Postharvest Biology and Technology 83 (2013) 5864

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Postharvest Biology and Technology
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Use of killer yeast in the management of postharvest papaya anthracnose
J.R. Lima
a,
, D.M.F. Gondim
e
, J.T.A. Oliveira
b
, F.S.A. Oliveira
d
, L.R.B. Gonc alves
c
, F.M.P. Viana
d
a
Programa de ps-graduac o em Biotecnologia, RENORBIO, Universidade Federal do Cear, Empresa Brasileira de Pesquisa Agropecuria, EMBRAPA Laboratrio de Patologia
Ps-colheita, Rua Dra. Sara Mesquita, 2270, Planalto do Pici, CEP 60511-110, Fortaleza, CE, Brazil
b
Universidade Federal do Cear UFC, Dep. de Bioqumica e Biologia Molecular, Campus do PICI, CEP 60451-970, Fortaleza, CE, Brazil
c
Departamento Engenharia Qumica, Universidade Federal do Cear UFC, Campus do PICI, CEP 60455-760, Fortaleza, CE, Brazil
d
Empresa Brasileira de Pesquisa Agropecuria, EMBRAPAAgroindstria Tropical, Laboratrio de Patologia Ps-colheita, Rua Dra. Sara Mesquita, 2270, Planalto do Pici, CEP 60511-110,
Fortaleza, CE, Brazil
e
Universidade de Fortaleza UNIFOR, Fortaleza, CE, Brazil
a r t i c l e i n f o
Article history:
Received 30 October 2012
Accepted 17 March 2013
Keywords:
Postharvest
Mycoparasitism
Scanning electron microscopy SEM
Hydrolytic enzymes
a b s t r a c t
The efciency of two killer yeast strains, Wickerhamomyces anomalus (strain 422) and Meyerozyma guillier-
mondii (strain 443), as biocontrol agents against Colletotrichum gloeosporioides, a postharvest anthracnose
agent of papaya and other tropical fruit, was assessed. These strains were previously selected through
in vitro assays, but in the present study, their in vivo action was assessed. In addition, the inuence of
phytopathogen inoculation time on the fruit in combination with the use of the biocontrol agent was
also assessed. We assessed mycoparasitism as an antagonistic mechanism of action by scanning electron
microscopy (SEM). In addition, two hydrolytic enzymes, chitinase and -1,3 glucanase, were assayed.
Our results indicated that W. anomalus (strain 422) and M. guilliermondii (strain 443) reduced disease
incidence by 24.62% and 20.68%, respectively, for up to 6 d after inoculation, when applied 3h before the
phytopathogen and incubated in a wet chamber (95% RH) at 28

C. The time of yeast inoculation had a

signicant effect on its antagonistic action. Application of the yeasts 12 or 24 h before the phytopathogen
inoculation resulted in 13.75% and 30% of disease reductions for W. anomalus (strain 422) and 31.35%
and 41.17% reductions for M. guilliermondii (strain 443), respectively. Electron micrographs conrmed
mycoparasitism by showing the interaction of the yeasts with C. gloeosporioides hyphae, causing in some
cases, a loss of turgor and yeast penetration of walls with marked concavity formation on hypha cell
walls.
2013 Elsevier B.V. All rights reserved.
1. Introduction
The susceptibility of papaya to postharvest diseases is high,
thus necessitating careful management. Among the postharvest
papaya diseases, anthracnose, caused by the fungal species Col-
letotrichumgloeosporioides, is consideredoneof themost signicant
in northeastern Brazil (Serra and Silva, 2004; Vida et al., 2004). C.
gloeosporioides strains are also associated with the occurrence of
the so-called chocolate spot disease, which is characterized by
small surface patches of reddish-browncolor (Andrade et al., 2007),
in addition to anthracnose.
Postharvest losses are economically more damaging than those
in the eld because they add to the costs of production, fruit
transportation and storage. Such losses are one of the factors that
inuence the decisiontouse chemical agents tocontrol postharvest
diseases. However, there are growing concerns about the safety of

Corresponding author. Tel.: +55 085 3391 7264; fax: +55 085 33917222.
E-mail addresses: jaquerabelo@hotmail.com, jaqueline.lima@uece.br (J.R. Lima).
chemical agents (Jamalizadehet al., 2008; Robiglio et al., 2011), and
the appearance of resistant phytopathogens (Pimenta et al., 2010;
Lima et al., 2011) has boosted the demand for control alternatives
with lowrisks (Spadaro et al., 2004; Kealewand Ayalew, 2008) to
humans and other animals (Jamalizadeh et al., 2011; Li et al., 2011;
Manso and Nunes, 2011), and that will not contaminate soil and
groundwater (Veiga et al., 2006; Neto and Sarcinelli, 2009).
Several alternative strategies to the use of fungicides has been
reported, among which physical control with the use of gamma
and ultra violet radiation(Cia et al., 2007), use of edible coatings
based on chitosan (Ali et al., 2010, 2012), use of essential oils alone
(Bosquez-Molina et al., 2010) or in combination with other non-
toxic compounds, such as gum arabic (Maqbool et al., 2011) and
biological control (Platania et al., 2012). The use of microorganisms
as biocontrol agents represents one of the most viable alternatives
(Janisiewicz and Korsten, 2002; Romeiro and Garcia, 2007; Rosa
et al., 2010; Zhang et al., 2010).
Yeasts stand out among microorganisms as potential agents to
control plant diseases due to their ability to colonize the surface of
leaves and fruit (Rosa-Magri et al., 2011), high reproductive rate,
0925-5214/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.postharvbio.2013.03.014
J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864 59
lownutritional requirement (Droby and Chalutz, 1994) and known
innocuity, widely recognized by the use of yeast in industrial fer-
mentation for hundreds of years (Coelho et al., 2003; Druvefors,
2004). Furthermore, yeasts do not produce spores or mycotoxins,
unlike other potential biocontrol microorganisms such as la-
mentous fungi (Fan and Tian, 2000). Yeast-based or yeast-like
commercial formulations for postharvest rot biocontrol are already
available in the market, such as Nexy

, whose active ingredient is

Candidaoleophilayeast strainO, andCandifruit

, registeredinSpain
for use as a fungicide onfruit andwhose active ingredient is Candida
sake yeast CPA-1 (Sundh and Melin, 2011).
In general, biological control occurs through a wide variety of
mechanisms in addition to competition for space and nutrients
(Wang et al., 2010; Rosa et al., 2010). Several studies indicate the
existence of other biocontrol mechanisms, such as production of
enzymes that degrade the pathogen cell walls (Chanchaichaovivat
et al., 2008), induction of resistance (Zhao et al., 2008; Nantawanit
et al., 2010), generation of reactive oxygen species (Macarisin et al.,
2010), mycoparasitism(Long et al., 2005) and production of toxins,
which act on the cell walls of phytopathogens (Coelho et al., 2003).
In the last two decades, several scientic studies have demon-
strated the efciency of yeasts as biocontrol agents against a
wide variety of phytopathogens (Wang et al., 2008; Hashem and
Alamri, 2009; Rosa et al., 2010; Zhang et al., 2010; Kong et al.,
2010; Li et al., 2011; Manso and Nunes, 2011; Robiglio et al.,
2011; Rosa-Magri et al., 2011; Zhang et al., 2011). However, most
studies have focused on the search for biocontrol agents that
act on the pathogens of temperate climate fruit, for example,
apples, pears, strawberries and grapes, but there remains only
limited research on biocontrol agents toward tropical climate fruit
pathogens.
The present study was aimed at assessing the in vivo effect of
two killer yeast strains, Wickerhamomyces anomalus (strain 422)
and Meyerozyma guilliermondii (strain 443), previously selected in
an in vitro study, on the control of C. gloeosporioides, a papaya
anthracnose-causing phytopathogen. In addition, the inuence of
inoculation time on the biocontrol effect was examined along with
the occurrence of mycoparasitismby scanning electronmicroscopy
(SEM). Lastly, the activities of the hydrolytic enzymes -1,3-
glucanase and chitinase were assayed.
2. Materials and methods
2.1. Fruit
Healthy Gold papayas (Carica papaya L.), with a uniform size
and ripening stage, and without spots or bruises, were harvested
at the Cacimbo Farm, located in the municipality of Paraibapa,
approximately 90km from Fortaleza, capital of the state of Cear,
Northeastern Brazil. The fruit were taken to the Laboratory of
Postharvest Pathologyat EmbrapaTropical Agroindustry, Fortaleza,
sanitized with 2.0% (v/v) sodiumhypochlorite for 3min and left to
dry naturally.
2.2. Phytopathogen
A fungal strain was isolated from papaya with anthracnose
symptoms and identied as C. gloeosporioides Penz. The phy-
topathogen was maintained on Potato Dextrose Agar (PDA; Difco)
at 4

C. Conidial suspensions (1.010

5
conidia/mL) were prepared
from new cultures incubated for 10d at 28

C. The cells were

counted using a Neubauer chamber under optical microscope
(Nikon E-200) and concentration adjusted to 1.010
8
cell/mL
(Zhao et al., 2008).
2.3. Killer yeast
The killer yeasts isolatedandidentiedina previous study(Lima
et al., 2012), W. anomalus (strain 422) and M. guilliermondii (strain
443), were maintained at 4

C on PDA (Difco) until use. The rRNA

D1/D2-region sequences were deposited into GenBank with the ID
numbers JN627213 and JN627210, for strain 422 of W. anomalus
and strain 443 of M. guilliermondii, respectively.
Yeast liquid cultures were grown in Falcon asks containing
30mL of yeast extract peptone dextrose (YEPD) media (10g/L yeast
extract, 20g/L peptone, 20g/L glucose). The cultures were incu-
batedfor 24hat 28

C. Medium-freesuspensioncells wereobtained
by cell culture centrifugation at 1.5g for 10min followed by suc-
cessive washing (twice) with sterile distilled water. The pellets
were resuspended in sterile distilled water up to a concentration of
1.010
8
cells/mL. The cells were counted using a Neubauer cham-
ber under optical microscope (Nikon E-200) and concentration
adjusted to 1.010
8
cells/mL (Zhao et al., 2008).
2.4. Efciency of W. anomalus (strain 422) and M.
guilliermondii (strain 443) in papaya anthracnose control
In order to assess the efciency of the yeasts as biocontrol
agents, themethoddescribedbyZhaoet al. (2008), withslight mod-
ications, was used. After washing the fruit, two uniformwounds,
3mm deep and 5mmin diameter, were performed on the equator
of papaya with a sterile corkscrew. Twenty microliter suspen-
sion aliquots, containing 1.010
8
cells/mL of each strain (strain
422W. anomalus and strain 443M. guilliermondii) were inoculated
inholes located8cmapart fromeachother. After 3h, 20L of the C.
gloeosporioides conidial suspension(1.010
5
cells/mL) wereadded
to each wound. The fruit were incubated under high temperature
(28

C) and 95% RHto provide favorable conditions for the posthar-

vest onset of the disease. Lesion progression was assessed daily, at
3, 4, 5, and 6d after C. gloeosporioides inoculation, by measuring the
diameter of the damaged area relative to the initial wound. Subse-
quently, the percentage of disease inhibition was calculated using
the following equation:
n =
a b
a
100 (1)
where n is the percentage of disease reduction, a the colony area
of C. gloeosporioides in untreated fruit and b the colony area of C.
gloeosporioides inkiller yeast-treatedfruit. Sterile distilledwater, in
lieu of killer yeast, was used as a negative control and the fungicide
TECTO

(1000ppm), which has the active ingredient thiabenda-

zole, was used as a positive control. Two replicates of 10 papayas
were used for each treatment (20 fruit/treatment). The experiment
was repeated twice.
2.5. Inuence of the inoculation time of W. anomalus (strain 422)
and M. guilliermondii (strain 443) in the control of anthracnose
Papaya fruit were harvested and processed as described in Sec-
tion 2.1. Subsequently they were wounded (Section 2.4) and 20L
suspension aliquots (1.010
8
cells/mL) of W. anomalus (strain
422) and M. guilliermondii (strain 443) were inoculated at the
injured sites prior (3, 12, and 24h) and after (12 and 24h),
respectively, inoculation with the C. gloeosporioides suspension
(1.010
5
conidia/mL). The fruit were incubated for 6d after the
onset of the treatments, at 95% RH and 28

C, and necrotic lesion

progression was daily assessed by measuring the diameter of the
necrotic area relative to the initial wound, and the percentage of
disease inhibition calculated using Eq. (1). Sterile distilled water
was usedas negative control andthe fungicide TECTO

(1000ppm),
containing the active ingredient thiabendazole, as the positive
60 J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864
control. Two replicates of 20 papayas each were used for each
treatment. The experiment was repeated twice.
2.6. W. anomalus (strain 422) and M. guilliermondii (strain 443)
populations in the wounds
The establishment of W. anomalus (strain 422) and M. guillier-
mondii (strain443) killer yeasts wereassessedonthesurfaceof fruit
wounds. A 20L aliquot containing 1.010
8
cells/mL of yeast was
inoculated in the wound sites on the papaya fruit. After 0, 1, 2, 3, 4,
5, 6, 7 and 8d of incubation at 28

C, a tissue sample was removed,

and serial dilutions were prepared using a 0.9% saline solution. Cell
counting was carried out by the spread plate method. Three repe-
titions with three fruit were used for each strain. The experiment
was conducted twice (Zhang et al., 2007).
2.7. Examination of W. anomalus (strain 422) and M.
guilliermondii (strain 443) parasitismon C. gloeosporioides by
scanning electron microscopy (SEM)
Yeasts were individually picked onto Petri dishes with PDA
mediumintheformof twoparallel lines 1cmapart fromeachother.
A C. gloeosporioides myceliumdisk of 6mmin diameter, cultured in
PDAfor 10d, was placedat the center of the plate preciselybetween
the two yeast strains streak spots. The plates were incubated
at 28

C until an interaction between the phytopathogen hyphae

and the test yeast (4d of incubation) was reached. Subsequently,
samples of approximately 1cm
2
containing both organisms were
extractedwitha scalpel, washedtwice withdistilledwater andpre-
pared for SEM imaging, according to Bozzola and Russell (1999).
The positive control was a myceliumportion (1cm
2
) taken froma
C. gloeosporioides culture, whichwas grownonPDAat 28

Cfor 10d.
The samples were placed in a modied Karnovsky xative for 48h
and post-xed in 1% osmiumtetroxide in sodiumcacodylate buffer
(0.1M, pH 7.2) for 1h. Afterwards, the samples were dehydrated
by immersion in crescent ethanol series (30%, 50%, 70% and 90%)
for 10min. Next, the samples were immersed in 3 pure ethanol
baths of 10min each and taken to a critical point dryer (Emitech
K850), coated with gold sputter coating (Emitech K550) and exam-
ined with a SEM(Zeiss DSM940 A) using an acceleration voltage of
15kV.
2.8. Chitinase and -1,3-glucanase activities in the culture
mediumof the W. anomalus (strain 422) and M. guilliermondii
(strain 443)
The yeast strains W. anomalus (strain 422) and M. guilliermondii
(strain 443) were cultured in assay tubes with 20mL of YEPD
medium (10g/L yeast extract, 20g/L peptone, 20g/L glucose) at
28

C for 24h. Subsequently, the cultures were ltered through a

0.22mmembrane and the cell-free ltrated was used to measure
chitinase and -1,3-glucanase activities.
-1,3-Glucanase (GLU) activity was determined by the rate
of glucose production from the degradation of laminarin
(SigmaAldrich) used as substrate (Boller, 1992). Laminarin was
dissolved in ultrapure water (Milli-Q), heated to 60

C for 10min,
and dialyzed exhaustively against ultrapure water (Milli-Q) for
removal of free glucose. In the assay, 0.1mL of the sample was
incubated with 0.9mL of laminarin (2.0mg/mL) at 50

C for 30min.
After adding the appropriate reagents to the reaction mixture,
absorbance readings were taken at 520nm. The quantity of glucose
monomers released was determined using a d-glucose standard
curve (330g/mL). The -1,3-glucanase activity was expressed
in nanokatal per milligram of protein (nkatGlu/mgP). One nkat is
equivalent to 1.0nmol glucose released/mL/s.
Fig. 1. Effect of the pre- (3, 12, and 24h) and post- (12 and 24h) treatments of
papaya fruits with W. anomalus (strain 422) and M. guilliermondii (strain 443) on
the progression of the lesion caused by C. gloeosporioides, 6d after the onset of the
treatments. The vertical bars represent the standard error of the means (n=20).
Different letters over the columns at each time point differ signicantly (Tukeys
test, P0.05).
Chitinase activity was assessed using a colorimetric method to
detect N-acetyl-d-glucosamine (NAG) produced by the combined
hydrolytic actionof chitinaseand-glucuronidase(SigmaAldrich)
on non-radioactive colloidal chitin (Molano et al., 1977) used as
substrate (Boller, 1992; Reissig et al., 1995). The concentration
of NAG was determined by spectrophotometry at 585nm against
a standard curve of NAG (SigmaAldrich), ranging from 100 to
600mM. The chitinase activity was expressed in nanokatal per mil-
ligramof protein (nkat/mgP). One nkat is equivalent to 1.0nmol of
NAG released/mL/s, under the assay conditions.
2.9. Statistical analyses
The data were subjected to analysis of variance (ANOVA) [SIS-
VAR software, package (version 5.3; Federal University of Larvas
[Universidade Federal de Lavras])] (Ferreira, 2000) and statisti-
cal signicance was set to the level P0.05. Replication averages
withsimilar results were analyzedtogether. Averages andstandard
errors were calculated and reported for each experiment. When
averages were signicantly different, a comparison was performed
using Tukeys test (P0.05).
3. Results
3.1. Inuence of the inoculation time of W. anomalus (strain 422)
and M. guilliermondii (strain 443) in the control anthracnose
The time of inoculation between the antagonist yeasts and the
pathogen had a signicant effect on control efciency. Through-
out the incubation period, the papaya fruit treated with the yeasts
12 and 24h before C. gloeosporioides had signicantly smaller
(P0.05) infection lesion sizes than those inoculated with the
yeasts 12 and 24h later (Fig. 1). Indeed, the fruit treated with W.
anomalus (strain 422) 24h before C. gloeosporioides inoculation had
a necrotic lesion size 31.35% smaller than control samples after 6d
incubation, while those treated with the yeasts 24h after the phy-
topathogen inoculation were only 4% smaller. Similar results were
found for strain 443, in which the yeast application 24h before the
phytopathogen reduced in 41.17% the lesion size after 6d incuba-
tion. However, when M. guilliermondii was applied 24h after the
phytopathogen, the lesion size reduction was less than 10% com-
pared to control fruit (Fig. 1).
3.2. Inhibitory effect of killer yeast W. anomalus (strain 422) and
M. guilliermondii (strain 443) on C. gloeosporioides
The killer yeasts W. anomalus (strain 422) and M. guilliermondii
(strain 443) demonstrated in vivo antagonistic activity against C.
gloeosporioides when inoculated 3h before the phytopathogen. The
J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864 61
Fig. 2. Effect of the 3-h pre-treatment of papaya fruits with W. anomalus (strain
422) and M. guilliermondii (strain 443) on the evolution of the lesions caused by C.
gloeosporioides measured at 3, 4, 5, and 6d after conidum inoculation. The vertical
bars represent the standard error of the means (n=20). Different letters over the
columns at the same time interval differ signicantly (Tukeys test, P0.05).
suspensions of both strains signicantly reduced (P0.05) the
injury caused by C. gloeosporioides in papaya when inoculated 3h
after the yeast treatments up to 6d of the experimental period
(Fig. 2).
Inoculation with antagonistic yeast delayed the onset of the
anthracnose symptoms, and the diameter of infection remained
signicantly lower (P0.05) than the control group up to the end
of the experimental period (6d) and similarly to the fungicide
thiabendazole. At this time, W. anomalus (strain 422) and M. guil-
liermondii (strain 443) killer yeast reduced the wound diameter by
24.62% and 20.68%, respectively, when inoculated 3h prior to the
inoculation with the phytopathogen (Fig. 2).
3.3. Progression of W. anomalus (strain 422) and M.
guilliermondii (strain 443) populations on papaya surface
wounds
The growth dynamics of W. anomalus (strain 422) and M. guil-
liermondii (strain 443) respectively on papaya surface wounds
are displayed in Figs. 3 and 4. In both cases, the populations
increased after an adjustment period, reaching the maximum
growth between 3 and 4d after inoculation, at which time they
tended to stabilize and thendecreased to levels similar to the initial
levels at the end of the eighth day of incubation.
3.4. Parasitismof W. anomalus (strain 422) and M.
guilliermondii (strain 443) on C. gloeosporioides evaluated by
scanning electron microscopy (SEM)
W. anomalus (strain 422) and M. guilliermondii (strain 443) col-
onized C. gloeosporioides hyphae, leading to a loss of turgidity (see
arrows in Fig. 5E and F). Moreover, mycoparasitism, characterized
Fig. 3. Population dynamics of W. anomalus (strain 422) killer yeast on papaya fruit
wound sites. Vertical bars represent the standard deviations of the means.
Fig. 4. Population dynamics of M. guilliermondii (strain 443) killer yeast on papaya
fruit wound sites. Vertical bars represent the standard deviations of the means.
by the strong colonization of the pathogen hypha by the yeast
cells (see arrows in Fig. 5C, D, G and H), was also observed. Myco-
parasitism is one of the mechanisms involved in phytopathogen
biocontrol andthepresent studyindicatedthat theyeasts parasitize
C. gloeosporioides hyphae and cause physical damage, possibly due
to the production of hydrolyte enzymes that degrade the hyphae
cell walls, such as -1,3-glucanase. The yeast penetration of walls
withmarkedconcavity formationonhypha cell walls is highlighted
by arrows in Fig. 5G and H.
3.5. Hydrolytic enzyme production
The activity of -1,3-glucanase for W. anomalus (strain 422) and
M. guilliermondii (strain 443) was 1.18 and 0.78nkat/mgP, respec-
tively. No chitinase activity was found for the two yeasts under the
conditions used in the assay.
4. Discussion
In recent years, several studies have been conducted aiming to
isolate microorganisms that protect postharvest fruit from fungal
diseases (Dal Bello et al., 2008; Hashemand Alamri, 2009; Liu and
Tsao, 2009). However, these studies have been restricted to a small
groupof fruit, suchas apple, pear, strawberry andcitrus. To the best
of our knowledge, this study is the rst report on the assessment
of W. anomalus and M. guilliermondii as biocontrol agents against
C. gloeosporioides in postharvest papaya and in the investigation of
the mechanisms involved. W. anomalus (strain 422) and M. guil-
liermondii (strain 443) delayed the onset of disease symptoms and
reduce the intensity of disease lesions, after 6dof incubation. These
traits make these yeasts efcient biocontrol agents, when applied
simultaneously with C. gloeosporioides fungus. Similar results were
described by Platania et al. (2012), who reported Penicilliumdigita-
tum biocontrol by using the killer yeasts W. anomalus, on Torocco
orange fruit. Wang et al. (2010) reported a reduction in infection
after 5d of incubation when assessing the efciency of Rhodospo-
rium paludigenum for the biocontrol of the green mold caused by
Botrytis cinereaintomatoes. Zhanget al. (2010) alsoreportedreduc-
tions by AP6- Pseudozyma fusiformata, AP47 Metschnikowia sp. and
PL5 Aureobasidium pullulans, respectively, in the control of brown
rot caused by Monilinia spp. in peaches.
Reduction of the infection of papaya caused by C. gloeospori-
oides might be related to the high reproductive rate of the yeasts
compared to the phytopathogen. Indeed the yeasts colonized the
wound surface faster than the fungus when they were inoculated
simultaneously, thus protecting the fruit.
Thus these yeasts should be employed as a pre-treatment tool
as a way to reduce the anthracnose disease in papaya fruit. Zhao
et al. (2008) described similar results when assessing the in vivo
efciency of Pichia guilliermondii against the phytopathogen Rhizo-
pus nigricans. They reported higher efciency for tomato infection
62 J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864
Fig. 5. Electron micrographs of healthy phytopathogen hyphae, still turgid and without wall rupture (A, B); interaction of the antagonistic yeasts W. anomalus (strain 422)
(C, E, G) and M. guilliermondii (strain 443) (D, F, H) with the C. gloeosporioides phytopathogen hyphae after 4d of incubation at 28

C; C. gloeosporioides hyphae colonized by

yeast (C and D), loss of turgidity of yeast-colonized hyphae (E and F); yeast penetration of walls with marked concavity formation on hypha cell walls (G and H).
J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864 63
control when the yeast was inoculated 24h prior to the pathogen.
They further reported that the inhibition of lesions by 75.5%
with simultaneous inoculation of both organisms, against 14.7% of
reduction with yeast inoculation 24h after phytopathogen inocu-
lation. Therefore, this supports the hypothesis that competition for
nutrients and space plays a key role in the biocontrol effect. When
yeasts are inoculated prior to the phytopathogen, they have more
time to colonize the fruit and, thus, to reduce the availability of
nutrients to the pathogen.
Wang et al. (2008) and Gholamnejad et al. (2010) reported
higher control efciencywhenemployingRhodotorulapaludigenum
in the control of Alternaria alternata on cherry tomatoes and Can-
dida membranifaciens and Rhodotorula mucilaginosa in the control
of Penicillium expansum, respectively, when the application of the
biocontrol agents preceded phytopathogen inoculation.
Inadditionto the establishment of methodologies for the imple-
mentation of selected agents, studies on the inuence of the
inoculation time on the effectiveness of the biocontrol agents are
essential for developing control strategies. In the present study,
the highest efciency of the yeasts used was observed when they
were applied prior to C. gloeosporioides establishment. This nding
demonstrates the importance of the application of the antagonistic
yeasts immediately after the fruit harvest. The loss of biocontrol
efciency when the pathogen is inoculated previously may be
related to the antagonistic yeast mechanismof action. Competition
for space and nutrients has a key role in disease control, as demon-
strated in several studies (Zhao et al., 2008; Chanchaichaovivat
et al., 2008; Droby et al., 2009; Rosa et al., 2010; Wang et al.,
2010; Jamalizadeh et al., 2011; Li et al., 2011). Such a mechanism
is advantageous for farming due to the lack of human exposure to
potentially toxic compounds.
The killer yeasts studied, W. anomalus (strain 422) and M. guil-
liermondii (strain 443), were able to survive quite well in papaya
wounds, reaching scores above the initial inoculum by approxi-
mately 24h after inoculation in the fruit. This nding conrms the
importance of carrying out a survey of biocontrol agents within
the pathogen niche, which increases their benecial effects due
to a greater adaptation to environmental conditions (Janisiewicz
and Korsten, 2002; Droby et al., 2009; Gholamnejad et al., 2010;
Robiglio et al., 2011).
Abundant colonization and strong yeast binding to C. gloeospo-
rioides hyphae was revealed by electron microscopy. This
observation conrmed the occurrence of mycoparasitism in the
relationship between C. gloeosporioides and the killer yeast W.
anomalus (strain 422) and M. guilliermondii (strain 443). Further-
more, SEM also revealed turgidity loss, the occurrence of concave
areas and, in some cases, complete hypha rupture with yeast pene-
tration into the pathogen hyphae. These results are consistent with
those of Hashemand Alamri (2009), who described the occurrence
of strong P. anomala Mob 93 yeast binding on Botryodiplodia theo-
bromae hyphae after 4d of incubation as well as concave areas on
the surface of hyphae and evidence of penetration. These authors
further reported the occurrence of extracellular matrix accumula-
tion around the colonized hyphae. Chan and Tian (2005) reported a
strong binding capacity of Pichia membranefaciens to Monilinia fruc-
ticola, P. expansum and Rhizopus stolonifer hyphae and suggested
that this binding was related to the production of lytic enzymes
demonstrated in their study.
The evidence of -1,3-glucanase production by W. anomalus
(strain 422) and M. guilliermondii (strain 443) reinforces the pos-
sibility of using both organisms as a biocontrol agent because this
enzyme hydrolyzes glucans, the most abundant component of fun-
gal cell walls (Peberdy, 1990; Iorio et al., 2008; Fleuri and Sato,
2008). This enzyme degrades the cell walls of lamentous fungi
and some yeasts, and its activity is likely related to the C. gloeospo-
rioides hyphae lysis and penetration observed in the present study.
Bauermeister et al. (2010) further noted that the commercial
product Aspire

, recommended for the biological control of the

postharvest rot of citrus and other fruits such as apple and pear,
is based on C. oleophila -glucanase production.
There are several putative mechanisms of biological control.
These mechanisms represent activities involving a variety of eco-
logical relationships not only between biocontrol agents and
phytopathogens but also the entire resident biota. Therefore,
according to Jamalizadeh et al. (2011), knowing the main mech-
anisms involved in the action of a specic agent is key for the
development of formulations based on these agents and method-
ologies of its application. Therefore, in the present study, we opted
to use yeasts with killer activity already reported in previous stud-
ies. Although these studies have not shown any direct toxin effect
on the pathogen, its action on the fruit-resident microbiota may act
synergically with yeast action on the pathogen.
The present study demonstrated that W. anomalus (strain 422)
and M. guilliermondii (strain 443) killer yeast, isolated frompapaya,
were able to reduce the wounds caused by C. gloeosporioides when
inoculated on a fruit wound. This action is inuenced by the
relation between the time of biocontrol agent application and phy-
topathogen inoculation. It was also possible to demonstrate that
the effect of W. anomalus (strain 422) and M. guilliermondii (strain
443) yeast was due to multiple factors, including competition for
space and nutrients, mycoparasitismand secretionof enzymes that
lyse the phytopathogen cell wall.
Acknowledgments
The authors wouldlike tothankthe Fundac oCearense de Apoio
ao Desenvolvimento Cientco e Tecnolgico (FUNCAP) (Cear
State Foundation for the Support of Scientic and Technologi-
cal Development) and the Conselho Nacional de Desenvolvimento
Cientico e Tecnolgico (CNPq) (National Council for Scientic and
Technological Development) for providing research grants and the
EMBRAPA Tropical Agroindustry, whose invaluable support made
this study possible.
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