Post-harvest biological control by Pantoea agglomerans (CPA-2) on Golden Delicious apples

AIMS: To investigate the potential of Pantoea agglomerans to control the major post-harvest diseases on Golden Delicious apples. METHODS AND RESULTS: In laboratory trials, a high level of control of Penicillium expansum, Botrytis cinerea and Rhizopus stolonifer was obtained with P. agglomerans. In semi-commercial trials at 1degrees C in air and a low oxygen atmosphere, the reduction of blue mould was 81% and 100%, respectively, and control of grey mould was achieved equally with P. agglomerans and imazalil. In trials at 1degrees C and seven atmosphere conditions, maximum reduction in decay was 80% obtained at 3% O2-6% CO2. The population of P. agglomerans on apples followed the same pattern under all three atmosphere conditions studied. CONCLUSIONS: Pantoea agglomerans could be used effectively on apples under a wide range of temperature and atmosphere conditions. SIGNIFICANCE AND IMAPCT OF THE STUDY Pantoea agglomerans can be used as a biocontrol agent on apples at 8 x 10(7) cfu ml-1, the same concentration as in pears. This will facilitate the application of this biological control agent by the growers in packing houses.     

Activity of the Yeasts Cryptococcus laurentii and Rhodotorula glutinis Against Post-harvest Rots on Different Fruits

More than 200 yeasts were selectively isolated from microbial populations on the surface of different fruits. Fifty of these isolates were tested against blue mould ( Penicillium expansum ) on wounded apples. Isolates LS-11 of Rhodotorula glutinis and LS-28 of Cryptococcus laurentii were the most effective antagonists. They were further evaluated at 20IC on different fruits (apples, pears, strawberries, kiwi fruits and table grapes) against several of the main post-harvest pathogens ( Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer and Aspergillus niger ) and at 4IC on apples inoculated with P. expansum . At 20IC the antagonists significantly reduced rot incidence and showed a wide range of activity on different hostpathogen combinations; isolate LS-28 exhibited a higher and more stable activity than LS-11. Both yeasts were also effective against P. expansum in cold storage conditions. Populations of the two yeasts were assessed on wounded and unwounded surfaces of apples kept at both 20 and 4IC. At either temperature, isolate LS-28 reached greater densities in wounded tissues than LS-11, but had a lower ability to colonize unwounded apple skin. The two yeasts were able to grow in culture at temperatures ranging from 0 to 35IC. In assays performed in vitro at 24IC, the antagonists showed low sensitivity towards several fungicides commonly applied on fruits and vegetables.     

Development of a microbial community of bacterial and yeast antagonists to control wound-invading postharvest pathogens of fruits.

Two antagonists, the bacterium Pseudomonas syringae and the pink yeast Sporobolomyces roseus, against blue mold (caused by Penicillium expansum) on apple controlled this disease more effectively when combined at approximately equal biomass (50:50 of the same turbidity) than in individual applications. Addition of L-asparagine enhanced the biocontrol effectiveness of P. syringae but decreased that of S. roseus and had no significant effect when the antagonists were combined. Populations of both antagonists increased in apple wounds and were further stimulated by the addition of L-asparagine. The carrying capacity of wounds for P. syringae was not affected by S. roseus. Populations of P. syringae in wounds inoculated individually or in a 50:50 mixture with S. roseus reached the same level after 3 days at 22 degrees C. However, populations of S. roseus recovered after applications of the mixture were consistently lower than those recovered after individual applications. Similar effects were observed in in vitro tests in which populations of S. roseus grown in mixtures with P. syringae were consistently lower than those grown alone, while the populations of P. syringae were not affected by the presence of S. roseus. A total of 36 carbon and 35 nitrogen compounds were tested for utilization by both antagonists. Fourteen nitrogenous compounds were utilized by both P. syringae and S. roseus, and an additional nine compounds were utilized by P. syringae. S. roseus and P. syringae utilized 17 and 13 carbon sources, respectively; 9 sources were common to both antagonists. Populations of these antagonists in apple wounds appear to form a relatively stable community dominated by P. syringae.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Thiabendazole resistance and mutations in the β-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay

Penicillium expansum is the causal agent of blue mold rot, a postharvest decay of stored fruits. This fungus also produces the mycotoxins patulin and citrinin. Control of P. expansum still relies mainly on the use of fungicides such as thiabendazole. Since its introduction, resistant strains have been reported. The aim of this work was to investigate the thiabendazole resistance and mutations in the β-tubulin gene of P. expansum strains isolated from apples and pears with blue mold decay from Spain. A total of 71 strains of P. expansum were scored for resistance to thiabendazole and the β-tubulin gene was sequenced. Out of 71 strains, 37 were sensitive and 34 were resistant to thiabendazole. Regarding the β-tubulin gene sequence, 10 different genetic types were determined, with a 99.7–100% similarity. When the amino acid sequence was deduced, five different amino acid sequences were found. All except one of the sensitive strains lacked mutations in the region sequenced. Of the 34 resistant strains, only eight had mutations that involved the residues 198 and 240. All the strains with mutations at position 198 always corresponded to resistant isolates. However, a high percentage of resistant strains had no mutations in the region of the β-tubulin gene sequenced, and so other mechanisms may be involved in thiabendazole resistance.     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病(Penicilium expansum Link)的丝孢酵母(Trichosporon pullulans (Lindner.) Diddens and Lodder)、罗伦隐球酵母(Cryptococcus laurentii (Kuffer.) Skinner)和粘红酵母(Rhodotorula glutinis (Fresen.) F. C. Harrison).其中,抑病效果最好的T. pullulans 是一种用于采后果实生物防治的新型拮抗菌.研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果.实验结果表明:酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C. laurentii的细胞悬浮液中加入0.18 mol/L 的CaCl2能显著提高其抑病能力,但增加CaCl2 对T. pullulans 和R. glutinis 的抑病效果却没有明显作用.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病（Peniclium expansum Link)的丝孢酵母（Trichosporon pullulans(Lindner.)Diddens and Lodder)。罗伦隐球酵母（Cryptococcus laurentii(Kuffer.)skin-ner)和粘红酵母（Rhodotorula glutinis(Fresen.)F.C.Harrison)。其中,抑病效果最好的T.pullulans是一种用于采后果实生物防治的新型拮抗菌,研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果。实验结果表明;酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C.laurentii的细胞悬浮液中加入0.18mol/L的CaCl2能显著提高其抑病能力。但增加CaCl2对T.pullulans和R.glutinis的抑病效果却没有明显作用。     

Effect of 2-deoxy-D-glucose on mycotoxins from apples inoculated with Penicillium expansum

Patulin concentration was not significantly different in Bramley and Cox's apples inoculated with Penicillium expansum, and treated with the biocontrol enhancer 2-deoxy-D-glucose (DOG) when compared to no DOG treatment, despite average numerical increases. Some additional small HPLC peaks were detected from some extracts, one of which corresponded to citrinin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima

The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10? cells·mL?1 favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.     

Development of a biological control method against postharvest diseases of citrus fruits

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.     

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.     

假单胞菌YL11对扩展青霉的抑制作用及其机理初探

【背景】苹果青霉病是由扩展青霉引起的一种重要的果实采后病害,影响果实品质导致苹果腐烂从而造成经济损失。【目的】研究假单胞菌YL11对扩展青霉的抑制作用和苹果采后青霉病的防治效果,并对抑菌机理进行初步探讨。【方法】以扩展青霉为供试菌株,研究不同浓度的假单胞菌YL11无菌发酵液对扩展青霉菌落直径、孢子萌发率、菌丝体干重、苹果损伤接种病斑直径扩展的影响,利用对电导率、核酸及蛋白释放量、AKP含量、SDH活性、ATP酶活性和ATP含量的影响对抑菌机理进行探究。【结果】假单胞菌YL11无菌发酵液能有效抑制扩展青霉生长,抑菌圈直径为22.33±0.27 mm,抑菌效价为71.67 mm/mL;能有效抑制孢子萌发,100%无菌发酵液对孢子萌发抑制率达到80.2%;对扩展青霉的生物量也有一定抑制作用,体积分数为100%时,菌丝体干重为4.7mg/mL,抑制率达到39.74%;无菌发酵液处理能有效抑制苹果青霉病病斑的扩展,3d时对病斑扩展的抑制率最大,达到47.1%;无菌发酵液处理均能引起电导率升高、胞内核酸和蛋白释放量增大、胞外AKP含量升高、SDH活性降低、ATP酶活性和ATP含量均降低,且随着发酵液浓度的增加效果越明显。【结论】假单胞菌YL11能显著抑制扩展青霉的生长,破坏细胞膜结构、降低能量代谢酶活性,从而扰乱扩展青霉的正常生长,对苹果青霉病有较好的生防效果,具有潜在的开发价值。     

Biological control of apple blue mold with Pseudomonas fluorescens

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.     

In vitro studies on the effect of some chemicals on the growth and sporification of Penicillium expansum and Botrytis cinerea

Penicillium expansum and Botrytis cinerea are among the pathogens most frequently affecting apples and grapes after harvest, respectively. We studied the behaviour of these moulds when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO) as a alternative method to fungicides in controlling postharvest decay of horticultural products. The experiments were performed with 5 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of the two tested chemicals. Freshly prepared conidia of B. cinerea and P. expansum were sown onto the media and then kept into an incubation chamber at 21 degrees C up to 3 and 6 days, respectively. Daily, the colony forming units (cfu), the colony diameter and the degree of sporification were monitored. Compared to the control, both chemicals affected the growth rate of the two pathogens. The P. expansum and B. cinerea cfu value was not significantly inhibited but the colony diameter and the sporification degree decreased when concentration was raised. B. cinerea cultured on DMSO showed a significant drop of sporification up to the tested concentration of 10 microL/mL, and a complete inhibition of cfu when the concentration was higher than 20 microL/mL.     

beta-3 adrenergic agonist restores skeletal muscle insulin responsiveness in Sprague-Dawley rats.

Between 7 and 14 weeks of age, male Sprague-Dawley rats develop a greater than 50% loss in insulin-stimulated glucose transport in skeletal muscle. We treated rats aged 14 weeks with the beta-3 adrenergic agonist CL316,243 (1 mg/kg/day by minipump for 14 days). Treatment resulted in a 56% reduction in visceral fat (P < 0.05). Muscle mass and body weight were unchanged. In strips of soleus muscle isolated from rats treated with CL316,243, basal transport of [(3)H]-2-deoxyglucose (2-DOG) was unchanged (105.8 +/- 7.5 nmol/g/min for vehicle vs 122.0 +/- 8.7 for CL316,243). However, in rats treated with CL316,243, the increase in 2-DOG transport in response to a maximal concentration of insulin was substantially increased (55.5 +/- 13.1 nmol/g/min for vehicle vs 102.4 +/- 13.5 for CL316,243, P < 0.03). CL 316,243 caused no significant changes in fasting glucose, insulin, or free fatty acids. Treatment of soleus muscle strips in vitro with CL316,243 (either 0.1 nM or 1.0 nM for 120 min at 37 degrees C) had no effect either on basal 2-DOG transport or on insulin-stimulated transport. We conclude that the CL316,243 causes a reduction in visceral fat and a reversal of muscle insulin resistance. The effect CL 316,243 on muscle insulin responses appears to be indirect, as it did not occur in vitro.     

Patulin Production in Apples Decayed by Penicillium expansum

Sixty isolates of Penicillium expansum were tested for patulin production in decaying apples. All the isolates were found to produce the mycotoxin patulin as determined by thin-layer chromatography. Since patulin is known to be stable in many apple products, the results indicate that apple products made partially from apples decayed by P. expansum will contain patulin which may present a health hazard. The results also suggest that patulin may be important in the decay of apples by P. expansum.     

Control by imazalil of fruit storage rots caused by Alternaria alternata

The fungicide imazalil has potential value for controlling post-harvest diseases caused by Alternaria alternata in pears, apples and persimmons. Imazalil was active in vitro on germination and hyphal growth (ED₅₀) at 1.4 and 0.5 μg/ml, respectively. Protectant action of the fungicide lasted only 1 day after treatment. At 1000 μg/ml the fungicide inhibited development of Alternaria rot in inoculated apples at 0 °C and in naturally infected pears and persimmons at–1 °C.     

Effect of vitamin on 2-deoxy-d-glucose uptake in human fibroblast cultures

Summary 2-Deoxy-d-glucose (2-DOG) uptake was tested in human fibroblast cultures in the presence and absence of vitamin E. Addition of 10 μg/ml vitamin E to the culture medium significantly reduced this uptake for 2-DOG concentrations of 0.005, to 10 mmol/liter (P≤0.01). The decrease of 2-DOG uptake was inversely proportional to the rise in 2-DOG concentration (P≤0.01). The presence of vitamin E reduced by 71% the average cellular level of lipid peroxides (expressed as thiobarbituric acid reactive substances) and caused a small but significant decrease in the cholesterol concentration (P≤0.01). These last results might explain the decrease in 2-DOG uptake observed in the presence of vitamin E.     

Effects of Trichosporon sp. in Combination with Calcium and Fungicide on Biocontrol of Postharvest Diseases in Apple Fruits

The capability of yeast Trichosporon sp., an antagonist isolated from peach fruit, in biological control was evaluated in apple ( Malus domestica Borkh. cv. Fuji) fruits, when inoculated with different concentrations of Botrytis cinerea Pers. and Penicillium expansum (Link) Thom, as well as in combination with calcium and fungicide. The concentrations of the yeast cells and pathogen spores obviously influenced disease incidence and lesion development in apples. There was a significant negative correlation between concentrations of the yeast cells and infectivity of the pathogens. When the yeast cell suspensions reached the concentration of 108 colony-forming units (CFU)/mL, there was no infection caused by B. cinerea and P. expansum with spore concentrations below 106 spores/mL in apple fruits. The yeast at concentrations of 106-107 CFU/mL in combination with fungicide (iprodione at 50 μL/L) provided control of decay caused by B. cinerea and P．expansum better than separate application. Effect of controlling gray mould and blue mould rots was enhanced when Trichosporon sp., even at low concentration of 105 CFU/mL, was applied in the presence of 1%-2% CaCl2 in an aqueous suspension.