degrees C. Neither ripeness degree nor fungicide treatment affected patulin accumulation. CONCLUSIONS: Cold storage periods of 6 weeks do not lead to patulin accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: Shortening preprocessing times at warm temperatures would result into a reduction in patulin content at initial steps of fruits entering the processing plants.     

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.