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Abstract

The biocontrol potential of four wild yeast strains (Meyerozyma guilliermondii – strain YS-1, Meyerozyma caribbica – strain YS-3, Cryptococcus albidus – strain YS-4, and Cryptococcus sp. – strain YS-5) against Penicillium expansum was studied in vivo (on Golden Delicious apples). The test yeasts were applied to the fruits alone as well as in combination with 2% CaCl₂. Treated apples were stored at room temperature (~21°C) for up to 2 weeks or under refrigeration (3°C) for up to 2 months. Candida oleophila was used as positive biocontrol agent. Biocontrol activities were expressed as percentages of lesion size reduction caused by the test yeasts or by test yeasts + CaCl₂ as compared with decays on apples treated with P. expansum alone. All strains tested during this study showed some degree of biocontrol activity against P. expansum. When the test yeasts were applied alone, they effected moderate pathogen inhibition reducing the decay size by 28% to 52% at day 7 and 11% to 27% at day 14 of incubation at room temperature. When the treated apples were stored at 3°C, lesion size reduction was between 48% and 63% after 1 month and 24% to 41% after 2 months of incubation. Addition of CaCl₂ to yeast suspensions facilitated much higher pathogen inhibition. At room temperature, lesion size reduction ranged between 74% and 77% during the first week. After 2 weeks of incubation, decays on yeast + CaCl₂-treated apples were still substantially smaller (49%-73% lower) than those on apples treated with P. expansum alone. At refrigeration, lesion size reduction ranged between 76% and 92% in the first month of storage and between 75% and 87% after 2 months of incubation. Decay incidence was 75% to 100% in apples stored at room temperature and 30% to 85% in those kept under refrigeration. The inhibitory activities of the wild yeast strains were similar to those exhibited by C. oleophila for the most part. These strains, when combined with CaCl₂, showed high potential as biocontrol agents against P. expansum on stored apples.

Keywords

apples postharvest disease biocontrol yeasts

Introduction

Apples are consumed in substantial amounts in the western societies due to their favourable sensory and nutritional attributes. Therefore, they constitute a big part of the fresh produce-related economy. Postharvest invasion and spoilage by Penicillium expansum is the cause of great economic losses.^1,2 More importantly, this mould is capable of producing the highly toxic and mutagenic mycotoxin, patulin (PAT) in apples and other fruits rendering them hazardous to human health. Past studies have reported the presence of PAT in apple products such as apple juice and apple purees at levels exceeding the limits established by the United States Food and Drug Administration (USFDA) and other regulatory agencies.^3-6 More troublesome about the occurrence of PAT in apple products is that high amounts of these products are consumed by the most sensitive populations, infants, and young children.

Mainly chemical pesticides have been used for the prevention of postharvest spoilage by P. expansum. Use of these compounds, however, has some drawbacks such as possibly harmful effects on human and animal health and the contamination of the environment.^7,8 Moreover, some P. expansum strains acquire resistance to synthetic fungicides and they can no longer be controlled by them.^9,10 To solve these problems, researchers turned their efforts towards finding alternative means and measures for controlling P. expansum and other postharvest invaders. A seemingly feasible approach is the use of non-pathogenic, non-toxigenic organisms that have the ability to inhibit or hinder the growth of the pathogen(s). These organisms are collectively known as biocontrol agents (BCAs). Several such BCAs have been tested and a few are commercially used.^1,11-14 Several possible mechanisms of inhibitory action of BCAs against the pathogens have been reported; those include competition for nutrients and space, antibiosis, production of cell wall-degrading enzymes by the BCAs, induction of host resistance to the pathogen and oxidative response.^15-18 BCAs, however, have shown some deficiencies in controlling P. expansum such as inability to control already established decays and inconsistency in antagonistic activities.^19,20

BCAs with improved and better-sustained antagonistic activities are needed. Past studies have showed that combination of treatments such simultaneous use of BCA yeasts and certain compounds (eg, calcium chloride, sodium bicarbonate, silicon, chitosan, ascorbic acid) achieved a synergistic inhibitory effect against postharvest pathogens in vivo.^21-31 Therefore, this study was undertaken in search of a more efficient biocontrol of P. expansum using a combination of treatments. In a previous study, we had isolated a few yeast strains that exhibited inhibitory potential against the pathogen. The current research was an effort to increase the antagonistic activity of those strains by the incorporation of CaCl₂ in the BCA yeast suspensions.

Materials and Methods

Materials

Four yeast strains (Meyerozyma guilliermondii – strain YS-1 from Red Delicious apples, Meyerozyma caribbica – strain YS-3 from pineapple fruit salad, Cryptococcus albidus – strain YS-4 from red seedless grapes, and Cryptococcus sp. – strain YS-5 from Pyrus calleryana fruit) were previously isolated in our laboratory and stored in 15% glycerol at −80°C until used. Organic, ripe, sound quality Golden Delicious apples were purchased from local supermarkets; P. expansum (ATCC 7861) and Candida oleophila (ATCC MYA 1208) were obtained from the American Type Culture Collection (Manassas, VA, USA). Culture media (PDA – DIFCO, Detroit, MI, USA) were prepared in house according to manufacturer’s instructions.

Preparation of yeast cell suspensions

Yeast cultures were freshly prepared by growing the isolates on PDA agar for 48 h at 25°C. Subsequently, yeast cell suspensions were prepared in sterile distilled water and cell concentration was determined using a hemocytometer; final yeast concentrations were adjusted to 1.0 × 10⁸ cells/ml. For determining the combined effects of yeast antagonists and CaCl₂, the yeast suspensions were prepared in sterile 2% CaCl₂ and adjusted to concentrations of 1.0 × 10⁸ cells/ml.

Penicillium expansum spore suspension

P. expansum (ATCC 7861) was cultured on PDA for 7 days at 25°C. Subsequently, its spores were collected by flooding the sporulating cultures with sterile distilled water containing 0.05% Tween 80. The spore concentration was determined by a hemocytometer and culturally and adjusted to 1.0 × 10⁴ spores/ml. Spore suspension was stored at 5°C until used. Fresh suspensions were made weekly.

In vivo testing for biocontrol activity of yeast isolates alone and yeast + CaCl₂

In vivo testing was conducted according to the method described by He et al³² with minor modifications as follows: Golden Delicious apples were surface disinfected with 10% commercial bleach in sterile distilled water for 2 min and subsequently rinsed twice with sterile distilled water. The apples were allowed to dry at room temperature and then they were wounded in the equator area (3 wounds per apple) with a sterile 6-mm cork borer. Next, each wound was inoculated with 30 µL yeast suspension in distilled water (1.0 × 10⁸ cells/ml) (treatment 1) or 30 µL yeast suspension in 2% CaCl₂ (treatment 2). Two hours after the yeast inoculation, 20 µL of P. expansum spore suspension (1.0 × 10⁴ spores/ml) was added to each wound and the apples were stored in covered containers at room temperature for up to 2 weeks or at 3°C for up to 2 months. Control apples inoculated with 2% CaCl₂ + P. expansum or sterile distilled H₂O + P. expansum were also included in the experiment. P. expansum-caused decays were measured at weekly intervals for fruits stored at room temperature and at monthly intervals for the ones incubated at 3°C. Lesion sizes were compared with those on apples infected with P. expansum alone. C. oleophila was used as positive BCA control. Comparisons were also made between P. expansum decays of the two treatments (comparing the activities of each yeast strain alone and respective strain combined with CaCl₂). Four fruits were inoculated per treatment and the experiment was repeated three times. Results are the averages of P. expansum decay diameters of 36 fruit wounds per treatment.

Statistical analysis

T-tests (equal variance) were conducted to compare the two treatments (application of yeast alone vs yeast + CaCl₂).³³

Results

This research focused on the examination of the effect of four wild yeast strains (M. guilliermondii – strain YS-1, M. caribbica – strain YS-3, C. albidus – strain YS-4, and Cryptococcus sp. – strain YS-5) and CaCl₂ on the growth of P. expansum on Golden Delicious apples. The results of the study are summarized in Tables 1 to 5. Lesion sizes on yeast-treated apples were compared with those on apples infected with P. expansum alone and biocontrol activity was measured as percent lesion size reduction. Application of these yeasts on fruit wounds prior to pathogen inoculation resulted in reduced growth of the pathogen, therefore smaller decays. Decrease in lesion size depended on yeast strain and incubation temperature and time (Tables 1 and 2). At room temperature, lesion size (decay severity) on yeast-treated apples was reduced by 28% to 52% after 1 week and by 11% to 27% after 2 weeks of incubation, while decay incidence (percent infected wounds) was 100% during both incubation periods (Table 1). At 3°C, yeast application facilitated lesion size reduction of 48% to 63% after 1 month of incubation and 24% to 41% after 2 months of incubation. Decay incidence ranged between 69% and 88% during the first month and between 94% and 100% after 2 months of incubation (Table 2).

Table 1.

Biocontrol activity of wild BCA yeasts against Penicillium expansum on Golden Delicious apples at 21°C.

Treatment	Incubation at 21°C
	7 days			14 days
	Decay diameter (mm)^a	Lesion reduction (%)^b	Decay incidence (%)	Decay diameter (mm)	Lesion reduction (%)	Decay incidence (%)
Meyerozyma guilliermondii (YS-1)	18.25	46	100	43.58	24	100
Meyerozyma caribbica (YS-3)	16.25	52	100	41.67	27	100
Cryptococcus albidus (YS-4)	24.17	28	100	51.08	11	100
Cryptococcus sp. (YS-5)	19.58	42	100	47.58	17	100
Candida oleophila ^c	17.83	47	100	46.58	19	100
P. expansum (alone)	33.50	N/A	100	57.17	N/A	100

Abbreviation: BCA, biocontrol agent; N/A, not applicable.

Decay diameter values are the means of 36 measurements.

Percent lesion reduction was calculated in relation to decays caused by P. expansum alone.

C. oleophila served as the positive BCA control.

Table 2.

Biocontrol activity of wild BCA yeasts against P. expansum on Golden Delicious apples at 3°C.

Treatment	Incubation at 3°C
	1 month			2 months
	Decay diameter (mm)^a	Lesion reduction (%)^b	Decay incidence (%)	Decay diameter (mm)	Lesion reduction (%)	Decay incidence (%)
M. guilliermondii (YS-1)	14.69	56	75	52.50	41	94
M. caribbica (YS-3)	17.25	48	81	66.13	26	100
C. albidus (YS-4)	17.38	48	88	67.50	24	100
Cryptococcus sp. (YS-5)	15.94	52	69	56.25	37	100
C. oleophila ^c	12.38	63	81	54.25	39	100
P. expansum (alone)	33.31	N/A	100	88.94	N/A	100

Abbreviation: BCA, biocontrol agent; N/A, not applicable.

Decay diameter values are the means of 36 measurements.

Percent lesion reduction was calculated in relation to decays caused by P. expansum alone.

C. oleophila served as the positive BCA control.

Table 3.

Inhibitory activity of wild BCA yeasts + CaCl₂ against P. expansum on Golden Delicious apples at 21°C.

Treatment	Incubation at 21°C
	7 days			14 days
	Decay diameter (mm)^a	Lesion reduction (%)^b	Decay incidence (%)	Decay diameter (mm)	Lesion reduction (%)	Decay incidence (%)
M. guilliermondii (YS-1) + CaCl₂	6.85	76	75	19.25	70	85
M. caribbica (YS-3) + CaCl₂	6.80	76	75	21.85	66	90
C. albidus (YS-4) + CaCl₂	7.20	74	80	28.15	56	90
Cryptococcus sp. (YS-5) + CaCl₂	7.15	75	75	32.25	49	100
C. oleophila^c + CaCl₂	6.50	77	75	17.35	73	80
P. expansum + CaCl₂	22.90	19	100	56.15	12	100
P. expansum (alone)	28.25	N/A	100	63.60	N/A	100

Abbreviation: BCA, biocontrol agent; N/A, not applicable.

Decay diameter values are the means of 36 measurements.

Percent lesion reduction was calculated in relation to decays caused by P. expansum alone.

C. oleophila served as the positive BCA control.

Table 4.

Inhibitory activity of wild BCA yeasts + CaCl₂ against P. expansum on Golden Delicious apples at 3°C.

Treatment	Incubation at 3°C
	1 month			2 months
	Decay diameter (mm)^a	Lesion reduction (%)^b	Decay incidence (%)	Decay diameter (mm)	Lesion reduction (%)	Decay incidence (%)
M. guilliermondii (YS-1) + CaCl₂	2.25	92	30	10.10	87	70
M. caribbica (YS-3) + CaCl₂	3.05	90	35	11.75	85	75
C. albidus (YS-4) + CaCl₂	7.10	76	60	18.05	77	85
Cryptococcus sp. (YS-5) + CaCl₂	4.25	86	50	15.95	80	75
C. oleophila^c + CaCl₂	4.50	85	45	19.60	75	70
P. expansum + CaCl₂	20.80	29	100	65.75	18	100
P. expansum (alone)	29.45	N/A	100	79.70	N/A	100

Abbreviation: BCA, biocontrol agent; N/A, not applicable.

Decay diameter values are the means of 36 measurements.

Percent lesion reduction was calculated in relation to decays caused by P. expansum alone.

C. oleophila served as the positive BCA control.

Table 5.

Statistical comparisons (t-test) of P. expansum decay severity (lesion diameters) on Golden Delicious apples between the two treatments (yeast alone vs yeast + CaCl₂).

Treatment	DECAY DIAMETER (mm)^a at 21°C				Decay diameter (mm)^a at 3°C
	7 days		14 days		1 month		2 months
	– CaCl₂	+ CaCl₂	– CaCl₂	+ CaCl₂	– CaCl₂	+ CaCl₂	– CaCl₂	+ CaCl₂
M. guilliermondii (YS-1)	18.25a	6.85b	43.58c	19.25d	14.69e	2.25f	52.50g	10.10h
M. caribbica (YS-3)	16.25a	6.80b	41.67c	21.85d	17.25e	3.05f	66.13g	11.75h
C. albidus (YS-4)	24.17a	7.20b	51.08c	28.15d	17.38e	7.10f	67.50g	18.05h
Cryptococcus sp. (YS-5)	19.58a	7.15b	47.58c	32.25d	15.94e	4.25f	56.25g	15.95h
C. oleophila ^b	17.83a	6.50b	46.58c	17.35d	12.38e	4.50f	54.25g	19.60h

Abbreviation: BCA, biocontrol agent.

Means in same row and incubation temperature and time period followed by different letters are significantly different (P < .01). The data from each incubation temperature and incubation period were analysed separately.

Decay diameter values are the means of 36 measurements.

C. oleophila served as the positive BCA control.

Addition of 2% CaCl₂ (final concentration) facilitated an increase in biocontrol activity of the tested yeasts. When incubation took place for 1 week at room temperature, lesion reduction caused by the wild yeasts combined with CaCl₂ ranged between 74% and 77% and was for the most part similar to that exhibited by the control BCA yeast, C. oleophila. When the incubation was extended for a second week, the inhibitory activities of all yeast strains were reduced resulting in lesion reduction between 49% and 73% as compared with lesions on apples inoculated with P. expansum only. When the treated apples were stored at 3°C, the test yeasts combined with CaCl₂ caused high inhibition of P. expansum with lesion size reduction ranging between 76% and 92% after a 1-month storage and between 75% and 87% after a 2-month refrigeration. Decay incidence was 30% to 60% during the first month and 70% to 85% after 2 months at refrigeration. T-test comparisons between the two treatments (yeast alone vs yeast + CaCl₂) showed that significant increase in biocontrol activity was achieved with the addition of CaCl₂ for all tested strains (Table 5).

Discussion

The aim of this research was to determine the combined effects of wild BCA yeasts (M. guilliermondii, M. caribbica, C. albidus and Cryptococcus sp.) and CaCl₂ on the growth of P. expansum on Golden Delicious apples. These strains, when applied to apple fruit wounds alone, exhibited substantial antagonistic activity against P. expansum; when they were inoculated onto the fruits in combination with CaCl₂, however, their inhibitory activities were increased markedly. Some of the wild yeast strains facilitated higher inhibition of the pathogen than the control BCA yeast, C. oleophila, during refrigerated storage; this indicates that those strains maintained higher viability and/or grew more abundantly under reduced temperatures. Inhibition of postharvest pathogens by BCA yeasts has been reported in past studies.^34-37 Restriction of P. expansum growth by M. guilliermondii was reported by Yan et al.³⁴ These investigators artificially inoculated pear fruit with the BCA yeast and the pathogen and incubated the treated fruits at 4°C and 20°C. The results of this experiment showed that the yeast colonized the wounds of the fruits quickly, thus inhibiting pathogen growth. In addition, activities of the antioxidant enzymes of the pear, catalase and peroxidase, and phenylalanine ammonia lyase were increased after addition of the yeast; these enzymes are associated with disease resistance in pear. Cao et al³⁵ studied the ability of Pichia caribbica (synonym of M. caribbica) to control P. expansum decays and PAT degradation in Fuji apples stored at 4°C and 20°C. These researchers concluded that P. caribbica colonized fruit wounds rapidly, significantly reduced the incidence of blue mould decays, and was able to degrade PAT at both temperatures. In vitro experiments showed that the yeast inhibited pathogen spore germination. Mbili,³⁶ on the other hand, postulated that YieldPlus, a biofungicide containing C. albidus, was very effective against P. expansum and Botrytis cinerea artificially inoculated on Golden Delicious apples. Guerrero et al³⁷ tested three C. oleophila strains and noted that all strains exhibited substantial inhibition against P. expansum artificially inoculated on Golden Delicious apples.

Enhancement of the biocontrol activities of C. oleophila, Candida guilliermondii (teleomorph M. guilliermondii) Pichia spp., and Cryptococcus laurentii with the addition of CaCl₂ was reported in previous studies.^25-29,38,39 Wisniewski et al²⁶ examined the effect of Ca⁺⁺ on the BCA activity of two C. oleophila isolates (strains 182 and 247) against P. expansum and reported a substantial increase in inhibitory activity of isolate 182. The biocontrol enhancement of this yeast was attributed to inhibition of spore germination and reduction of the pectinolytic activity of P. expansum caused by calcium ions, while these ions had no adverse effect on the yeast survival and growth. McLaughlin et al²⁵ tested the inhibitory activity of two Candida strains combined with CaCl₂ and found that addition of this salt improved the biocontrol activity of the yeasts. Yu et al^27,28 reported increased antagonistic activity of a C. laurentii strain against P. expansum and B. cinerea when it was combined with CaCl₂. These investigators found that CaCl₂ increased the biocontrol of mould rots by educing fruit resistance to pathogens when it was applied on the fruit wounds 24 h prior to mould inoculation. Scherm et al,³⁸ on the other hand, showed that inclusion of CaCl₂ to C. guilliermondii (strains 3C-1b and F1) suspensions enhanced P. expansum decay control on Fuji and Golden Delicious apples. Gramisci et al³⁹ studied the effects of various nutrients and additives (including CaCl₂) on the biocontrol activities of Pichia membranifaciens and Vishniacozyma victoriae against P. expansum and found that CaCl₂ significantly improved the antagonistic activity of the yeasts. Our research demonstrated that addition of CaCl₂ substantially increased the antagonistic activities of M. guilliermondii, M. caribbica and the two Cryptococcus species studied here against P. expansum. Integration of these yeast strains with CaCl₂ could provide a significant improvement in long-term storage of fruits like apples.

Conclusions

The wild yeast strains isolated in our laboratory and studied here showed substantial inhibitory activity against P. expansum in vivo (on Golden Delicious apples) during the first week of incubation at 21°C. The highest activity was observed in strains isolated from apples and a fruit salad. These strains had activities similar to that of C. oleophila. Biocontrol activity was higher and better preserved during refrigerated storage (at 3°C) of the treated apples. Incorporation of CaCl₂ in the yeast suspensions increased the inhibitory activities markedly. Testing of these strains for pathogenicity is necessary before considering them for use in food commodities. Therefore, future research efforts will include rigorous toxicological testing to determine whether these BCA yeasts are safe for use in products destined for human consumption, and studies to ascertain whether they possess antagonistic activity against other postharvest apple pathogens.

Footnotes

Acknowledgements

Special thanks are due to Jeffrey Kohn (Office of Regulatory Affairs, FDA) for his constructive comments during the preparation of the manuscript.

Funding:

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests:

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author Contributions

VHT conceived and designed the experiments, conducted the experimental work, analysed the data, and wrote the first draft. EJK made significant contributions to manuscript writing. Both authors agreed with the manuscript results and conclusions, and reviewed and approved the final version.

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Effect of CaCl 2 and Various Wild Yeasts From Plant Origin on Controlling Penicillium expansum Postharvest Decays in Golden Delicious Apples

Abstract

Keywords

Introduction

Materials and Methods

Materials

Preparation of yeast cell suspensions

Penicillium expansum spore suspension

In vivo testing for biocontrol activity of yeast isolates alone and yeast + CaCl2

Statistical analysis

Results

Discussion

Conclusions

Footnotes

Acknowledgements

Funding:

Declaration of conflicting interests:

Author Contributions

References

In vivo testing for biocontrol activity of yeast isolates alone and yeast + CaCl₂