Enhancement of the performance of
Candida saitoana
by the
addition of glycolchitosan for the control of postharvest
decay of apple and citrus fruit
Ahmed El-Ghaouth
a,c, Joseph L. Smilanick
b, Charles L. Wilson
c,*
aFaculte des Sciences et Techniques,Uni
6ersite De Nouakchott,Nouakchott,B.P. 5026, Mauritania bUSDA-ARS,2021 South Peach A
6enue,Fresno,CA 93727, USA cUSDA-ARS Appalachian Fruit Research Station,45 Wiltshire Road,Kearneys
6ille,WV 25430, USA Received 29 June 1999; accepted 15 January 2000
Abstract
At a concentration of 0.025% (w/v) chitosan-chloride inhibited spore germination ofBotrytis cinerea,Penicillium expansum, andCandida saitoana. In contrast, at 0.5% (w/v) glycolchitosan inhibited spore germination ofB.cinerea andP.expansum, but not the growth ofC.saitoanain vitro or in apple wounds. The combination ofC.saitoanawith 0.2% glycolchitosan was more effective in controlling gray and blue mold of apple caused by B. cinerea and P. expansum, respectively, and green mold of oranges and lemons caused by P. digitatumthan C. saitoana or 0.2% glycolchitosan alone. The level of control was similar to that obtained with the fungicide imazalil on oranges and lemons.C.saitoanain combination with 0.2% glycolchitosan reduced green mold incidence equally on light green and yellow lemons, whileC.saitoanawas more effective on light green lemons than on yellow lemons. When applied as a pretreatment, sodium carbonate enhanced the efficacy of all treatments tested against green mold with the greatest effect on light green lemons. Of the treatments tested, pretreatment with sodium carbonate followed by the combination ofC.saitoanawith 0.2% glycolchitosan was the most effective in controlling green mold of both light green and yellow lemons. © Published by Elsevier Science B.V.
Keywords:Antagonist; Apple fruit; Gray, blue and green mold;Botrytis cinerea;Candida saitoana; Citrus; Chitosan; Glycolchitosan; Imazalil;Penicillium expansum;Penicillium digitatum; Sodium carbonate; Thiabendazole
www.elsevier.com/locate/postharvbio
1. Introduction
Microbial biocontrol agents have shown great potential as an alternative to synthetic fungicides
for the control of postharvest decay of fruits and vegetables (Roberts, 1990; Wilson and Wis-niewski, 1994; Filonow et al., 1996; Bull et al., 1997; Chand-Goyal and Spotts, 1997; Droby et al., 1998; El Ghaouth et al., 1998; Janisiewicz, 1998). At present, a yeastCandida oleophila Mon-trocher, and two strains of a bacterium Pseu
-domonas syringae Van Hall are registered for
* Corresponding author. Tel.: +1-304-7253451; fax: + 1-304-7282340.
E-mail address:[email protected] (C.L. Wilson)
postharvest use and are commercially available
under the trade names Aspire (Ecogen,
Langhorne, PA), Biosave-100, and Biosave-110 (EcoScience, Worchester, MA), respectively.
Despite the efforts that have focused on the development of microbial biocontrol agents, their widespread utility as a postharvest treatment has not been fully realized. Apparently, microbial an-tagonists offer a level of control equivalent to synthetic fungicides only when supplemented with low doses of the recommended fungicide (Droby et al., 1993, 1998; Chand-Goyal and Spotts, 1997). This is because of their variable perfor-mance under commercial conditions (Brown and Chambers, 1996; Wilson et al., 1996).
Recently, several additives have been shown to augment the biocontrol activity of selected antag-onists (McLaughlin et al., 1990; Janisiewicz, 1994; Wisniewski et al., 1995). A combination of an antagonist with either CaCl2, nitrogenous
com-pounds, or the sugar analog 2-deoxy-D-glucose,
was shown to increase the effectiveness of the antagonists and reduce the microbial population required to give effective control (McLaughlin et al., 1990; Janisiewicz, 1994; Wisniewski et al., 1995). Among potential additives, chitosan (b -1,4-glucosamine polymer) could be a useful additive to antagonistic microorganisms. Chitosan and its derivatives such as glycolchitosan and car-boxymethylchitosan are known to form a semi-permeable film and are inhibitory to a number of pathogenic fungi, and also induce host-defense responses (Allan and Hadwiger, 1979; El Ghaouth et al., 1994). Combining chitosan with antagonists will make it possible to exploit the antifungal and eliciting property of chitosan-chloride and the bi-ological activity of the antagonist. In the present study we report on the effect of the combination of two chitosans with Candida saitoana Nakase and Suzuki on postharvest diseases of apple and citrus fruit.
2. Materials and methods
2.1. Reagents, microorganisms, and fruit material
Chitosan-chloride and glycolchitosan were
ob-tained from Sigma (St Louis, MO). Thiabenda-zole (2-4-thiazolylbenzimidaThiabenda-zole; Merck, Rahway, NJ) and imazalil were obtained from GreenChem-icals (Winchester, VA). C.saitoana was grown at 24°C for 48 h in shake-flask cultures of nutrient-yeast broth. Yeast cells were pelleted by centrifu-gation with a Sorval RC-58 centrifuge (Dupont Instruments, Wilmington, DE) at 3000×g for 20 min, resuspended in sterile distilled water, and centrifuged again. The resulting pellets were dis-persed in sterile distilled water or different chi-tosan-chloride solutions and the concentration of the yeast was adjusted to 108
colony-forming units (CFU) ml−1
using a hemacytometer.
Penicillium expansum Link, Penicillium digi
-tatum (Pers.:Fr.) Sacc., and Botrytis cinerea
Pers.:Fr. were isolated from infected fruit and maintained on potato dextrose agar (PDA). A spore suspension was obtained by flooding 2-week-old cultures of B.cinerea, P.digitatum, and
P.expansumwith sterile distilled water containing 0.1% (v/v) Tween 80. Spores were counted with a hemacytometer, and spore concentrations from the pathogens were adjusted with sterile distilled water to obtain 1×106
spores per ml for B.
cinerea and 1×104
spores per ml forP.digitatum
and P. expansum.
Tree-ripe apple (Malus domesticaBorkh.) culti-var ‘Red Delicious’ were hand-harvested at com-mercial maturity at the Appalachian Fruit Research Station, Kearneysville, WV and stored at 4°C under regular air for at least 4 months before their use in biocontrol tests. ‘Washington’ navel oranges (Citrus sinens (L.) Osbeck), ‘Eu-reka’ lemons (Citrus lemon (L.) Burm) grown in the San Joaquin Valley of California were har-vested within 2 days before treatment. Selected lemons were divided into two classes on the basis of fruit surface color (green to light green and yellow). Fruit were sorted to remove any with apparent injuries or infections and were randomly assigned to different treatments.
2.2. Effect of different chitosans on antagonistic yeasts and pathogens
against B.cinerea, P.expansum, and P.digitatum
was determined on one tenth strength potato dex-trose broth (PDB) in 24-well microtiter plates. Chitosan-chloride and glycolchitosan solutions were prepared as described previously (El Ghaouth et al., 1994) and added to PDB to obtain concentrations of 0, 0.025, 0.05, 0.1, 0.2, and 0.5% (w/v). The solutions were autoclaved and dispensed immediately into microtiter wells. Each well was inoculated with 500 spores of B.
cinerea, P. expansum, or P. digitatum. For each fungus, four replicates of four wells were used at each concentration and the microtiter plates were incubated at 24°C. Spore germination was determined periodically over a period of 5 days.
The long-term effect of various chitosan-chlo-rides on the survival of C. saitoana was also assessed. A range of concentrations (0, 0.025, 0.05, 0.2, and 0.5%) of chitosan-chloride and gly-colchitosan solutions were supplemented with 5% yeast maltose broth (YMB), autoclaved, and im-mediately dispensed into sterile 50-ml Erlenmeyer flasks. Shake-flask cultures were started with
105
CFU of yeast cells and incubated at 24°C. Samples were collected every week over a period of 55 days and dilution-plated in triplicate on a yeast-maltose agar medium. Plates were in-cubated at 24°C and colonies were counted after 48 h.
2.3. Biocontrol acti6ity of the combination of C. saitoana with chitosan compounds
2.3.1. Experiment c1
The effect of the combination of C. saitoana
with chitosan-chloride or glycolchitosan on apples inoculated with B. cinerea or P. expansum was investigated. Apple fruit were wounded (3 mm wide by 5 mm deep) as previously described (McLaughlin et al., 1990). Fruit wounds were treated with 35ml of one of the following: (1) cell suspensions of C. saitoana at 108 CFU ml−1
containing 0, 0.05, 0.2, or 0.5% of glycolchitosan and chitosan-chloride; (2) 0.05 and 0.5% of gly-colchitosan or chitosan-chloride; or (3) sterile water. Treated wounds were challenge-inoc-ulated with 30 ml of conidial suspensions of B.
cinerea or P. expansum. Fruits were stored at
24°C under high humidity (95% RH) in
enclosed plastic trays. Each treatment was applied to four replicates of 18 fruit each. Fruits were evaluated daily for symptoms over a period of 14 days.
2.3.2. Experiment c2
Lemon and orange fruit were wounded by puncturing each fruit with a stainless steel probe with a tip 2 mm long and 1 mm wide (Smilanick et al., 1997). Wounded fruit were treated with a yeast cell suspension containing 0 or 0.2% of glycolchitosan, 0.2% of glycolchitosan, sterile wa-ter, or 1000 mg/ml imazalil. Subsequently, the wounds were challenge-inoculated with a spore suspension of P. digitatum. The different treat-ments, including inoculum, were applied using a handheld sprayer, and 0.5 ml was applied to
each fruit. Each treatment was applied to four replicates of 60 oranges or 80 lemons each. Fruit were stored 14 days under high humidity (95% RH) in enclosed plastic trays at 20°C, then evaluated for percentage of fruit with decay le-sions.
2.4. Wound colonization by C. saitoana
The effect of glycolchitosan on the survival of
C. saitoana in apple wounds was determined. Apples were wounded as described before, treated with 35ml of a yeast cell suspension (107CFU/ml)
containing 0 or 0.2% of glycolchitosan. Treated fruit were stored at 24°C as described previously. Each treatment was applied to three replicates of six fruit each. Tissue samples from the different treatments were collected 0, 1, 3, and 7 days after treatment. At each sampling time, tissue samples containing the wounds were removed with a no. 7 cork borer (6 mm diameter) from three apples selected randomly from each treatment. Tissue samples were homogenized in 5 ml of sterile wa-ter, vortexed, dilution-plated in triplicate on
yeast-maltose agar medium and the plates
2.5. Effect of sodium carbonate on the
combination of glycolchitosan with C. saitoana
In California packinghouses, lemon fruit are often treated with sodium carbonate (Na2CO3,
sodium carbonate) to improve cleaning and to reduce postharvest decay (Eckert and Eaks, 1989). Lemon fruit were selected by hand from field bins, divided into two groups on the basis of color (light green and pale yellow to yellow), washed with water on a processing line, and randomly assigned to different treatments. Selected fruit were inoculated by puncturing each fruit with a stainless steel probe that had been dipped into a water suspension of 106
spores ml−1of
P.digitatum. Inoculated fruit were stored at 20°C for 1 or 24 h before treatment with sodium carbonate.
Glass tanks containing 22 l of 3% (w/v) sodium carbonate or water were placed in a 300-l heated water tank and the temperature of the sodium carbonate solution was maintained at 37°C. The sodium carbonate concentration in the solution and the temperature were monitored periodically. Inoculated fruit were immersed in a sodium car-bonate solution for 2 min, rinsed with fresh water for 5 s, and air dried for 10 – 20 min. Dried fruit were sprayed using a handheld spraying system with either a yeast suspension containing 0 or 0.2% of glycolchitosan. Inoculated controls were treated with water or dipped in 1000mg/ml imazalil. Fruits were stored at 20°C under high humidity (95% RH) in enclosed plastic trays. Each treatment was ap-plied to four replicates of 25 lemons each. Fruits were evaluated daily for decay symptoms over a period of 14 days and diseased fruit were discarded at each inspection.
2.6. Statistical analyses
All the biocontrol experiments were repeated three times and the incidence of decay determined in all experiments was based on the percentage of fruit infected. An arcsine-square root transforma-tion was applied to the data from the different trials prior to analyses of variance. Homogeneity of variance for different trials of each experiment was evaluated by Hartley’sF-Max test atP=0.05 of the arcsine-square root transformation of the
per-centage of infected fruit. Data from separate trials were combined because statistical analysis deter-mined homogeneity of variances. Duncan’s new multiple range test (P=0.05) to separate means was applied to compare treatments.
3. Results
3.1. Effect of two chitosans on pathogen and yeast growth
The two chitosans tested were effective in inhibit-ing the spore germination of B. cinerea and P.
expansum, with the greatest effect at the higher concentrations. At low concentrations chitosan-chloride was more effective in inhibiting spore germination of B. cinerea and P. expansum than glycolchitosan. At 0.025%, chitosan-chloride com-pletely inhibited spore germination of B. cinerea
and P.expansum, whereas complete inhibition by glycolchitosan was obtained at a concentration of 0.05%. Chitosan-chloride also adversely affected the growth of C. saitoana. At a concentration of 0.025%, chitosan-chloride completely inhibited the growth of C. saitoana. In contrast, glycolchitosan at 0.5% showed no adverse effect on the growth of
C. saitoana (Fig. 1).
In vitro, the rate of growth of C. saitoana was not influenced by glycolchitosan. Increasing the concentration of glycolchitosan from 0.2 to 0.5% showed no apparent effect on the growth of C.
saitoana. Within 7 days, the population size ofC.
saitoanaincreased nearly tenfold and remained so for 55 days (Fig. 1). A similar growth pattern ofC.
saitoanawas observed in the presence of glycolchi-tosan in apple wounds (Fig. 2). In fruit wounds, the population dynamic ofC.saitoanawas not affected by the addition of glycolchitosan. After 24 h of incubation, the population size of C. saitoana
increased nearly tenfold with and without glycol-chitosan and stabilized thereafter (Fig. 2).
3.2. Biocontrol acti6ity of the combination of chitosans with C. saitoana
the efficacy ofC. saitoana in controlling posthar-vest decay of apples, lemons, and oranges (Tables 1 and 2). In apple fruit, the biocontrol activity of
C. saitoana was adversely affected by chitosan-chloride (Table 1). In contrast, the combination of
C. saitoana with 0.2% glycolchitosan was more effective in controlling postharvest decay than C.
saitoana or 0.2% glycolchitosan alone. The effi-cacy of the combination of C. saitoana with gly-colchitosan did not appear to increase by increasing the concentration of glycolchitosan
from 0.2 to 0.5%. Apple fruit treated with the combination of C. saitoana and 0.2% glycolchi-tosan and then inoculated with B. cinerea or P.
expansum showed no visible symptoms of infec-tion for up to 10 days of storage at 24°C, while in control fruit lesions were visible by the 4th day of storage (data not shown).
A similar level of control of decay by the combination of C. saitoana and 0.2% glycolchi-tosan was observed in green lemons and oranges challenge-inoculated with P. digitatum (Table 2).
Fig. 1. Population dynamics ofC.saitoanagrown in glycolchitosan and chitosan-chloride. Solutions were supplemented with 5.0% yeast maltose broth (YMB). Bars represent standard deviations.
Table 1
Effectiveness ofC. saitoana, glycolchitosan or chitosan-chlo-ride, alone or in combination on decay of ‘Red Delicious’ apple caused byB.cinereaandP.expansum
Infected fruit (%)b,c
Treatmenta
0.5% Glycolchitosan 70e
81e
0.2% Chitosan-chloride 80e
56e
C.saitoana 47f
77e
0.2% Chitosan-chloride+ 83e C.saitoana
suspension. Glycolchitosan or chitosan-chloride were applied at 0.2 and 0.5% with and without yeast cells.B.cinereaandP. expansumspore concentrations were 106 and 104 conidia/ml,
respectively.
bMeans are averaged values of three trials. Each trial
contained four replicates of 18 fruits each per treatment. Decay was evaluated after 14 days of storage at 24°C.
cValues followed by the same letter are not significantly
different at P=0.05, according to Duncan’s multiple range test.
slight effect on yellow lemons (Table 3). When applied as a pretreatment, sodium carbonate en-hanced the efficacy of all treatments tested against green mold with the greatest effect on light green lemons. Of the treatments tested, pretreatment with sodium carbonate followed by the combina-tion of C. saitoana with 0.2% glycolchitosan was the most effective in controlling green mold of both light green and yellow lemons (Table 3). Decay control using the combination of sodium carbonate with eitherC.saitoanaor glycochitosan was higher on the light green lemons than on yellow lemons but significantly lower than with the combination of C. saitoana and 0.2% glycol-chitosan (Table 3).
4. Discussion
We have found recently that C. saitoana was compatible with chemically-modified chitosan, glycolchitosan (El Ghaouth and Wilson, 1997).
Table 2
Effectiveness of C. saitoana and glycolchitosan, alone or in combination on decay of ‘Washington’ navel orange and ‘Eureka’ lemon fruit caused byP.digitatum
Treatmenta Infected fruit (%)b,c
Green lemon Yellow lemon Orange
suspension. Glycolchitosan was applied at 0.2% with and without yeast cells. P.digitatumspore concentration was 104
conidia/ml.
bMeans are averaged values of three trials/experiments.
Each trial contained four replicates of 60 lemons and 80 oranges fruits each per treatment. Decay was evaluated after 14 days of storage at 20°C.
cValues followed by the same letter are not significantly
different at P= 0.05, according to Duncan’s multiple range test.
Control of decay by the combination of C.
saitoana and 0.2% glycolchitosan on green and yellow lemons and oranges was significantly supe-rior to that observed following C. saitoana and 0.2% glycolchitosan treatment alone. C. saitoana
in combination with 0.2% glycolchitosan was equally effective in reducing green mold incidence on light green and yellow lemons. When used alone, however,C.saitoana was more effective in reducing decay on light green lemons than on yellow lemons.
3.3. Compatibility of sodium carbonate with biocontrol treatments
Table 3
The effect of sodium carbonate pre-treatment on the biocon-trol ofP.digitatumin ‘Eureka’ lemon with the combination of C.saitoanaand glycolchitosan
Infected fruit (%)b,c
Treatmenta
Green lemon Yellow lemon
99d
98d
Control
Sodium carbonate 41e 88e
20e 75f
Sodium carbonate+ gly-colchitosan
Sodium carbonate+C. 19e 53f saitoana
32g
Sodium carbonate+gly- 5f
colchitosan+C. saitoana
aLemons were immersed for 2 min in 3% (w/v) sodium
carbonate solution, washed with water, and then treated with yeast cell suspension (108 CFU/ml) containing 0 or 0.2% of
glycolchitosan, 0.2% glycolchitosan alone, or sterile water. All fruit were inoculated 24 h before treatment with spore suspen-sion ofP. digitatum. Decay was evaluated after 14 days of storage at 20°C.
bMeans are averaged values of three trials. Each trial
contained four replicates of 25 fruits each per treatment.
cValues followed by the same letter are not significantly
different at P=0.05, according to Duncan’s multiple range test.
the biocontrol agents. Similarly, in the present study the effectiveness of the combination of C.
saitoana with 0.2% glycolchitosan may be due to the interplay of the biological activity of C.
saitoana and the antifungal property of 0.2% gly-colchitosan. Glycolchitosan was inhibitory to spore germination of major postharvest pathogens but showed no effect on the growth ofC.saitoana
in vitro and in planta. The antifungal and eliciting activity of chitosan is well established and is believed to stem from its polycationic nature (El Ghaouth et al., 1994). This may in part explain the higher inhibitory effect of chitosan-chloride in comparison to chemically-modified chitosan (gly-colchitosan) which has a lower percentage of free amino groups.
Variability in the performance of antagonists as biocontrol agents has been ascribed to several factors including the fruit physiological status (Droby et al., 1993; Wilson et al., 1996). Micro-bial antagonists often display a protective effect that diminishes with an increase in tissue ripeness which has been ascribed to an increase in the exflux of nutrients (Wilson et al., 1996). In citrus fruit, the biocontrol activity of antagonists can be overcome by the addition of nutrients (Droby et al., 1989). The increase in nutrient availability as a result of biochemical changes associated with ripening could explain the poor level of control achieved by C. saitoana against green mold of yellow lemon. The observed increased perfor-mance of the combination of C. saitoana with 0.2% glycolchitosan against decay of yellow lemons may be due to both the antifungal and film-forming property of glycolchitosan. Because of its filmogenic property, glycolchitosan may act as a barrier to the outward flux of nutrients and consequently may reduce the availability of nutri-ent to a level that will not sustain growth of the pathogen. This contention is supported by the fact that fungal cells exposed to chitosan often display signs of nutrient depravation (El Ghaouth et al., 1994).
In conclusion, this study demonstrates that the biocontrol activity ofC.saitoanaagainst decay of apple, lemon, and orange caused byB.cinerea,P.
expansum, and P. digitatumwas enhanced signifi-cantly by the addition of glycolchitosan. The level This makes it possible to exploit both the
antifun-gal and eliciting property of chitosan, and the biological activity of the antagonist. The results from the present study confirm that the combina-tion ofC.saitoanaand 0.2% glycolchitosan offers better control of decay of apple and citrus fruit than C. saitoana and 0.2% glycolchitosan alone. Furthermore, the combination ofC.saitoana and 0.2% glycolchitosan was shown to confer a level of disease control equivalent to a commercially recommended fungicide, imazalil, and to be patible with sodium carbonate, a compound com-monly used as a postharvest treatment in packinghouses.
Enhancement of microbial biocontrol agents has been reported with chemical and physical additives such as CaCl2, 2-deoxy-D-glucose, and
of control conferred by the combination of C.
saitoana and 0.2% glycolchitosan was superior to that conferred byC.saitoanaand 0.2% glycolchi-tosan and appears to be due to additive interac-tions between the yeast and glycolchitosan. Combining antagonistic yeasts with glycolchi-tosan can be expected to provide better control of decay than the use of biocontrol agents alone. This combination could be useful as part of a strategy to reduce losses caused by pathogen iso-lates resistant to currently used postharvest fungi-cides (Eckert et al., 1994).
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