Sensitivity of Penicillium digitatum and P. italicum to Imazalil and Thiabendazole in Morocco
Green and blue molds (caused by Penicillium digitatum
and P. italicum, respectively) are the main postharvest diseases
of citrus fruits in Morocco. Following packing houses reports of reduced
efficacy of fungicides used to control theses diseases, a survey was conducted
during 2005-2006 packing season to characterize, both qualitatively and
quantitatively, the resistance of P. digitatum and P. italicum
to imazalil (IMZ) and thiabendazole (TBZ). Isolates of P. digitatum
and P. italicum were obtained from decayed citrus fruits collected
from commercial citrus packing houses located in the Souss-Massa-Draa
(SMD), South of Morocco and were evaluated in-vitro for their sensitivity
to IMZ and TBZ. Of the 290 P. digitatum isolates, 19% (55/290)
were resistant to IMZ and 37% (107/290) were resistant to TBZ tested at
discriminatory concentrations of 0.1 or 20 μg mL-1, respectively.
In contrast, only 2.5 (5/204) and 21% (44/204) of P. italicum isolates
collected from packing houses were resistant to IMZ and TBZ, respectively.
No resistance to TBZ and IMZ were detected in Penicillium sp.,
isolates collected from a citrus orchard which has no known history of
fungicide use. The proportion of collected isolates that were resistant
to both fungicides was 1.5% for P. italicum and 10% for P. digitatum.
The mean EC50 values for in vitro inhibition of mycelial
growth of the P. digitatum resistant-isolates were between 0.81
and 0.98 μg mL-1 for IMZ, whereas those of TBZ were between
39.23 and 50.84 μg mL-1. The mean EC50 values
for P. italicum resistant-isolates ranged from 0.53 to 0.61 μg
mL-1 for IMZ and from 52.97 to 59.92 μg mL-1
for TBZ, whereas the mean EC50 values for orchard collected
isolates were 0.04 μg mL-1 for IMZ and 0.16 μg mL-1
for TBZ. The data will provide a baseline for monitoring resistance to
IMZ and TBZ in populations of P. digitatum and P. italicum
in the SMD commercial citrus packing houses in the future.
Citrus fruit cultivation is very important in Morocco, being the first exporting agricultural sector and playing a major role in the national economic development. The largest volume of citrus fruit for fresh consumption and export in the Morocco is grown and shipped from packing houses in Souss-Massa-Draa (SMD), Valley. Postharvest decay is one of the major factors affecting fresh citrus fruit quality and marketing value and it is essential to control decay in order to maintain the quality and prolong the shelf life of citrus fruit, particularly in a market where transport from producer to consumer may take several weeks.
The most important diseases that cause commercially significant losses, in Morocco (Elkhamass et al., 1994) and worldwide (Eckert and Eaks, 1989; Holmes and Eckert, 1999; Zhu et al., 2006), are green mold, caused by Penicillium digitatum (Pers.: Fr.) Sacc. and blue mold, caused by P. italicum Wehmer. These Penicillium species are ubiquitous and produce copious amounts of asexual conidia that are efficiently dispersed via air currents (Holmes and Eckert, 1995). Their inoculums are found in citrus growing-areas and in fruit handling environment. These fungi only infect fruit through injuries created by harvesting and subsequent handling (Brown and Miller, 1999).
Control of green and blue molds is mainly based on the use of fungicides treatments
principally thiabendazole (TBZ) and imazalil (IMZ) sprayed, alternately or simultaneously,
on fruit during waxing operation in the packing house. In most commercial packing
houses in SMD, pre-storage chemical control with benomyl (BEN) or thiophanate-methyl
(THM) is commonly applied, as postharvest drench, to reduce the incidence of
green and blue molds in citrus fruit that are stored in cold before processing.
Unlike IMZ, the postharvest fungicides, BEN, THM and TBZ belong to the chemical
class of benzimidazole and have a similar mode of action. The imidazole fungicide
IMZ is effective for controlling TBZ-resistant isolates of P. digitatum
and P. italicum (Wild, 1994; Smilanik et al., 2005).
Several reports (Staub, 1991; Hewitt, 1998) indicated that when populations of fungal pathogens are repeatedly exposed to site-specific fungicides, resistant strains could be readily selected. The result of this selection has been documented in cases in which TBZ and IMZ were used extensively in controlling P. digitatum and P. italicum in commercial packing houses (Bus, 1992; Wild, 1994; Holmes and Eckert, 1999; Besri et al., 1983; Kinay et al., 2007).
However, although study has been done in several other countries (Eckert et al., 1994; Holmes and Eckert, 1995; Brown and Miller, 1999), little is known about the fungicide sensitivity of P. digitatum and P. italicum populations in the SMD citrus packing houses. Thus, the research reported herein was conducted to determine, under packing house conditions, the proportion and the level of resistance of P. digitatum and P. italicum to imazalil and thiabendazole in the SMD Valley, South of Morocco.
MATERIALS AND METHODS
Fungicides: The commercial citrus postharvest fungicides TBZ (50% a.i., Tecto 500 SC, RODA, Morocco) and IMZ (80% a.i., Magnate 80 EC, SAOAS, Morocco) were used. Fungicide solutions were first prepared by dissolving each commercially-formulated fungicide in acetone. An aqueous stock solution of each fungicide was then prepared. To obtain desired concentrations, samples from stock solutions of fungicides were added aseptically to molten (50°C) sterile Potato Dextrose Agar (PDA) medium at pH 5.9±0.2. Control plates contained PDA and acetone and the final concentration of acetone in the medium did not exceed 1% (v/v). All concentrations were expressed as microgram of active ingredient (a.i.) per mililiter of growth medium.
Sampling locations and fungal isolations: The isolates of P. digitatum
(290) and P. italicum (204) used in this study were recovered from
infected citrus fruit collected in 4 packing houses located in SMD Valley, South
of Morocco, during the 2005-2006 packing season. These packing houses are referred
to as packing houses I, II, III and IV, respectively, throughout the remainder
of the article. Isolates of Penicillium sp., were evaluated for fungicide
sensitivity in comparison with reference isolates recovered from a citrus orchard
where TBZ and IMZ are not used. At least five infected citrus fruits, with symptoms
of green and blue molds, were collected monthly (from November 2005 to May 2006)
from each packing house. Fruits were placed in separate plastic bags and transported
to the laboratory.
Using the procedure described by Bus et al. (1991), conidia were collected from rotted citrus fruits, previously surface-disinfected with 90% ethanol, using a sterile cotton swabs that were stored in sterile test tubes. The conidia were transferred to Petri plates containing potato dextrose agar acidified with 0.5 mL of lactic acid (80%) per liter (pH 5.3±0.2). After a four days incubation period at 25°C, plates were examined under a stereomicroscope to determine colonies identities. Isolates were confirmed to belong to P. digitatum and P. italicum species, according to Domsch et al. (1980) identification key. Pure subcultures of P. digitatum and P. italicum were stored on PDA slants at 5°C in the dark, until needed.
Fungicide sensitivity tests: The isolates of P. digitatum and P. italicum were in vitro-tested for sensitivity to IMZ and TBZ at a discriminatory concentration of 0.1 or 20 μg mL-1, respectively; a concentration completely inhibitory for the sensitive isolates but not for the resistant isolates. Hyphal plugs (5 mm diameter) from the periphery of actively growing colony were inverted and placed in the centers of 3 replicate Petri plates containing PDA medium supplemented with fungicide. After a ten days incubation period at 25°C, the isolates were considered either resistant if any growth was observed on fungicide-amended medium or sensitive if no growth has occurred.
Pathogenicity tests: Isolates under study, which included both sensitive isolates obtained from citrus fruit collected in an orchard and resistant isolates recovered from citrus fruit collected in packing houses, were inoculated into citrus fruit to ensure the pathogenicity of P. digitatum and P. italicum isolates. Orange fruit were washed, surface-disinfected with 90% ethanol and then inoculated by the injection of 10 μL of a spore suspension (105 spores mL-1) of each Penicillium sp., isolate about 1 cm below the rind surface. Three oranges constituted a single replicate and each treatment was replicated two times. After inoculation, all fruit were stored for one week at 20°C and 95% RH, after which they were examined for decay development and sporulation.
Measurement of fungicide sensitivity: For each packing house P. digitatum and P. italicum resistant-isolates were used to determine the effective concentration for inhibition of 50% of mycelium radial growth (EC50). These isolates were arbitrary chosen and represent the 4 sampled packing houses.
A 5 mm diameter disk was taken from the margin of a seven day-old culture of each isolate of P. digitatum and P. italicum and placed at the center of PDA plate. Fungicide concentrations in amended PDA medium were 0, 0.1,0.2, 0.5 and 1.0 μg of TBZ per mL; 0, 0.015, 0.025, 0.035, 0.045 and 0.05 μg of IMZ per mL, for the sensitive isolates collected from a citrus orchard. For the TBZ-and IMZ-resistant isolates the following concentrations were used: 0, 0.2, 0.4, 0.5, 0.6, 0.8 and 1.0 μg of IMZ per mL; 0, 10, 20, 40, 50 and 70 μg of TBZ per mL. All isolates were collected from commercial packing houses. Three Petri plates were used for each fungicide concentration. Colony diameters were measured after seven days at 25°C. The experiment was performed twice. Percent growth inhibition at each fungicide concentration was calculated according to the following formula:
Statistical analysis: The EC50 values were calculated for
each isolate and each fungicide by regression analysis of the percent inhibition
of fungal growth versus the log of the fungicide concentration (μg mL-1)
(Mondal et al., 2005). The data were subjected to statistical Analysis
of Variance (ANOVA) using the STATISTICA software, version 6, StatSoft, 2001,
France. Mean separation was performed following the method of Newman and Keuls
test at p = 0.05.
Fungicide sensitivity: In this study, 290 isolates of P. digitatum
were collected from 4 commercial citrus packing houses located in SMD Valley.
All isolates were tested in vitro for resistance to either TBZ or IMZ
at discriminatory concentrations of 20 or 0.1 μg per mL, respectively.
Present results show that 3.79, 40, 4.41 and 12.9% of P. digitatum isolates
collected from packing houses I, II, III and IV, respectively, were resistant
to IMZ (Table 1). Whereas, all P. digitatum isolates
collected from a citrus orchard were sensitive to IMZ. The percentage of TBZ-resistant
isolates of P. digitatum collected from the four packing houses ranged
from 9 to 75%. In contrast, all 25 isolates of P. digitatum recovered
from infected fruit collected in a citrus orchard (nonexposed population) were
sensitive to TBZ tested at a discriminatory concentration of 20 μg mL-1
||Evaluation of the sensitivity to imazalil and thiabendazole
of Penicillium digitatum and P. italicum isolates from 4 locations
in the Souss-Massa-Drâa Valley, Morocco
|xSensitivity of isolates to fungicide was tested
on PDA medium amended with TBZ at 20 μg mL-1 or IMZ at 0.1
μg mL-1. Isolates which failed to grow on the amended medium
were considered sensitive. YNo. of isolates resistant to fungicide,
ZTotal No. of Penicillium isolates per packing house
A discriminatory concentration of 0.1 μg of IMZ per mL was used for testing
P. italicum resistance. As shown in Table 1, IMZ-resistant
isolates of P. italicum were relatively rare. Among 204 isolates collected
during 2005-2006 packing season only 5 were resistant to IMZ; resistance was
found at two packing houses (II and IV). Whereas, all P. italicum isolates
collected in a citrus orchard were sensitive to IMZ. Based on a discriminatory
concentration of 20 μg of TBZ per mL, resistant isolates of P. italicum
were recovered from decayed fruit collected in the 4 citrus commercial packing
houses (Table 1). However, none of the P. italicum
isolates, recovered from fruit collected in a citrus orchard, showed growth
at 20 μg mL-1 of TBZ, thus all isolates were considered sensitive
to TBZ. Four groups of resistant isolates were identified among all Penicillium
sp., isolates recovered from packing houses:
sensitive to both IMZ and TBZ
resistant to IMZ only
resistant to TBZ only
resistant to both IMZ and TBZ
The in vivo pathogenicity test indicated that the IMZ-and TBZ-resistant isolates of P. digitatum and P. italicum were not significantly different from IMZ-and TBZ-sensitive isolates in their growth and sporulation on untreated fruit (data not shown).
Level of resistance: Quantification of the sensitivities of P. digitatum
and P. italicum to IMZ and TBZ was based on in vitro inhibition
of mycelial growth. As shown in Table 2, the mean EC50
values of 25 TBZ-and IMZ-sensitive isolates of P. digitatum collected
from a citrus orchard on fungicide-amended PDA were 0.14 and 0.046 μg mL-1,
(EC50) of wild-type citrus orchard isolates of P. digitatum
and P. italicum to imazalil and thiabendazole
|xEC50: Effective concentration of fungicide
in the culture medium needed to inhibit 50% of mycelial growth. Mean values
were determined for ten isolates
(EC50) of P. digitatum and P. italicum resistant-isolates
collected from 4 commercial packing houses to imazalil and thiabendazole
|xEC50: Effective concentration of fungicide
in the culture medium needed to inhibit 50% of mycelial growth. yNo.
of isolates used per sampling location are given in parentheses. zValues
within columns followed by the same letter(s) (a or A) are not significantly
different (p = 0.05). Values are means of three replicates per isolate followed
by the standard deviation of the mean
The mean EC50 values for mycelial growth reduction for 14 isolates
of P. italicum obtained from infected fruit collected from an orchard,
with no history of fungicides use, were 0.07 μg of imazalil per mL and
0.15 μg of thiabendazole per milliliter. These values are much lower than
the values recorded by isolates recovered from fruit collected from commercial
citrus packing houses. The citrus orchard populations were therefore considered
as being indicative of wild-type populations.
The mean EC50 values of P. digitatum resistant-isolates, sampled from the four packing houses, varied from 39.23 to 50.84 μg mL-1 of TBZ and from 0.81 to 0.98 μg mL-1 of IMZ and were not significantly different (p = 0.05, Table 3). For P. italicum, the mean EC50 values for mycelial growth reduction for resistant-isolates were from 52.97 to 59.92 μg mL-1 for TBZ and from 0.53 to 0.61 μg mL-1 for IMZ, with no significant difference (p = 0.05, Table 3). On the basis of these results, it appears that mycelial growth of P. digitatum and P. italicum was more sensitive to IMZ than to TBZ.
In the present study, the baseline sensitivity to IMZ and TBZ was determined using 25 and 14 wild-types isolates of P. digitatum and P. italicum, respectively, collected in a citrus orchard without a previous history of fungicide exposure. Their sensitivity was compared to a representative P. digitatum and P. italicum populations isolated from decayed citrus fruits (oranges and mandarins) collected from commercial packing houses located in the SMD Valley. The development of fungal resistance to IMZ and TBZ has been well documented (Holloman, 1993; Holmes and Eckert, 1999; Ghosoph et al., 2007). The results presented here for SMD citrus packing houses isolates of P. digitatum and P. italicum also concur with these findings.
All P. digitatum and P. italicum isolates collected in a citrus orchard were sensitive in vitro to IMZ and TBZ. This result was consistent with earlier study (Holmes and Eckert, 1995, 1999; Kinay et al., 2007) and suggests that naturally occurring resistant isolates of P. digitatum and P. italicum are rare to absent in citrus orchards, especially in those orchards without a prior history of fungicides usage. Nevertheless, isolates of P. digitatum resistant to TBZ, to IMZ, or to both fungicides were detected in all packing houses sampled. Resistance to IMZ was less common than resistance to TBZ in the SMD citrus packing houses. This result was consistent with earlier published data for P. digitatum populations from China (Zhu et al., 2006) and from Sao Paulo (Brazil) citrus packing houses (Fischer et al., 2009). The proportion of P. digitatum resistant isolates was as high as 19% (55/290) for IMZ and 37% (107/290) for TBZ. Considering the extensive and heavy use of both fungicides in commercial citrus packing houses, it is not surprising that resistance to TBZ and IMZ was so prevalent in P. digitatum populations sampled from SMD, Valley. As reported in a similar study (Bus et al., 1991), among 115 P. digitatum isolates collected from citrus fruits at the wholesale markets in Rotterdam (Holland) and originating from Morocco, 30% of the isolates showed in vitro resistance to TBZ at 10 μg mL-1 and only 2% showed resistance to IMZ tested at 0.2 μg mL-1. Unsatisfactory control of green mold has been observed in some commercial citrus packing houses in SMD. However, no relationship has been established between unsatisfactory control of the disease and a decrease in sensitivity to IMZ and TBZ in P. digitatum isolates recovered from fruit collected in packing houses. Present results demonstrate that the high proportion of isolates with reduced sensitivity to IMZ and TBZ may account for the unsatisfactory control of green mold in commercial citrus packing houses in the SMD Valley.
In contrast, only 2.5 (5/204) and 21% (44/204) of the P. italicum isolates collected in citrus packing houses showed growth on PDA amended with IMZ at 0.1 μg mL-1 or TBZ at 20 μg mL-1, respectively. Similar proportion of P. italicum resistant isolates to IMZ and TBZ have been observed in Rotterdam from mandarin and orange fruits produced in Morocco (Bus et al., 1991). The low occurrence of resistant P. italicum isolates to IMZ may be due to the low frequency of this species among Penicillium sp., populations in citrus packing houses (Bus et al., 1991; Holmes and Eckert, 1999).
In this study, among 290 P. digitatum isolates tested 29 were resistant to both IMZ and TBZ; whereas only three out of 204 P. italicum isolates tested were resistant to both fungicides. This phenomenon of double resistance to IMZ and TBZ has also been reported on P. digitatum (Holmes and Eckert, 1999) and P. italicum (Bus et al., 1991). The mutations in CYP51 gene and in β-tubulin gene have been described as the most common mechanisms of resistance to IMZ and TBZ, respectively (Ma and Michailides, 2005; Schmidt et al., 2006; Zhu et al., 2006; Ghosoph et al., 2007). The number of resistant isolates collected from the 4 packing houses varied greatly, indicating that many factors influencing the development of resistance were involved, such as sanitation measures, layout of the packing house and fungicide-spray programs used in each packing house.
Present study indicated that the resistance of P. digitatum and P. italicum to IMZ and TBZ was not linked with any decrease in pathogenicity. This finding is consistent with eariler study, in which increased resistance to these fungicides was not associated with reduced pathogenicity (Gutter et al., 1981; De Waard and van Nistelrooy 1990; Seidel et al., 1990; Holmes and Eckert, 1995).
The mean EC50 values of P. digitatum sensitive-isolates were 0.036 μg mL-1 for IMZ and 0.14 μg mL-1 for TBZ and were of the same order as those reported by Holmes and Eckert (1999) and Kinay et al. (2007) for nonexposed population. The 14 P. italicum isolates that were tested from citrus orchard were sensitive to IMZ and TBZ with mean EC50 values of 0.04 and 0.16 μg mL-1, respectively. Establishing the level of sensitivity of P. digitatum and P. italicum is critical for monitoring the evolution of resistance to TBZ and IMZ in populations of these important pathogens in the SMD citrus packing houses. This information is of great importance in planning efficient measures for the postharvest control of green and blue molds of citrus fruit and for implementing effective anti-resistance strategies in the SMD commercial citrus packing houses.
In resistant P. digitatum isolates collected from packing houses, the mean EC50 values were from 39.23 to 50.84 μg mL-1 for TBZ and from 0.81 to 0.98 μg mL-1 for IMZ. Despite inhibitory differences, the EC50 values for TBZ and IMZ were consistent with other reports. For example, as reported by Holmes and Eckert (1991), the EC50 value for TBZ was greater than 47 μg mL-1, whereas EC50 values for IMZ varied from 0.87 to 0.92 μg mL-1, in isolates of P. digitatum collected in California citrus packing houses. In the case of P. italicum resistant isolates, the mean EC50 values for TBZ (52.97-59.92 μg mL-1) were higher than reported by Bus in 1992 (22.6 μg mL-1). In contrast, the mean EC50 values (0.53-0.61 μg mL-1) for P. italicum IMZ-resistant isolates used in this study were slightly higher compared to the mean EC50 value (0.52 μg mL-1) for 4 IMZ-resistant isolates collected in California citrus packing houses (Holmes and Eckert, 1999). According to Holmes and Eckert (1999), variability in the EC50 values reported by various researchers can be explained by differences in experimental procedures (e.g., inoculum age, medium pH) and the calculation of the EC50 value. Present observation that the levels of resistance to IMZ and TBZ did not differ significantly within populations of P. digitatum and P. italicum recovered from the 4 packing houses may be explained by the fact that these populations are subjected to a constant selection pressure, due to repeated application of the same fungicide. Imazalil and thiabendazole are applied in the SMD citrus packing houses at relatively high dosage rates (2,000 μg mL-1 for IMZ and 4,800 μg mL-1 for TBZ) in a wax formulation that covers the surface of fruit.
The favorable environmental conditions in packing house, the abundant sporulation
of the Penicillium sp., and the fact that all SMD packing houses use
common fungicide-spray programs are factors contributing to the development
of fungicide resistance (Dekker, 1986). Nevertheless, there has been no report
of complete failure of green and blue molds control in any of the commercial
citrus packing houses from which these resistant P. digitatum and P.
italicum isolates were recovered. However, the sole reliance on TBZ and
IMZ for more than 26 years and since, there is currently no effective chemical
or biological alternatives available for the control of green and blue molds,
could put the citrus industry in the SMD in a precarious position. Continuous
monitoring programs for fungicide sensitivity determination are required in
order to indicate the need to change the applied spray programs and thus, prevent
failures of control. To our knowledge, this report constitutes the first mention
of resistance in populations of P. digitatum and P. italicum
in SMD citrus packing houses to IMZ.
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