# Biocontrol Activity of the Local Strain ofMetschnikowia pulcherrima on Different Postharvest Pathogens

Authors: Sezai Türkel; Mihriban Korukluoğlu; Mümine Yavuz
Journal: Biotechnology Research International
 (2014)

---

## Abstract

The strains of the yeastMetschnikowia pulcherrima have strong biocontrol activity against various microorganisms. Biocontrol activity of M. pulcherrima largely depends on its iron immobilizing pigment pulcherrimin. Biocontrol activity of pulcherrimin producing strain, M. pulcherrima UMY15, isolated from local vineyards, was tested on different molds that cause food spoilage. M. pulcherrima UMY15 was a very effective biocontrol agent against Penicillium roqueforti, P. italicum, P. expansum, and Aspergillus oryzae in in-vitro plate tests. However, the inhibitory activity of M. pulcherrima UMY15 was less effective on Fusarium sp. and A. niger species in biocontrol assays. In addition, M. pulcherrima UMY15 strain completely inhibited the germination and mycelia growth of A. oryzae, A. parasiticus, and Fusarium sp. spores on artificial wounds of apples when they coinoculated with M. pulcherrima UMY15. Moreover, when coinoculated, M. pulcherrima UMY15 strain also inhibited the growth of P. roqueforti, P. italicum, P. expansum, A. oryzae, Fusarium sp., and Rhizopus sp. in grape juice, indicating that M. pulcherrima UMY15 can be used as a very effective biocontrol yeast against various species of postharvest pathogens, including   Penicillium, Aspergillus, Fusarium, and Rhizopus.


---

## Body

## 1. Introduction

Postharvest spoilage of fruits and vegetables by various molds results in substantial economic loss every year [1]. Certain species ofAspergillus,Penicillium,Fusarium, andRhizopus are the major molds that can be found regularly on food stuff throughout the world. Some species or varieties of these molds also produce mycotoxins [2, 3]. Use of traditional fungicidal chemicals in postharvest disease control results in the formation of drug resistant microorganisms and large scale environmental pollution, which also causes severe health problems in human populations [4, 5]. Biological control of postharvest diseases caused by various fungal pathogens seems to be the best alternative to chemical fungicidal agents [6–8].Different yeast species have been used as effective biocontrol agents against certain fungal pathogens [8–11]. Some of these yeasts areMetschnikowia pulcherrima, Trichosporon pullulans, Rhodotorula glutinis,Pichia membranifaciens,andPichia anomala. Each one of these biocontrol or antagonistic yeasts can effectively inhibit growth of the various fungal pathogens on different fruits and vegetables. Some of these yeasts or their products have been commercially produced by different companies as biocontrol agents [9, 12]. Biocontrol yeasts inhibit the growth of targeted pathogens by different mechanisms. Competition for nutrient and space, secretion of specific lytic enzymes, and synthesis and secretion of specific inhibitory secondary metabolites are the only few examples of the mechanisms of the action for the biocontrol yeasts [8, 13].Different strains of the yeastM. pulcherrima have been used as a highly effective biocontrol agent against different fungal species such asPenicillium expansum andBotrytis cinerea [14, 15]. Antagonistic effects of differentM. pulcherrima strains on different species ofCandida,Aspergillus,E. coli,Proteus vulgaris,Trichosporon mucoides, and onTrichodermaspp., were also reported [16].M. pulcherrima produces a secondary metabolite pulcherrimin and secretes it to the growth medium [17]. Pulcherrimin forms a chelate complex and immobilizes the iron ions in the growth medium [15, 18]. Hence, it seems thatM. pulcherrima strains exert their antagonistic effects on the other microorganisms by the depletion of iron in the growth medium [15, 16, 18]. In addition to the pulcherrimin pigment, there is evidence indicating thatM. pulcherrima also secretes lytic enzymes such as chitinase that contribute to the overall antagonistic effects of relatedM. pulcherrima strains [19].In this study, we have tested the antagonistic effects of the recently isolatedM. pulcherrima strain on different fungal species involved in food spoilage. We have found that theM. pulcherrima UMY15 strain, isolated from a local vineyard in Turkey, is a very effective biocontrol agent against different species ofPenicillium,Aspergillus, andFusarium on synthetic growth medium and also on apple.


## 2. Materials and Methods

### 2.1. Microorganisms and Growth Medium

Isolation and characterization ofM. pulcherrima UMY15 strain were described previously [16].M. pulcherrima UMY15 strain was cultivated in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) for antagonistic activity tests.Food spoilage molds used in this study areP. roqueforti,P. italicum,P. expansum,Fusariumsp.,Rhizopussp.,A. niger,A. oryzae, andA. parasiticus. All of the molds are held in the culture collection of the Food Engineering Department of Uludag University (Bursa, Turkey). Molds were sporulated on malt extract agar (MEA) plates for 7 days at 30°C. Then, spores were collected aseptically into filter-sterilized 10 mL 0.1% Tween 80. The numbers of mold spores in Tween suspensions were adjusted to 105 spores/mL using sterile 0.1% Tween solution [20]. Spore concentrations were determined microscopically using Thoma slides. Spore suspensions were used immediately after preparations for antagonistic activity tests.


### 2.2. Antagonistic Activity Tests

First,M. pulcherrima UMY15 strain was cultivated in 10 mL YPD medium overnight at 30°C in an incubator shaker with 130 rev/min at 30°C to obtain saturated precultures. Then, from these precultures, 200 μL of yeast sample was inoculated into 10 mL YPD medium and grown to the midlog stage (OD600 : 1.0) under same growth conditions. Yeast cells were harvested by centrifugation (1500 g for 5 min) and washed once with 10 mL of sterile distilled water and resuspended in 10 mL of sterile distilled water. ThisM. pulcherrima suspensions are used in antagonistic activity tests as described below.For antagonistic activity tests, 100μL of spore suspensions from each mold species (approximately 104 spores) was taken from the stock spore suspensions and spread evenly on synthetic dextrose (SD) medium (1.67 g/L yeast nitrogen base, 5 g/L ammonium sulfate, 20 g/L glucose, and 20 g/L agar). When the surface of spore-spread plates dried, 4 μL ofM. pulcherrima UMY15 strain (prepared as described) was planted on the plates in duplicates, and then the plates were incubated at 30°C for spore germination and growth for 1-2 days. All of the antagonistic activity tests were repeated at least twice. Inhibition zones were measured manually and defined as the distance extending from the edges of theM. pulcherrima UMY15 colonies to the beginning of the fungal lawn on the plates and expressed in millimeters.Antagonistic effects ofM. pulcherrima UMY15 on germination of fungal spores were also tested when these molds were grown on apple. For this assay, apple (Malus domestica Borkh, cv. Golden Delicious) slices with dimensions of 4 cm L × 2 cm W × 1 cm H were prepared aseptically from the fully matured Golden Delicious apples. Then 3 holes (3-4 mm diameter, 3-4 mm deep) were prepared on these slices with sterile pipette tips. To these three holes on apple slices, 10 μL of spore suspensions only, 10 μL ofM. pulcherrima UMY15 together with 10 μL of spore suspensions, and 10 μL ofM. pulcherrima UMY15 samples were added, respectively. Apple slices were placed in sterile petri dishes and incubated at 25°C for 2 days for spore germination and fungal growth. At the end of incubation period, spoilage zones on the apple slices caused by the germination and the growth of molds were measured manually. Percentage of the biocontrol activity ofM. pulcherrima UMY15 strain on the above given fungal species was expressed as the ratio of infection zones (mm) developed on apples when these molds coexist withM. pulcherrima UMY15 strain over the zone of infection formed by these molds alone on apple wounds [21].Antagonistic effects ofM. pulcherrima on the germination and hyphal growth of various molds in grape juice were also analyzed in shake-flask culture. For these assays, approximately 105 of mold spores and 104 colony forming unit (CFU) fromM. pulcherrima samples were coinoculated into 20 mL of additive-free grape juice in 100 mL flask. Grape juice samples inoculated withM. pulcherrima and/or mold spores were incubated at 30°C in an incubator shaker with 130 rev/min. Cultures were visually inspected for mold sporulation after 48 hours. Efficacy of spore germination in grape juices was estimated by comparing the amount of mycelia growth in the grape juice cultures to the cultures that contain only spores of relevant molds (control group withoutM. pulcherrima).The numbers given for inhibition and spoilage zones in Tables1 and 2 are the average values of at least 4 independent experiments. The standard deviations for the zones of inhibitions were less than 10%.Table 1
Antagonistic effects ofM. pulcherrima UMY15 strains on the spore germination and the mycelia growth of different fungal species.
Fungal species
Zone of inhibitions (mm)a
24 h
48 h
P. roqueforti
4
3
P. italicum
3
3
P. expansum
3
3
Fusariumsp.
1
0.5
Rhizopussp.
1
1
A. niger
1
0
A. oryzae
3
2
A. parasiticus
2
1.5
aInhibition zones were measured as the distance from the edges of M. pulcherrima colonies to the beginning of the fungal lawns on SD plates at the 24th and 48th hours of incubations. Standard deviations were less than 10% in zones of inhibition.Table 2
Inhibition of spore germination and fungal growth on the artificial wounds on apple byM. pulcherrima UMY15 strain.
Fungal species
Inoculations and zones (mm) of fungal growtha
% Biocontrol
Fungal spores only
Fungal spores +M. pulcherrima
P. roqueforti
NG
NG
NA
P. italicum
15
5
67
P. expansum
NG
NG
NA
Fusariumsp.
12
0
100
Rhizopussp.
Full growth
Full growth
0
A. niger
15
6
60
A. oryzae
8
0
100
A. parasiticus
10
3
70
aZones of fungal growth were measured as the distance from the center of artificial wounds to the ends of fungal growths (rots) on apples at the end of 48th hours of incubations.
NG: no growth on apple wounds, NA: not applicable. Standard deviations were less than 10% in zones of inhibition.


## 2.1. Microorganisms and Growth Medium

Isolation and characterization ofM. pulcherrima UMY15 strain were described previously [16].M. pulcherrima UMY15 strain was cultivated in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) for antagonistic activity tests.Food spoilage molds used in this study areP. roqueforti,P. italicum,P. expansum,Fusariumsp.,Rhizopussp.,A. niger,A. oryzae, andA. parasiticus. All of the molds are held in the culture collection of the Food Engineering Department of Uludag University (Bursa, Turkey). Molds were sporulated on malt extract agar (MEA) plates for 7 days at 30°C. Then, spores were collected aseptically into filter-sterilized 10 mL 0.1% Tween 80. The numbers of mold spores in Tween suspensions were adjusted to 105 spores/mL using sterile 0.1% Tween solution [20]. Spore concentrations were determined microscopically using Thoma slides. Spore suspensions were used immediately after preparations for antagonistic activity tests.


## 2.2. Antagonistic Activity Tests

First,M. pulcherrima UMY15 strain was cultivated in 10 mL YPD medium overnight at 30°C in an incubator shaker with 130 rev/min at 30°C to obtain saturated precultures. Then, from these precultures, 200 μL of yeast sample was inoculated into 10 mL YPD medium and grown to the midlog stage (OD600 : 1.0) under same growth conditions. Yeast cells were harvested by centrifugation (1500 g for 5 min) and washed once with 10 mL of sterile distilled water and resuspended in 10 mL of sterile distilled water. ThisM. pulcherrima suspensions are used in antagonistic activity tests as described below.For antagonistic activity tests, 100μL of spore suspensions from each mold species (approximately 104 spores) was taken from the stock spore suspensions and spread evenly on synthetic dextrose (SD) medium (1.67 g/L yeast nitrogen base, 5 g/L ammonium sulfate, 20 g/L glucose, and 20 g/L agar). When the surface of spore-spread plates dried, 4 μL ofM. pulcherrima UMY15 strain (prepared as described) was planted on the plates in duplicates, and then the plates were incubated at 30°C for spore germination and growth for 1-2 days. All of the antagonistic activity tests were repeated at least twice. Inhibition zones were measured manually and defined as the distance extending from the edges of theM. pulcherrima UMY15 colonies to the beginning of the fungal lawn on the plates and expressed in millimeters.Antagonistic effects ofM. pulcherrima UMY15 on germination of fungal spores were also tested when these molds were grown on apple. For this assay, apple (Malus domestica Borkh, cv. Golden Delicious) slices with dimensions of 4 cm L × 2 cm W × 1 cm H were prepared aseptically from the fully matured Golden Delicious apples. Then 3 holes (3-4 mm diameter, 3-4 mm deep) were prepared on these slices with sterile pipette tips. To these three holes on apple slices, 10 μL of spore suspensions only, 10 μL ofM. pulcherrima UMY15 together with 10 μL of spore suspensions, and 10 μL ofM. pulcherrima UMY15 samples were added, respectively. Apple slices were placed in sterile petri dishes and incubated at 25°C for 2 days for spore germination and fungal growth. At the end of incubation period, spoilage zones on the apple slices caused by the germination and the growth of molds were measured manually. Percentage of the biocontrol activity ofM. pulcherrima UMY15 strain on the above given fungal species was expressed as the ratio of infection zones (mm) developed on apples when these molds coexist withM. pulcherrima UMY15 strain over the zone of infection formed by these molds alone on apple wounds [21].Antagonistic effects ofM. pulcherrima on the germination and hyphal growth of various molds in grape juice were also analyzed in shake-flask culture. For these assays, approximately 105 of mold spores and 104 colony forming unit (CFU) fromM. pulcherrima samples were coinoculated into 20 mL of additive-free grape juice in 100 mL flask. Grape juice samples inoculated withM. pulcherrima and/or mold spores were incubated at 30°C in an incubator shaker with 130 rev/min. Cultures were visually inspected for mold sporulation after 48 hours. Efficacy of spore germination in grape juices was estimated by comparing the amount of mycelia growth in the grape juice cultures to the cultures that contain only spores of relevant molds (control group withoutM. pulcherrima).The numbers given for inhibition and spoilage zones in Tables1 and 2 are the average values of at least 4 independent experiments. The standard deviations for the zones of inhibitions were less than 10%.Table 1
Antagonistic effects ofM. pulcherrima UMY15 strains on the spore germination and the mycelia growth of different fungal species.
Fungal species
Zone of inhibitions (mm)a
24 h
48 h
P. roqueforti
4
3
P. italicum
3
3
P. expansum
3
3
Fusariumsp.
1
0.5
Rhizopussp.
1
1
A. niger
1
0
A. oryzae
3
2
A. parasiticus
2
1.5
aInhibition zones were measured as the distance from the edges of M. pulcherrima colonies to the beginning of the fungal lawns on SD plates at the 24th and 48th hours of incubations. Standard deviations were less than 10% in zones of inhibition.Table 2
Inhibition of spore germination and fungal growth on the artificial wounds on apple byM. pulcherrima UMY15 strain.
Fungal species
Inoculations and zones (mm) of fungal growtha
% Biocontrol
Fungal spores only
Fungal spores +M. pulcherrima
P. roqueforti
NG
NG
NA
P. italicum
15
5
67
P. expansum
NG
NG
NA
Fusariumsp.
12
0
100
Rhizopussp.
Full growth
Full growth
0
A. niger
15
6
60
A. oryzae
8
0
100
A. parasiticus
10
3
70
aZones of fungal growth were measured as the distance from the center of artificial wounds to the ends of fungal growths (rots) on apples at the end of 48th hours of incubations.
NG: no growth on apple wounds, NA: not applicable. Standard deviations were less than 10% in zones of inhibition.


## 3. Results

### 3.1. Testing the Inhibitory Effects ofM. pulcherrima UMY15 Strain on Fungal Growth

The inhibitory or antagonistic effects ofM. pulcherrima UMY15 strain on the spore germination and the growth of eight different mold species were first investigated by plate tests.M. pulcherrima UMY15 strain’s suspensions were applied onto the spores of molds used in this study as described. Germinations and hyphal growth ofP. roqueforti,P. italicum, andP. expansum spores were significantly inhibited byM. pulcherrima UMY15 strain on SD petri plates (Table 1 and Figures 1(a) and 1(b)). At the end of 24 hours incubation period, 3-4 mm of inhibition zones were clearly visible on the spore germination plates ofPenicillium species used in this study. However, unlikePenicillium species, germination ofFusariumsp.,Rhizopussp., andA. niger spores was inhibited at low levels byM. pulcherrima strain. Inhibition zones on the spore germination plates of these three fungal strains were approximately 1 mm (Table 1). Nonetheless, there is a significant level of inhibitory effect ofM. pulcherrima strain on two otherAspergillus species,A. oryzae andA. parasiticus,respectively (Table 1 and Figures 1(c) and 1(d)). Inhibition zones on the spore germination plates ofA. oryzae andA. parasiticus were 2-3 mm after 24 hours of incubation period.M. pulcherrima inhibits the growth of different molds on SD plates. Lawns on plate (a)P. expansum, plate (b)P. roqueforti, plate (c)A. oryzae, and plate (d)A. parasiticus.
(a)
(b)
(c)
(d)


### 3.2. Inhibition of Fungal Growth on Apple byM. pulcherrima UMY15 Strain

We have shown thatM. pulcherrima UMY15 strain has a significant inhibitory effect on the spore germination and mycelial growth of certain molds such asPenicillium andAspergillus on petri plates. Next, we wanted to test whetherM. pulcherrima strain will also inhibit the germination of these fungal species when their spores seeded on artificial wounds on Golden Delicious apples. Hence, spore suspensions of certain species ofPenicillium,Fusarium,Rhizopus, andAspergillus were applied on small holes on apples with or withoutM. pulcherrima strain.Coexistence or cocultivation ofM. pulcherrima strain UMY15 withFusariumsp. spores completely inhibited the germination ofFusariumsp. spores and spoilage of artificially wound apples (Table 2, Figure 2(a)). In the absence ofM. pulcherrima strain,Fusariumsp. spores germinated and mycelia growth led to the formation of 12 mm spoilage zones on apple wounds. However, whenM. pulcherrima strain andFusarium spores coinoculated on same wound, there were no spoilage zones on artificial wounds on apples (Table 2, Figure 2(a)). Biocontrol activity ofM. pulcherrima onFusarium was determined as 100%.Examples of the inhibitory effects ofM. pulcherrima UMY15 strain on different fungal species on artificially wound apples. Infection assays forFusariumsp. (a),A. Oryzae (b),A. niger (c), andA. parasiticus (d) were done on artificial wounds on apple slices. Inoculations on artificial wounds are 1: mold spores only, 2:M. pulcherrima only, and 3: mold spores andM. pulcherrima inoculations.
(a)
(b)
(c)
(d)Cocultivation ofM. pulcherrima strain withA. oryzae on apple wounds also prevented the spore germination, mycelia growth, and spoilage of apple slices. As seen in Figure 2(b) and Table 2, when spore suspensions ofA. oryzae were applied on apple wounds, spore germination and mycelia growth resulted in the development of an 8–10 mm wide zone of infection. However, in the presence ofM. pulcherrima strain together with the spores of these molds, there were no spoilage zones on apples, indicating thatM. pulcherrima strain has very strong inhibitory effects (100% biocontrol) on the germination of the spores of these fungal pathogens.We have also tested the inhibitory effects ofM. pulcherrima UMY15 strain on the germination ofP. roqueforti,P. italicum,P. expansum,Rhizopussp.,A. parasiticus, andA. niger’s spores’ germinations and their efficacy in the formation or development of rotting zones on artificial apple wounds. Since apple is not a natural habitat forP. roqueforti, there was no spore germination and fungal growth on apple holes that containP. roqueforti spores at the end of a 2-day incubation period. In a similar manner, we were unable to detect any spore germination and fungal growth on apple wounds which containP. expansum spores at the end of a 2-day incubation period. However,M. pulcherrima strain had a significant level of biocontrol activity (50–70% inhibition) on the germination and mycelia development ofP. italicum,A. niger, andA. parasiticus (Figures 2(c) and 2(d)). Germination and the development ofP. italicum andA. niger spores on apple wounds in the absence ofM. pulcherrima strain resulted in the formation of an approximately 15 mm wide zone of infection. However, cocultivation of the spores of these molds withM. pulcherrima strain led to the formation of about a 5-6 mm wide zone of infection on apple wounds (Table 2, Figure 2(c)). In addition, we further tested the inhibitory effects ofM. pulcherrima on spore germination in grape juice (Table 3). The results indicated thatM. Pulcherrima is also very effective onP. roqueforti,Fusarium sp., andA. oryzae, with 100% biocontrol activity (Table 3).Table 3
Inhibition of spore germination and fungal growth in grape juice byM. pulcherrima UMY15 strain.
Fungal species
% mycelial growtha
% Biocontrol
Fungal spores only
Fungal spores +M. pulcherrima
P. roqueforti
100
0
100
P. italicum
100
50
50
P. expansum
100
25
75
Fusariumsp.
100
0
100
Rhizopussp.
100
25
75
A. niger
100
70
30
A. oryzae
100
0
100
A. parasiticus
100
40
60
aMycelial growth in grape juice was determined visually. Spore germination and growth of mycelia in control groups that do not contain M. pulcherrima UMY15 were accepted as %100 growth. %biocontrol activity was estimated by comparing the mycelial growth of mold spores coinoculated with M. pulcherrima UMY15 strains to the growth of controls.


## 3.1. Testing the Inhibitory Effects ofM. pulcherrima UMY15 Strain on Fungal Growth

The inhibitory or antagonistic effects ofM. pulcherrima UMY15 strain on the spore germination and the growth of eight different mold species were first investigated by plate tests.M. pulcherrima UMY15 strain’s suspensions were applied onto the spores of molds used in this study as described. Germinations and hyphal growth ofP. roqueforti,P. italicum, andP. expansum spores were significantly inhibited byM. pulcherrima UMY15 strain on SD petri plates (Table 1 and Figures 1(a) and 1(b)). At the end of 24 hours incubation period, 3-4 mm of inhibition zones were clearly visible on the spore germination plates ofPenicillium species used in this study. However, unlikePenicillium species, germination ofFusariumsp.,Rhizopussp., andA. niger spores was inhibited at low levels byM. pulcherrima strain. Inhibition zones on the spore germination plates of these three fungal strains were approximately 1 mm (Table 1). Nonetheless, there is a significant level of inhibitory effect ofM. pulcherrima strain on two otherAspergillus species,A. oryzae andA. parasiticus,respectively (Table 1 and Figures 1(c) and 1(d)). Inhibition zones on the spore germination plates ofA. oryzae andA. parasiticus were 2-3 mm after 24 hours of incubation period.M. pulcherrima inhibits the growth of different molds on SD plates. Lawns on plate (a)P. expansum, plate (b)P. roqueforti, plate (c)A. oryzae, and plate (d)A. parasiticus.
(a)
(b)
(c)
(d)


## 3.2. Inhibition of Fungal Growth on Apple byM. pulcherrima UMY15 Strain

We have shown thatM. pulcherrima UMY15 strain has a significant inhibitory effect on the spore germination and mycelial growth of certain molds such asPenicillium andAspergillus on petri plates. Next, we wanted to test whetherM. pulcherrima strain will also inhibit the germination of these fungal species when their spores seeded on artificial wounds on Golden Delicious apples. Hence, spore suspensions of certain species ofPenicillium,Fusarium,Rhizopus, andAspergillus were applied on small holes on apples with or withoutM. pulcherrima strain.Coexistence or cocultivation ofM. pulcherrima strain UMY15 withFusariumsp. spores completely inhibited the germination ofFusariumsp. spores and spoilage of artificially wound apples (Table 2, Figure 2(a)). In the absence ofM. pulcherrima strain,Fusariumsp. spores germinated and mycelia growth led to the formation of 12 mm spoilage zones on apple wounds. However, whenM. pulcherrima strain andFusarium spores coinoculated on same wound, there were no spoilage zones on artificial wounds on apples (Table 2, Figure 2(a)). Biocontrol activity ofM. pulcherrima onFusarium was determined as 100%.Examples of the inhibitory effects ofM. pulcherrima UMY15 strain on different fungal species on artificially wound apples. Infection assays forFusariumsp. (a),A. Oryzae (b),A. niger (c), andA. parasiticus (d) were done on artificial wounds on apple slices. Inoculations on artificial wounds are 1: mold spores only, 2:M. pulcherrima only, and 3: mold spores andM. pulcherrima inoculations.
(a)
(b)
(c)
(d)Cocultivation ofM. pulcherrima strain withA. oryzae on apple wounds also prevented the spore germination, mycelia growth, and spoilage of apple slices. As seen in Figure 2(b) and Table 2, when spore suspensions ofA. oryzae were applied on apple wounds, spore germination and mycelia growth resulted in the development of an 8–10 mm wide zone of infection. However, in the presence ofM. pulcherrima strain together with the spores of these molds, there were no spoilage zones on apples, indicating thatM. pulcherrima strain has very strong inhibitory effects (100% biocontrol) on the germination of the spores of these fungal pathogens.We have also tested the inhibitory effects ofM. pulcherrima UMY15 strain on the germination ofP. roqueforti,P. italicum,P. expansum,Rhizopussp.,A. parasiticus, andA. niger’s spores’ germinations and their efficacy in the formation or development of rotting zones on artificial apple wounds. Since apple is not a natural habitat forP. roqueforti, there was no spore germination and fungal growth on apple holes that containP. roqueforti spores at the end of a 2-day incubation period. In a similar manner, we were unable to detect any spore germination and fungal growth on apple wounds which containP. expansum spores at the end of a 2-day incubation period. However,M. pulcherrima strain had a significant level of biocontrol activity (50–70% inhibition) on the germination and mycelia development ofP. italicum,A. niger, andA. parasiticus (Figures 2(c) and 2(d)). Germination and the development ofP. italicum andA. niger spores on apple wounds in the absence ofM. pulcherrima strain resulted in the formation of an approximately 15 mm wide zone of infection. However, cocultivation of the spores of these molds withM. pulcherrima strain led to the formation of about a 5-6 mm wide zone of infection on apple wounds (Table 2, Figure 2(c)). In addition, we further tested the inhibitory effects ofM. pulcherrima on spore germination in grape juice (Table 3). The results indicated thatM. Pulcherrima is also very effective onP. roqueforti,Fusarium sp., andA. oryzae, with 100% biocontrol activity (Table 3).Table 3
Inhibition of spore germination and fungal growth in grape juice byM. pulcherrima UMY15 strain.
Fungal species
% mycelial growtha
% Biocontrol
Fungal spores only
Fungal spores +M. pulcherrima
P. roqueforti
100
0
100
P. italicum
100
50
50
P. expansum
100
25
75
Fusariumsp.
100
0
100
Rhizopussp.
100
25
75
A. niger
100
70
30
A. oryzae
100
0
100
A. parasiticus
100
40
60
aMycelial growth in grape juice was determined visually. Spore germination and growth of mycelia in control groups that do not contain M. pulcherrima UMY15 were accepted as %100 growth. %biocontrol activity was estimated by comparing the mycelial growth of mold spores coinoculated with M. pulcherrima UMY15 strains to the growth of controls.


## 4. Discussion

Crop protection is the major problem in the production of fresh fruits and vegetables. Large amounts of fruits and vegetables (up to 40%) are rotten by food pathogens after harvesting from the production fields [22]. One of the commonly used methods for the postharvest protection of fresh fruits is to apply antifungal chemicals. Apart from their hazards to human health, fungal pathogens develop resistance to fungicidal chemicals [4]. Hence, biocontrol of postharvest diseases by antagonistic yeasts is the best alternative to antifungal chemicals [4, 12]. Several yeast species are currently used as biocontrol agents for postharvest preservation of fruits and vegetables [23].M. pulcherrima is one of the best biocontrol yeasts that are used in the prevention of the postharvest spoilage of fresh fruits [14, 24]. Biocontrol activity ofM. pulcherrima largely depends on its pulcherrimin pigment that immobilizes free iron ions in the growth medium [18, 21].Previously, we had isolated a new strain ofM. pulcherrima from the local vineyards of the Düzce province of Turkey [16]. Antagonistic effects of this newM. pulcherrima strain on human pathogen yeasts and bacteria have been shown in that previous study [16]. Certain species ofPenicilliumsp.,Fusariumsp.,Rhizopussp., andAspergillussp. are the major cause of spoilage of fresh fruits. In addition, certain species ofAspergillus also produce highly toxic aflatoxins [3]. In this study, we have analyzed the biocontrol activity of one of these newM. pulcherrima strains (UMY15) on aforementioned postharvest pathogens both on plate tests and also on artificial wounds on apples. Our biocontrol activity tests showed that theM. pulcherrima UMY15 strain has very effective antagonistic activities onP. roqueforti,P. italicum, andP. expansum on plate tests. However,M. pulcherrima UMY15 is less effective on other fungal pathogens such asFusariumsp. andRhizopussp. Furthermore,M. pulcherrima UMY15 did not have any inhibitory effect on the growth ofA. niger at the end of a 48 hour incubation period. Although there was an inhibitory zone on theA. niger lawn at the end of a 24 h incubation period,A. niger overcomes the antagonistic effects ofM. pulcherrima UMY15 at the end of 48 h of growth on plates. Different fungal species transport and acquire free iron from the growth medium by different mechanisms [25]. Hence it is possible that the differential antagonistic effects ofM. pulcherrima UMY15 on different pathogenic molds might result from the differences of iron requirements of these molds for their growth and development.Cocultivation ofM. pulcherrima UMY15 strain with different mold spores on artificial wounds of apples indicates that this yeast is also a very effective inhibitor of fungal spore development.M. pulcherrima UMY15 completely inhibited (100% biocontrol) the development ofFusariumsp. andA. oryzae spores on apple wounds (Figures 2(a) and 2(b), Table 2). It is less effective onA. parasiticus,A. niger, andP. italicum spore germination and mycelia development on apple. However,M. pulcherrima UMY15 is a better inhibitor ofA. niger on apples than the petri tests. This indicates thatM. pulcherrima UMY15 is a better competitor for pathogenic molds when it grows on apples. This was also shown by Janisiewicz et al. [26], thatM. pulcherrima is a highly effective antagonistic yeast for long term storage of apples (up to 6 months). In our in-vivo analyses, we have used only one dose ofM. pulcherrima UMY15 samples (approximately 103 yeasts cells/inoculums on apple wounds). Efficacy ofM. pulcherrima can be further improved by increasing the concentration of yeast samples applied on mold spores. In addition to our results, Oro et al. [27] reported that different isolates ofM. pulcherrima have significant lethal effects on the non-Saccharomyces yeast when they are coinoculated into grape must. Sisti and Savini [28] also showed that local isolates ofM. pulcherrima strain are highly effective as antifungal agents on human related dermatophytes. They have pointed out that strains ofM. Pulcherrima have great potential to be used in the natural treatment of certain fungal infections.


## 5. Conclusion

Results of this study clearly indicated thatM. pulcherrima UMY15 strain is a very effective biocontrol agent that can be used in the prevention of postharvest diseases caused by molds. ThisM. pulcherrima strain can be developed as a commercial product for postharvest protection of fruits and vegetables from fungal pathogens.



---

*Source: 397167-2014-04-17.xml*