Resistance of Verticillium theobromae to Benzimidazole Fungicides in Morocco
A. Ait Benaoumar
In response to growers report of reduced efficacy of
benzimidazole fungicides for control of cigar end-rot, a survey was conducted
from 2002 to 2005 in four banana-growing locations, with various histories
of benzimidazole use, in the Souss-Massa-Drâa Valley, Agadir to
determine the proportion of isolates that were in vitro resistant
to benomyl and thiophanate-methyl. Of the 274 isolates, collected in Biougra,
Belfâa and Ouled-teima locations with more than 10 years of benzimidazole
use, 65% (180/274) were resistant to benomyl, 67% (184/274) were resistant
to thiophanate-methyl and 65% were resistant to both fungicides tested
at a discriminatory concentration of 10 μg mL-1. Only
1.5% of isolates exhibited a differential reaction to the two fungicides.
No resistance to benomyl and thiophanate-methyl were detected in isolates
collected from banana plantations in the Tamri location which has no known
history of benzimidazole use. The mean effective concentrations that reduced
growth by 50% (EC50) for resistant-isolates of V. theobromae
were between 80 and 97 μg mL-1 for benomyl and between
194 to 233 μg mL-1 for thiophanate-methyl. In contrast,
wild-type isolates exhibited mean EC50 values for benomyl and
thiophanate-methyl of 0.47 and 0.91 μg mL-1, respectively.
All 305 isolates from the four locations sampled in this study were sensitive
in vitro to chlorothalonil at 10 μg mL-1. Conidial
germination of sensitive-isolates collected from banana plantations never
exposed to benzimidazole fungicides were completely inhibited by 1000
μg mL-1 of benomyl or thiophanate-methyl. However, conidial
germination of resistant-isolates was not affected by both fungicides
tested at 1000 μg mL-1.
In Morocco, the greenhouse culture of bananas (Musa spp.) began
in the 1980s and soon became one of the fastest-growing segments of agriculture,
especially after a government-imposed ban on foreign banana imports went
into effect in 1978. Currently, Morocco and Spain are the world`s largest
greenhouse banana-producing countries (Galan Sauco et al., 2004).
In 2006, banana cultivated area in Morocco spanned some 4400 ha, of which
more than 98% was represented by plastic greenhouse plantations, with
the Souss-Massa-Drâa (SMD) Valley as the main area of production
(60 to 70% of total production).
In Morocco, the culture of bananas in plastic greenhouses is being hindered
by nematodes and fungal pathogens, as the main causes of disease of this
crop (Janick and Ait-Oubahou, 1989; Guedira et al., 2004). Among
fungal diseases, cigar-end rot is one of the most serious infections in
greenhouses banana cultures. This disease is caused by two fungi, Verticillium
theobromae (Turc.) Mason and Hughes and Trachysphaera
fructigena Tabor and Bunting. T. fructigena, is known to cause
a destructive rot in the banana plantations of West and Central Africa
(Snowdon, 1990), whereas, V. theobromae is more widespread,
occurring in most banana-growing regions (Bhangale and Patil, 1983; Janick
and Ait-Oubahou, 1989). Unlike others soil-borne Verticillium species
that colonize the vascular tissues of plants, V. theobromae is
mainly a banana fruit-rotting fungus. This fungus is also a member of
a pathogenic complex which causes a rotting of the crowns during shipment
and transit of boxed bananas (Snowdon, 1990; Alvindia et al., 2006).
In the SMD Valley, cigar-end rot is controlled primarily by multiple
applications of benomyl and/or thiophanate-methyl, because non chemical
alternatives, such as the manual removal of the pistil and perianth, do
not represent commercially-viable means of control. Both benomyl and thiophanate-methyl
belong to the benzimidazole class of fungicides known as a multiplication
inhibitor during fungal mitosis that share a similar site specific mode
of action (Ma and Michailides, 2005). Most growers in the SMD manage the
disease by spraying systemic fungicides (e.g., benomyl, thiophanate-methyl)
or combinations of systemic and protectant fungicides (e.g., chlorothalonil,
mancozeb). The fungicide regime commonly used includes two to eight benzimidazole
sprays per season of either benomyl or thiophanate-methyl at rates of
0.25 g to 1.5 g a.i. L-1 and it is not uncommon for a grower
to use the same fungicide for the entire production cycle. In recent years
unsatisfactory control of cigar-end rot has been observed in some banana
greenhouses, with a previous history of benzimidazole fungicides use.
The growers commonly attribute this unsatisfactory control to application
method, or frequency and rate of application. However, this failure to
achieve control of cigar-end rot in banana greenhouses treated with benomyl
and/or thiophanate-methyl may be the result of acquired resistance to
benzimidazole among populations of V. theobromae. Several reports
(Hewitt, 1998; Staub, 1991) indicated that when populations of fungal
pathogens are repeatedly exposed to site-specific fungicides, resistant
strains can be readily selected. The result of this selection has been
widely documented in cases in which benzimidazole fungicides were used
extensively in controlling fungal pathogens during field as well as greenhouse
production (Johnson et al., 1994; Murray, 1996; Hanson et al.,
1996; van de Graaf et al., 2003). However, little is know about
the benzimidazole fungicides sensitivity of V. theobromae populations
in banana greenhouses in Morocco.
Therefore, the objectives of this study were to determine if benzimidazole
fungicides resistance exists in SMD banana greenhouses and to determine
the levels of sensitivity of V. theobromae isolates collected from
four banana-growing locations of the SMD Valley, with various histories
of benzimidazole use, to both benomyl and thiophanate-methyl.
MATERIALS AND METHODS
Sampling locations: Samples of banana fruits exhibiting cigar-end
rot symptoms were collected from four geographically-isolated locations
in SMD Valley, Agadir between 2002 and 2005. Benzimidazole fungicides
use varied among sampling locations, with three of the locations, namely,
Biougra, Belfâa and Ouled-teima totalling more than 10 years of
benzimidazole fungicides use and representing the main commercial banana
plastic greenhouse production areas. The fourth location, Tamri, is isolated
from the above-mentioned areas and consists of open field banana plantations,
with no history of benzimidazole use. This fourth location was used to
collect baseline (wild-type) isolates of V. theobromae. A
representative sample of at least 30 fruits was collected from each location.
Fruits from individual bunches were picked, placed in separate polyethylene
bags and transported to the laboratory.
Pathogen isolation: Infected fruits were surface disinfected with
a solution of 0.5% sodium hypochlorite (NaClO), rinsed with sterile water
and then allowed to air dry. Small pieces of fruit tissue were aseptically
excised from the advancing edge of the rot and placed on Water-Agar (WA,
2%) containing 50 μg mL-1 of rifampicin. Cultures were
incubated at 23°C for 2 to 5 days. When fungal growth from the tissue
became visible, the fungi were subcultured on PDA and reincubated as above.
Identification of V. theobromae was verified by examination under
a compound microscope. A total of 305 V. theobromae isolates were
collected from fruits sampled at the four studied locations, with each
isolate originating from a different fruit. All isolates were single-spored
and maintained on PDA at 5°C.
Fungicides: The fungicides used in this study were benomyl (50%,
Benlate 50WP, AMAROC, Maroc), thiophanate-methyl (70%, Pelt 44WP, BAYER,
Maroc) and chlorothalonil (75%, Daconil 75WP, CPCM, Maroc). The chlorothalonil
was included in this study for comparison. Fungicide solutions were first
prepared by dissolving each commercially-formulated fungicide in acetone.
An aqueous suspension of each fungicide was then prepared and added aseptically
to molten (50°C) sterile Potato Dextrose Agar (PDA). Control dishes
contained PDA and acetone and the final concentration of acetone in the
medium did not exceed 1% (v/v). All concentrations were expressed as active
Fungicide sensitivity tests: The isolates of V. theobromae
were in vitro-tested for sensitivity to benomyl, thiophanate-methyl
and chlorothalonil at a discriminatory concentration of 10 μg mL-1
of medium. Agar plugs (5 mm diameter) were cut from the periphery of actively
growing colonies and transferred, mycelium down, to three replicate Petri
dishes containing PDA medium supplemented with fungicide. After a ten
days incubation period at 23°C, the isolates were considered either
resistant if growth was observed on fungicide-amended medium or sensitive
if no growth has occurred.
Determination of fungicide sensitivity (concentration producing 50%
growth inhibition EC50 values): For each location, ten
V. theobromae isolates were used to determine the fungicide concentration
producing about a 50% growth inhibition (EC50). These isolates
were arbitrary chosen and represent different locations of bananas production
(Biougra, Belfâa, Ouled-teima and Tamri).
A 5 mm diameter disk was taken from the margin of a seven day-old culture
of each isolate of V. theobromae and placed, upside down,
at the center of a PDA dish. The PDA medium was amended with the following
concentration of benomyl or thiophante-methyl: 0, 0.1, 1, 10, 100 and
1,000 μg mL-1 for the benzimidazole-resistant isolates,
collected from Biougra, Belfâa and Ouled-Teima locations and 0,
0.01, 0.1, 0.5, 1 and 5 μg mL-1 for the benzimidazole-sensitive
isolates of V. theobromae collected from Tamri location. Three
Petri dishes were used for each fungicide concentration. Colony diameters
were measured after seven days at 25°C. Percent growth inhibition
at each fungicide concentration was calculated according to the following
Growth inhibition (%) = [(unamendedfungicideamended)/unamended]
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). The
experiment was performed twice.
Effect of fungicides on conidial germination: The inhibitory effect
of fungicides on spore germination was tested on 2% Water-Agar (WA) amended
with 0, 100 and 1,000 μg mL-1 of benomyl, thiophanate-methyl
or chlorothalonil. A spore-suspension from a mixture of either four resistant-isolates
(Biougra, Belfâa and O-Teima locations) or four sensitive-isolates
(Tamri location) was obtained from one-week-old cultures by flooding the
cultures with sterile distilled water containing 0.05% (v/v) Tween 80
and filtered through two layers of sterile cheesecloth to remove hyphal
fragments. The spore concentration of these suspensions was adjusted to
1x105 conidia mL-1 with the aid of a haemocytometer.
Aliquots (100 μL) of spore suspensions were aseptically-spread in
triplicate onto WA dishes. After a 20 h incubation at 23°C, germination
was determined by observing at least 100 conidia for each concentration
under a light microscope. A spore was scored as germinated if the germ
tube length was equal to at least one time that of the conidium. Each
treatment was replicated twice.
Statistical analysis: 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 tests: In this study, we collected 95, 88,
91 and 31 V. theobromae isolates from banana plantations located
in Biougra, belfâa, Ouled-teima and Tamri, respectively.
||Sensitivity of Verticillium theobromae isolates
to benomyl, thiophanate-methyl and chlorothalonil collected from four
locations in the Souss-Massa-Drâa Valley, Morocco
R: No. of isolates resistant to fungicide, T: No. of
isolates tested, *Sensitivity of isolates to fungicide was tested
on PDA medium amended with 10 μg mL-1. Isolates which
failed to grow on the amended medium were considered sensitive
All isolates were tested in vitro for resistance
to either benomyl or thiophanate-methyl at a discriminatory concentration
of 10 μg mL-1. Results of Table 1 show
that 64, 61 and 71% of V. theobromae isolates from Biougra, Belfâa
and Ouled-teima locations, respectively, were resistant to benomyl, whereas
isolates from the Tamri location were sensitive to benomyl. The percentage
of thiophanate-methyl resistant isolates from Biougra, Belfâa and
Ouled-teima locations ranged from 64 to 74%. Whereas all 31 field-collected
V. theobromae isolates from the Tamri location were sensitive to
thiophanate-methyl tested at a discriminatory concentration of 10 μg
mL-1 (Table 1). It has often been observed
in other plant-pathogenic fungi that isolates resistant to one benzimidazole
fungicide showed reduced sensitivity to the other benzimidazole fungicides
(Keinath and Zitter, 1998). In this study, the majority of V. theobromae
isolates exhibited cross-resistance to both fungicides. Of 305 single-conidium
isolates tested, 59% were resistant to both benomyl and thiophanate-methyl,
while 40% of these isolates were sensitive to both compounds. Only, four
V. theobromae isolates exhibited a differential sensitivity
to the two fungicides: resistance to thiophanate-methyl but sensitivity
to benomyl. We also found that all 305 V. theobromae isolates
were sensitive to the chlorothalonil when tested at a discriminatory concentration
of 10 μg mL-1 (Table 1).
Level of resistance: As shown in Table 2, the
mean EC50 values of 10 benzimidazole-sensitive isolates of
V. theobromae collected from the Tamri location on benomyl-or thiophanate-methyl
amended PDA were 0.47 and 0.91 μg mL-1, respectively.
These values were significantly lower than those observed for isolates
from the other locations. The Tamri population was therefore considered
as being indicative of a wild-type population (Table 2).
The mean EC50 values of V. theobromae isolates sampled
from commercial banana greenhouses at Biougra (97 μg mL-1),
Belfâa (90 μg mL-1) and Ouled-teima (80 μg
mL-1) to benomyl were not significantly different (p = 0.05).
Effect in vitro
of fungicides on conidial germination
of V. theobromae
isolates collected from Biougra, Belfâa,
Ouled-teima and Tamri locations. The fungicides tested were benomyl
(A), thiophanate-methyl (B) and chlorothalonil (C) at the following
concentrations: 0, 100 and 1,000 μg mL-1
bars represent standard deviations of the mean
||Sensitivity (EC50) to benzimidazole fungicides
of Verticillium theobromae isolates collected in banana plantations
at four locations in Souss-Massa-Drâa Valley, Morocco
xEC50: Effective concentration
of fungicide in the culture medium needed to inhibit 50% of mycelial
growth, yResistance Factor (RF) = mean EC50
resistant-isolates/mean EC50 sensitive-isolates, zValues
within columns followed by the same letter are not significantly different
(p = 0.05). Mean values were determined for 10 isolates per sampling
For thiophanate-methyl, the mean EC50 values for isolates collected from the three locations ranged between 194 and
233 μg mL-1 and no significant difference was apparent
between these locations (Table 2). Compared to benomyl
sensitivity, isolates of V. theobromae collected in the same banana
grown locations were less sensitive to thiophanate-methyl than to benomyl.
Resistance factors for both fungicides were higher and ranged between
170 and 206 for benomyl and from 210 and 256 for thiophanate-methyl, indicating
the presence of V. theobromae isolates that were highly insensitive
to both fungicides.
Effect of fungicides on conidial germination: A spore-suspension
mixture of four resistant-isolates to benomyl or thiophanate-methyl collected
from Biougra, Belfâa and Ouled-Teima and four sensitive-isolates
collected in Tamri, were used to analyze the effects of fungicides on
conidial germination of V. theobromae. The results obtained (Fig.
1B, C) showed that benomyl and thiophanate-methyl
did not prevent spore germination of isolates resistant to both fungicides,
even at the highest concentration tested (1000 μg mL-1).
In contrast, conidial germination of V. theobromae isolates sensitive
to benomyl and thiophanate-methyl was totally inhibited at 1000 μg
mL-1. For both fungicides, conidium germination rates for sensitive
isolates were only slightly decreased in the presence of 100 μg mL-1
as compared to resistant isolates and were above 50%. The chlorothalonil
at 100 μg mL-1 completely inhibited spore germination
of both benzimidazole-resistant and benzimidazole-sensitive isolates of
V. theobromae collected from the four locations studied (Fig.
The benzimidazole fungicides have been used quite extensively during
the last 20 years to control cigar-end rot in the SMD Valley, Agadir,
Morocco. Benzimidazole are systemic fungicides that acts as a multiplication
inhibitor during fungal mitosis (Ma and Michailides, 2005) and resistance
to these compounds has been detected in many fungal species. The present
study showed that all V. theobromae wild-type isolates collected
from an open-field banana plantation in the Tamri region were sensitive,
in vitro, to both benomyl and thiophanate-methyl tested at the
discriminatory concentration of 10 μg mL-1. These results
suggest that naturally occurring benzimidazole-resistant strains of V.
theobromae are absent or present at very low frequency from locations
without any prior history of benzimidazole exposure. However, among 274
V. theobromae isolates collected from banana locations with a history
of prolonged benzimidazole use, 65% (180/274) and 67% (184/274) were resistant
to benomyl and thiophanate-methyl, respectively. Considering the extensive
and heavy use of both fungicides in commercial banana greenhouses, it
is not surprising that resistance to benomyl and thiophanate-methyl was
so prevalent in V. theobromae populations sampled from SMD, Valley.
The high incidence of resistance to both fungicides was similar to that
reported for others fungal pathogens (Malathrakis and Vakalounakis, 1983;
Romero and Sutton, 1998; Keinath and Zitter, 1998).
It has been observed in other plant-pathogenic fungi that isolates resistant
to one benzimidazole also exhibit reduced sensitivity to the other benzimidazole
fungicide (Bus et al., 1991; Kawchuk et al., 1994). In this
study, we found that all isolates resistant to benomyl were also resistant
to thiophanate-methyl. Present results indicate that, in all likelihood,
the unsatisfactory control of cigar-end rot in the SMD, Valley banana
greenhouses stems from the emergence and the spread of V. theobromae
strains highly resistant to benzimidazole fungicides. This is in agreement
with previous reports indicating that decreased sensitivity of fungal
pathogens to benzimidazole is directly correlated with reduced performance
of the fungicides in the field and in the greenhouse (Moorman and Lease,
1992; De Lapeyre et al., 1997; Romero and Sutton, 1998; Errampalli
et al., 2001).
The majority of V. theobromae resistant-isolates in our collection exhibited
high levels of resistance to both thiophanate-methyl and benomyl. The average
EC50 of isolates resistant to benomyl was between 80 and 97 μg
mL-1 and between 194 and 233 μg mL-1 for thiophanate-methyl.
The median EC50 values for benzimidazole fungicides tested did not
differ significantly (at p = 0.05) among locations with previous history of
benzimidazole use. In contrast, the range of EC50 values for
V. theobromae sensitive-isolates was between 0.09 and 1.4 μg mL-1
for benomyl and between 0.1 and 2.2 μg mL-1 for thiophanate-methyl.
The sensitivity of V. theobromae isolates, collected from the Tamri location,
to benomyl was of the same order as that reported by Igeleke and Ayanru (2007).
In another study, among eight isolates of V. theobromae isolated from
rotted banana crowns, three were less sensitive to the benzimidazole fungicide
thiabendazole, with EC50 values higher than 10 μg mL-1
(Johanson and Blazquez, 1992). The EC50 values for benomyl and thiophanate-methyl
were consistent with other reports. For example, benomyl and thiophanate-methyl
EC50 ranged from 27 to 251 μg mL-1 and from 27 to
more than 1,000 μg mL-1, respectively, in isolates of Botrytis
cinerea (LaMondia and Douglas, 1997). The different levels of resistance
to benomyl among V. theobromae populations may be due to the presence
of a different allele for benomyl resistance as observed with Venturia inaequalis
populations (Koenraadt et al., 1992).
The resistance factors, calculated by dividing mean EC50 values of resistant-isolates collected in Biougra, Belfâa and Ouled-Teima
by the mean EC50 values of sensitive-isolates collected in
Tamri location, were between 170 and 206 for benomyl and between 210 and
256 for thiophanate-methyl. The difference in resistance factors reflects
greater variation in sensitivity to benomyl and thiophanate-methyl within
populations of V. theobromae and indicates differences in sensitivity
to benzimidazole fungicides between the wild-type population and the exposed
populations of V. theobromae.
With regard to the effect of fungicides on conidial germination, the
data presented here show that the benomyl and the thiophanate-methyl tested
at a concentration of 1000 μg mL-1 were without effect
on conidial germination of V. theobromae resistant-isolates collected
from locations with a history of benzimidazole use. However, these two
fungicides totally inhibited conidial germination of sensitive-isolates
at a concentration of 1000 μg mL-1.
In the present study, at 10 and 100 μg mL-1, the multi-site
fungicide chlorothalonil completely inhibited mycelial growth and germination
of conidia, respectively, of both benzimidazole-sensitive and benzimidazole-resistant
isolates of V. theobromae, collected from the four locations sampled.
Therefore, an appropriate strategy for limiting the spread of V. theobromae
resistance to benomyl and thiophanate-methyl in the SMD Valley banana
greenhouses would involve the use of mixtures of benzimidazole fungicides
and contact fungicides such as chlorothalonil. Fungicide insensitivity
is generally not a concern with protectant fungicides such as chlorothalonil
because of their multi-site mode of action (Vincent and Sisler, 1968).
In conclusion, the data presented in this study reveal, to our knowledge
for the first time, that a majority of V. theobromae isolates collected
in commercial banana-greenhouses in SMD Valley between 2003 and 2005 were
resistant to benomyl and thiophanate-methyl. This high proportion of isolates
with reduced sensitivity to benzimidazole fungicides may account for the
unsatisfactory control of cigar-end disease in the SMD. On the basis of
these data, the use of benzimidazole fungicides for the control of cigar-end
rot should be seriously questioned. Moreover, these results emphasize
the need for new control strategies involving lower pesticide use, which
would be in line with consumer requirements.
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