Mycol. Res. 109 (6): 741–745 (June 2005).

f The British Mycological Society 741

doi:10. 1017/S095375620500242X Printed in the United Kingdom.

Reduced sensitivity of the mushroom pathogen Verticillium

fungicola to prochloraz-manganese in vitro

Francisco J. GEA1, Marı́a J. NAVARRO1 and Julio C. TELLO2

1
Centro de Investigación, Experimentación y Servicios del champiñón, Apdo. 8, ES-16220 Quintanar del Rey (Cuenca), Spain.
2
Dpto. de Producción Vegetal, Escuela Polite´cnica Superior II, Universidad de Almerı´a, ES-04120 Almerı´a, Spain.
E-mail : fjgea.cies@dipucuenca.es

Received 13 April 2004; accepted 4 January 2005.

105 isolates of Verticillium fungicola from Spanish mushroom crops collected between 1992 and 1999 were tested in vitro
for their sensitivities to prochloraz-manganese. Dose response relationships for inhibition of mycelial growth by the
fungicide were assessed in radial growth experiments on fungicide-amended malt extract agar. The ED50 values recorded
for all 105 isolates studied ranged between 0.8 ppm in 1992 and 8.8 in 1998, with an average of 2.9. 86% of the isolates
tested were more sensitive to prochloraz-manganese and had ED50 values below 5 ppm, while the other 14 % were slightly
tolerant with ED50 values equal or above 5 ppm. Of those tested from 1999, 60 % (21 isolates) grew with 50 ppm and
40 % (14) also at 100 ppm, although mycelial growth was inhibited at least by 82 and 87 %, respectively. The resistance
factor calculated ranged from low fungicide resistance (RF=3.0) in 1992 to moderate resistance (RF=12.6) in 1998.
These data confirm that the sensitivity of V. fungicola to the prochloraz-manganese gradually diminishes.

INTRODUCTION fungicide was also limited due to the development of

Verticillium fungicola var. fungicola, the causal agent of
dry bubble disease in Agaricus bisporus, causes serious
losses of production in Spanish mushroom farms
(Gea & Tello 1997), as in other mushroom growing
countries (Desrumeaux et al. 1998, Grogan, Keeling &
Jukes 2000). Recently, this fungal pathogen has also
been identified as the causal agent of dry bubble in
A. bitorquis crops (Gea, Tello & Navarro 2003).
Dry bubble has been controlled through the use of
chemicals, cultural practices and sanitation. Among the
chemicals, dithiocarbamates were first used to control
diseases caused by Cladobotryum dendroides, Mycogone
perniciosa and Verticillium fungicola (Yoder, Sinden &
Hauser 1950). Methylbenzimidazole carbamate (MBC)
fungicides were also used in the mushroom industry
and provided excellent control of V. fungicola (Wuest &
Cole 1970, Holmes, Cole & Wuest 1971, Snel &
Fletcher 1971), until resistance to these fungicides was
reported in this pathogen soon after they were released
(Wuest, Cole & Sanders 1974, Bollen & van Zaayen
1975, Fletcher & Yarham 1976). Chlorothalonil acted
an alternative to the MBC fungicides (Gandy &
Spencer 1976), although the effectiveness of this
resistance (Gea, Tello & Honrubia 1996, Bonnen &
Hopkins 1997).
Prochloraz, a member of the sterol demethylation
inhibitor (DMI) fungicides, was introduced in the
mushroom industry in the early 1980s and achieved
good control of V. fungicola (van Zaayen & van
Adrichem 1982, Fletcher, Hims & Hall 1983). Gea,
Tello & Honrubia (1996) showed that prochloraz-
manganese was more effective for the control of
V. fungicola than other fungicides such as benomyl,
chlorothalonil, formaldehyde, iprodione and the mix
prochloraz+carbendazim. However, the widespread
use of prochloraz has raised concerns regarding a
decrease in sensitivity to this fungicide. The resistance
to DMIs has been reported in the case of other patho-
gens (Huggenberger, Collins & Skylakakis 1984, Stanis
& Jones 1985, Elad 1992). However, the resistance of
V. fungicola isolates to prochloraz-manganese has not
been demonstrated conclusively, although there is in
vitro evidence which points to a decrease in sensitivity
to this fungicide (Geels 1996, Desrumeaux et al.
1998, Grogan et al. 2000). However, Grogan et al.
(2000) demonstrated that prochloraz-manganese still
managed a reasonable degree of control of dry bubble
Prochloraz sensitivity in Verticillium fungicola
caused by two isolates showing different degrees of
sensitivity to this fungicide.
The disease is still controlled in Spanish mushroom
crops by prochloraz-manganese, although some farms
have reported unsatisfactory levels of control (depend-
ing on seasons and crops) in recent years. However, no
relationship has been established between unsatisfac-
tory control of the disease and loss of sensitivity to
prochloraz-manganese in field isolates of V. fungicola.
Monitoring pathogen populations for fungicide sensi-
tivity over time is a good way of determining whether
resistance is developing, and so this study was initiated
to compare the sensitivity of field isolates collected
over several years. In this way, the possibility of a
gradual increase in the frequency of DMI-resistant
isolates in fungal populations would point to any
important limitation for the efficient use of prochloraz-
manganese. It was thought that this information may
help in attempts to track the disease as well in
developing better control measures.
MATERIALS AND METHODS
Isolates
105 isolates of Verticillium fungicola were recovered
from diseased fruit bodies of Agaricus bisporus and
A. bitorquis showing dry bubble symptoms. Sam-
ples were collected between 1992 and 1999 from 31
prochloraz-treated farms situated in two intensive
mushroom-growing areas of Spain (Castilla-La Mancha
and La Rioja). A strain from the Centraalbureau
voor Schimmelcultures (CBS 992.69), isolated in 1969
prior to the introduction of prochloraz-manganese,
was used as reference organism.
The isolates were identified according to the
taxonomic criteria described by Gams & van Zaayen
(1982). The isolates were submitted to temperatures of
20, 27 and 30 xC for 10 d, using four repetitions per
isolate.
Representative isolates are deposited in the Spanish
Type Culture Collection (CECT).
Sensitivity tests to prochloraz-manganese
A commercial formulation of prochloraz-manganese
46 % WP (Sporgon1 , AgrEvo, Valencia) was used as
active ingredient (a.i.). This was dissolved in ethanol
and added as solutions to molten sterile malt extract
agar (45 x) to give prochloraz-manganese concen-
trations of 0, 0.1, 0.5, 1, 5, 10, 20, 50 and 100 ppm, and
an ethanol concn. of 1% (v/v). All fungicide-amended
media were prepared within 24 h of use. A 5 mm diam
malt agar plug was taken from the growing edge of an
active mycelium, inverted and placed in the centre of
each malt agar plate amended with fungicide. There
were four replicates per combination of isolate and
fungicide concentration. The size of the colonies was
742
determined by measuring two mutually perpendicular
diameters after 12 d of incubation at 20 x in the dark.
Data analysis
The radial growth rate for each isolate was determined
on unamended control plates. The colony diameter,
minus the diameter of the agar plug, was calculated as
an average of four plates for each isolate and divided by
the number of days, to give average mycelial growth
rate.
The sensitivity distribution of the population of
V. fungicola was estimated by ED50 values (ppm of a.i.
inhibiting radial mycelial growth by 50 %). In plant
pathogens, this method is the most suitable for DMI
fungicides because mycelial growth is related to the
formation and proliferation of subcuticular stromata,
the developmental stage most effectively inhibited by
these fungicides under practical conditions. In contrast,
spore germination, appressoria formation and cuticle
penetration are not affected by DMI fungicides
(Scheinpflug & Kuch 1987).
ED50 values were calculated for each isolate by in-
terpolation from computer-generated log-probit plots
of fungicide concentration and relative inhibition :
100 – (colony diameter on amended medium/colony
diameter on control)r100. For each period of time, a
resistance factor was determined. According to Delp &
Dekker (1985), the resistance factor was expressed as
the ratio between the ED50 of the isolates tested and the
ED50 of the reference isolate CBS 992.69, which was
prochloraz-manganese sensitive. This factor can be
used as an indicator of the risk of developing resistance.
In all the cases, normality and homoscedasticity were
previously checked by the Kolmogorov–Smirnov and
Cochran tests, respectively. When the data obtained
were not normally distributed and a closer approxi-
mation to normality could not be accomplished by
transformation, a Kruskal–Wallis test was performed
to ascertain whether there were significant differences
between the years considered. The method used to dis-
criminate among the medians for each combination of
years was Wilcoxon–Mann–Whitney’s two-sample test.
Data (growth rates, ED50 values, resistance factors)
were analyzed using Statgraphics1 Plus v. 4.1
(Statistical Graphics Corp., Princeton, NJ).
RESULTS AND DISCUSSION
All the isolates were identified as Verticillium fungicola
var. fungicola, since all grew at 20 x but none at 27
or 30 x.
The mean ED50 values of prochloraz-manganese
and resistance factors are depicted in Table 1 and the
frequency distributions of ED50 values are shown in
Fig. 1. The ED50 values were not normally distributed
(Kolmogorov–Smirnov’s test: 0.1506 ; P<0.01) and
did not show homoscedasticity (Cochran’s test: 0.5547 ;
F. J. Gea, M. J. Navarro and J. C. Tello 743

Table 1. ED50 values of prochloraz-manganese and resistance factors for isolates of Verticillium fungicola var. fungicola collected over the
years 1992–99 in Spain.

ED50 (ppm) Number of Number of

isolates with isolates with Resistance
Year Isolates Meana Range ED50<5 ppm ED50o5 ppm factorb

1992 17 1.5 a 0.8–2.1 17 0 3

1995 11 3.3 b 1.7–4.2 11 0 6
1996 14 3.4 b 1.7–5.0 13 1 7.1
1998 28 3.2 b 1.3–8.8 22 6 12.6
1999 35 3.0 b 1.4–5.7 30 5 8.1
Total 105 2.9 0.8–8.8 93 12
a
Different letters indicate significantly different medians from those in other years at the 95.0% confidence level (Wilcoxon–Mann–Whitney
two-sample test).
b
The resistance factor was expressed as the ratio of the highest ED50 of the isolates tested to the ED50 of the reference isolate CBS 992.69
(0.7 ppm).

1992 1995 1996 1998 1999 tolerant to the fungicide and had ED50 values of
25
5–8 ppm while 30% of isolates were more sensitive,
20
20 with ED50 values of 1–4 (Grogan et al. 2000). From
Number of isolates

18
16 1996 the first isolates of V. fungicola began to be de-
15 tected with ED50 values o5 ppm. Between 1998 and
10 1999, 21 and 14 %, respectively, of the isolates analysed
10 99

4 4
5
2 2
1 1
0
0-1 1-3 3-5
ED50 (ppm)
Fig. 1. Frequency distribution of ED50 values to prochloraz-
manganese for isolates of Verticillium fungicola var. fungicola
collected over the years 1992–99 in Spain.
P<0.05). The highest mean ED50 value (3.4 ppm) was
recorded in 1996 and the lowest (1.5 ppm) in 1992. The
ED50 values recorded for all 105 isolates studied ranged
between 0.8 and 8.8 ppm, with an average of 2.9. 86 %
of the isolates tested were more sensitive to prochloraz-
manganese and had ED50 values below 5 ppm, while
the other 14% were slightly tolerant, with ED50 values
equal or above 5 ppm. Statistically significant differ-
ences (Kruskal–Wallis’ test, H=30.8316 ; P<0.05)
were found between the ED50 values from 1992 and
those from the other years.
The data obtained with the 17 isolates from 1992
(ED50 : 0.8–2.1 ppm) were similar to those described by
Nair & Macauley (1987) for 9 isolates of V. fungicola
from Australian cultures (ED50 : 0.1–2.0 ppm). In
no case were the Australian isolates tolerant to
prochloraz-manganese detected (ED50 o5 ppm). Sub-
sequent studies in the Netherlands showed that a
certain level of resistance to prochloraz-manganese
among isolates of Verticillium existed, and these were
inhibited at a 40–80 ppm (Geels 1996). In Belgium,
Desrumeaux et al. (1998) also identified Verticillium
isolates which were less sensitive to prochloraz-
manganese in vitro. Lastly, a survey of Verticillium
isolates from British mushroom farms carried out in
1997 indicated that 64 % of isolates tested were weakly
5
showed ED50 values o5 ppm (Table 1, Fig. 1). These
4
results clearly show that the sensitivity of V. fungicola
to prochloraz-manganese has gradually decreased.
5-7 >7 The ED50 of the reference isolate CBS 992.69 for
prochloraz-manganese was 0.7 ppm, based on which
the resistance factor calculated ranged from 3 in 1992
to 12.6 in 1998 (Table 1). The higher resistance factor
for isolates from 1998 (12.6) does not necessarily imply
that resistance will become a problem. The wide range
of ED50 values (1.3–8.8 ppm) and a low mean ED50
value (3.2 ppm) for that year indicate a wider range in
sensitivity among isolates within that population
(Fig. 1). As the range of sensitivity increases, the risk
of individuals becoming less sensitive to the fungicide
than the rest of the population also increases. The
gradual selection pressure exerted on the pathogen by
the fungicide, and the fact that not many applications
are required each year to control dry bubble, contrib-
ute to increasing the resistance of V. fungicola to
prochloraz-manganese. Although the risk of resistance
developing in V. fungicola seems low to moderate, the
use of practices to reduce the incidence of the disease
(farm hygiene) should be utilized to minimize that risk.
Table 2 shows the percentages by which mycelial
growth of V. fungicola was inhibited by different con-
centrations of prochloraz-manganese and the number
of isolates showing mycelial growth at these con-
centrations. All 105 isolates tested grew at 1 ppm, while
only 85 % (89 isolates) grew at 10 ppm. Of the 17 iso-
lates from 1992, only one grew at 10 ppm and none
showed mycelial growth at 50 and 100 ppm. All the
isolates from 1995 grew at 10 ppm and none at 50 and
100 ppm. However, from 1996 onwards some isolates
of V. fungicola were capable of growing in the presence
of both 50 and 100 ppm of fungicide. From 1998, 13
isolates (46 %) grew with 50 ppm and 6 (21 %) with
Prochloraz sensitivity in Verticillium fungicola 744

Table 2. Inhibition of radial mycelial growth of Verticillium fungicola var. fungicola isolates by several concentrations of
prochloraz-manganese.

1 ppm 10 ppm 50 ppm 100 ppm

Year Isolates na % inhibition na % inhibition na % inhibition na % inhibition

1992 17 17 25–67 1 90–100 0 100 0 100

1995 11 11 14–29 11 60–81 0 100 0 100
1996 14 14 12–39 14 60–85 3 84–100 2 90–100
1998 28 28 10–45 28 59–100 13 84–100 6 91–100
1999 35 35 13–37 35 51–85 21 82–100 14 87–100
Total 105 105 10–67 89 51–100 37 82–100 22 87–100
a
Number of isolates with mycelial growth.

100 ppm. Of those tested from 1999, 60 % (21) grew
with 50 ppm and 40% (14) also at 100 ppm, although
mycelial growth was inhibited at least by 82 and 87%,
respectively. In summary, 37 of the 105 isolates tested
showed mycelial growth with 50 ppm fungicide and 22
also with 100 ppm. It can also be seen how the per-
centage of inhibition of mycelial growth for the same
concentration of fungicide fell during the years
studied, so that in the presence of 10 ppm prochloraz-
manganese the isolates from 1992 showed 90–100 %
inhibition, while those from 1999 only showed
51–85 % inhibition.
These data confirm that the sensitivity of V. fungicola
to prochloraz-manganese gradually diminishes.
However, the changing patterns of prochloraz toler-
ance in vitro did not appear to be associated with loss of
control in the mushroom crops in Spain, but, rather,
with a relaxation of the hygiene practices normally used
in controlling the disease. Previous studies (Geels 1996,
Desrumeaux et al. 1998, Grogan et al. 2000) pointed to
a similar tendency in other countries. However, Grogan
et al. (2000) demonstrated that prochloraz-Mn still
significantly controlled dry bubble disease up to the end
of the second flush. Cropping experiments carried out
with a sensitive and a tolerant isolate showed that the
more sensitive isolate was in fact a more aggressive
pathogen in the absence of any fungicide. However, in
the presence of prochloraz-manganese, both isolates
were significantly inhibited.
Mushroom growers can no longer exclusively rely on
prochloraz-manganese, as a primary management tool.
Hygiene is probably the major control mechanism,
aided sustantially by efficient fungicide usage. In short,
management strategies for dry bubble should focus on
integrated disease control.
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Corresponding Editor: J. I. Lelley