Neus Teixidó

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake.

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.

Biological control of major postharvest pathogens on apple with Candida sake

Epiphytic microorganisms isolated from apples, pears and the surfaces of apple leaves were screened for antagonistic activity against Penicillium expansum (blue-mold), Botrytis cinerea (gray-mold) and Rhizopus nigricans (Rhizopus rot) on apple (Malus domestica). A total of 933 bacteria and yeasts were tested in primary screening against P. expansum. Ninety-two strains reduced the lesion size on apples by more than 50%, 72 of which were isolated from the surface of apples. For secondary screening against P. expansum, B. cinerea and R. nigricans, 31 strains were selected. The most promising isolate, CPA-1, was identified as Candida sake. This yeast, isolated from apples in storage season was very effective against all three diseases. Wounded Golden Delicious apples protected with the yeast suspension at a concentration of 2.6 x 10(6) CFU/ml and inoculated with conidia of B. cinerea and R. nigricans of 10(5) and 10(4) conidia/ml, respectively, did not develop rot. Complete control of P. expansum was obtained at the same concentration of the antagonist with a pathogen inoculum concentration of 10(3) conidia/ml. This strain, also provided excellent control of rot development under cold storage conditions. The strain of Candida sake can grow actively in aerobic conditions. In drop-inoculated wounds of apples, the populations of C. sake increased by more than 50-fold during the first 24 h at 20 degrees C. The maximum population of C. sake on apple wounds was the same at 20 as at 1 degrees C and was recovered after three and twenty days, respectively.

Biological control of postharvest pear diseases using a bacterium, Pantoea agglomerans CPA-2

Epiphytic microorganisms isolated from the fruits and leaf surfaces of apples and pears were screened for antagonistic activity against Penicillium expansum on pears. From 247 microorganisms tested for antagonistic properties against P. expansum, a bacterium strain identified as Pantoea agglomerans (CPA-2) was selected. This bacterium was very effective against Botrytis cinerea, P. expansum and Rhizopus stolonifer. Complete control at the three tested concentrations (2 x 10(7), 8 x 10(7) and 1 x 10(8) CFU ml(-1)) was obtained on wounded pears inoculated with 10(3), 10(4) and 10(5) conidia ml(-1) of P. expansum and R. stolonifer. At 8 x 10(7) CFU ml(-1), Pan. agglomerans reduced B. cinerea decay by more than 80% at the three concentrations of the pathogen. In over 3 years of experiments in semicommercial trials, Pan. agglomerans provided excellent control against B. cinerea and P. expansum under cold storage, either in air or in low oxygen atmospheres. Equal control was obtained with Pan. agglomerans at 8 x 10(7) CFU ml(-1), as with the fungicide imazalil at commercial doses, against both pathogens. Pan. agglomerans grew well inside wounds on pears at both room and cold temperatures and under modified atmospheres. In contrast, it grew poorly on the surface of intact fruit.

Improving control of green and blue molds of oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate

The potential of using Pantoea agglomerans (strain CPA-2) alone, or in combination with sodium bicarbonate or sodium carbonate solutions, for control of Penicillium digitatum (green mold) and Penicillium italicum (blue mold) on oranges was investigated under ambient (20 °C) and cold storage (3 °C) conditions. P. agglomerans controlled both pathogens on oranges at 2 × 108 cfu ml-1. The biocontrol agent was found to be completely tolerant to 2% sodium bicarbonate at room temperature, although its culturability was reduced by > 1000-fold after 30 min in 2% sodium carbonate. The efficacy of P. agglomerans for control of green mold was improved when combined with sodium bicarbonate, resulting in complete and 97.6% reduction of decay incidence at 3 °C and 20 °C, when compared to untreated controls. Satisfactory results were also obtained with the combined treatment for control of blue mold. P. agglomerans grew well inside wounds on oranges at both 20 °C and 3 °C. In contrast, it showed a reduced growth on the surface of intact fruit. Sodium bicarbonate at 2% concentration did not noticeably affect antagonist population development. Thus, use of bicarbonate treatment at 2% followed by the antagonist P. agglomerans CPA-2 could be an alternative to chemicals for control of postharvest diseases on oranges.

Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media.

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.

Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions.

The biocontrol potential of the yeast Candida sake (CPA-1) against Penicillium expansum decay of apples under several controlled atmosphere conditions was investigated. In a laboratory trial under different commercial cold storage conditions, increasing concentrations of C. sake improved decay control. A maximum reduction of decay was achieved at 3% O2-3% CO2 atmosphere. It amounted to a 97% lesion reduction after treatment with a suspension containing 2.4 x 10(6) CFU/ml of C. sake (CPA-1). In a semi-commercial trial at 1 degree C with wounded fruits, the reduction in decay diameter caused by C. sake exceeded 80% after 60 days at 21% O2 and 60% after 120 days of storage under controlled atmosphere conditions. For seven controlled atmosphere conditions studied, a significant influence by C. sake on the P. expansum decay was observed, and the lesion size was reduced more than 70% by C. sake at 10(7) CFU/ml. The populations of C. sake (CPA-1) on the apple surface followed the same pattern under all controlled atmosphere conditions studied. They decreased 4-10-fold during the first 2 weeks, followed by an increase to the initial level after 45 days, and thereafter the count remained constant for the period of 90 days examined. This indicated the capacity of C. sake (CPA-1) to colonize the surface of apples under various storage conditions. The ability to colonize was even higher in apple wounds.

Pilot tests of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit

Abstract The yeast Candida sake (strain CPA-1) was tested as a biocontrol agent of postharvest diseases, primarily blue mold, caused by Penicillium expansum on apple fruits. In a semi-commercial trial with non-injured fruits stored in air at 1°C, a concentration of 1.6×10 6 colony forming units/ml (CFU/ml) of C. sake reduced the incidence of decayed fruits by more than 70%. Over a period of three seasons, in commercial trials the efficacy of CPA-1 applied in a drench was evaluated and compared with the fungicides imazalil and thiabendazole+folpet. The application of C. sake at 10 7 CFU/ml resulted in a reduction in the incidence of decay to a level equal to that with imazalil (375 ppm) and higher than that with thiabendazole (425 ppm)+folpet (1000 ppm). Population of the biocontrol agent increased on the surface of wounded fruits 5-fold and decreased on the surface of non-wounded fruits more than 10-fold in the first 60 days in storage at 1°C. The viability of C. sake was not reduced after 30 min immersion in benomyl, sulfur, flusilazol, ziram, thiabendazole or diphenylamine. Conversely, captan, imazalil, and ethoxyquin decreased C. sake viability and would not be compatible with it used at commercial rates. The yeast was able to grow in culture at temperatures from 1 to 34°C.

Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides

Bacillus subtilis CPA-8, a strain with demonstrated ability to control Monilinia spp. in peaches, was studied to elucidate its mechanisms of antifungal activity. Growth inhibition assays using cell-free supernatants and butanolic extracts showed strong antifungal activities against Monilinia laxa and Monilinia fructicola. By comparison with the reference B. subtilis strains UMAF6614 and UMAF6639, fengycin, iturin and surfactin lipopeptides were identified by thin layer chromatography in butanolic extracts from cell-free supernatants, indicating that antibiosis could be a major factor involved in the biological control ability of CPA-8. TLC-bioautography analysis confirmed the presence of fengycin, iturin and surfactin lipopeptides but strong antifungal activity could be associated only with fengycin lipopeptides. These results were definitively supported by mutagenesis analysis targeted to suppress fengycin biosynthesis by disruption of the B. subtilis fenB gene. By TLC-bioautography analysis it was possible to identify transformants from CPA-8 with reduced or suppressed antifungal activity, and this phenotype was associated with the lack of fengycin bands. Fruit trials confirmed that fengycin-defective mutants and their cell-free supernatants lost their ability to control peach brown rot disease in comparison with CPA-8 wild type strain or Serenade Max®, a commercial formulation based on B. subtilis. Furthermore, population dynamics studies determined that CPA-8 fengycin-deficient mutants survived in wounds in peach fruit equally well as the CPA-8 wild type. Taken together our data indicate that fengycin-like lipopeptides play a major role in the biological control potential of B. subtilis CPA-8 against peach brown rot.

Liquid formulation of the postharvest biocontrol agent Candida sake CPA-1 in isotonic solutions

ABSTRACT Viability of the postharvest biocontrol agent Candida sake CPA-1 stored as liquid formulation was evaluated by studying the effect of growth, preservation medium, and temperature. C. sake was grown in molasses medium with unmodified water activity (a(w)) and in the same with a(w) modified to 0.98 with the addition of several solutes. Cells were preserved with isotonic solutions of different substances. Efficacy of liquid formulations stored for different periods was tested against infection by Penicillium expansum on apples. The best growth media were the (unmodified one and those modified to 0.98 a(w) with the addition of glycerol or sorbitol. For all growth media, the best preservation medium was the isotonic solution prepared with trehalose. When the effect of trehalose concentration in the preservation medium was studied, generally, at trehalose concentrations below the isotonic one, C. sake viabilities increased with increased trehalose. However, the best results were obtained when cells were preserved with the trehalose solution which was isotonic with cells. After 7 months of storage at 4 degrees C, cells that were grown in the sorbitol-modified medium and preserved with the isotonic solution of trehalose (0.96 M) maintained their viability and efficacy against P. expansum infection of apples.

Efficacy of preharvest and postharvest Candida sake biocontrol treatments to prevent blue mould on apples during cold storage

This study compared the biocontrol efficiency of preharvest and postharvest applied yeast cells of Candida sake to apples (cv. Golden Delicious) wounded before and after harvest and inoculated with Penicillium expansum prior to cold storage conditions in two seasons, 1994/95 and 1995/96. The establishment of populations of C. sake during this period also was determined. In both years, postharvest treatment with the antagonist resulted in significant (P<0.05) and effective control of Penicillium rot whether pre- or postharvest wounds were made. Maximum disease control achieved, in terms of incidence and severity, was greater than 80% reduction in lesion diameter and 50% reduction in the incidence of lesions. However, preharvest application of the antagonistic yeast at a concentration of 3·106 CFU/ml was less effective against Penicillium rot than postharvest treatment. No advantages in biocontrol were observed when apples were treated with the yeast antagonist both pre- and postharvest. Candida sake population levels during cold storage of apples receiving only preharvest application of the antagonist decreased prior and more rapidly than levels in apples receiving a postharvest application. High populations of C. sake were present in postharvest-treated apples, even after 90 days in cold storage.