Plam Pathology (1992) 41, 41-46

Multiple fungicide resistance to benzimidazoles, dicarboximides

and diethofencarb in field isolates of Botrytis cinerea in Israel

Y, ELAD, H, YUNIS and T, KATAN

Department of Plant Pathologv. Agricultural Research Organization, The Volcani Center. Bet Dagan
50250, Israel

During the period Jatiuary-March 1989, 15 greenhouses at 12 sites in Israel were surveyed for the
presence of fungicide-resistant strains of Botrytis cinerea, using a fungicide-amended Botrytis-se\ecn\e
tnedium. Resistance to benzimidazoles (Ben'*) and to dicarboximides (Die") was frequent in most sites.
Resistance to carbendazim + diethofencarb (Ben'* NPC*) was found in all eight sites in which a mixture
of these fungicides had been used against grey mould, but not in other sites, A new phenotype of multiple
fungicide resistance was found among these isolates. The new phenotype, designated Ben'* Die'* NPC*,
combines the three previously described characteristics of resistance to benzimidazole, dicarboximide
and A'-phenylcarbamate fungicides. It was found only in cucumber greenhouses that had been sprayed
with the fungicide mixture carbendazim + diethofencarb against grey mould. Isolates of this phenotype
were pathogenic in artificial inoculation of cucumber cotyledons treated with carbendazim, iprodione or
carbendazim + diethofencarb.

INTRODUCTION NPC* isolates obtained in 1988 was resistant to

rea, is a dicarboximides, although Die'* strains have been
Grey mould, caused by Botrvtis cinerea, is a
, , , , c u e II II frequent in the B. cinerea population of the

B. cinerea strains resistant to the benamidazoles

tively c
(Ben'*) and the dicarboximides (Die"*) greatly gy ,. ,
, , , „, „ , „ . .^ f. The purposes of the study conducted m the
d
reduced h usefulness
the fl off these
h f d
fungicides ffor ^ ^ determine- (i) if fhe Ren'*
rol of grey mould. The fungicidal ~ " °l ' ^ ^ ^ "^^'^ ^° determine, (i) ,f the Ben
mixture of carbendazim (MBC) plus dietho- ^^^ '
subsequ
fencarb was introduced by Sumitomo Chemical ^
Company in an attempt to exploit the 'negative ^'^^'"^ «^'^''" " ' ' ' f ,^"^^ ^''P'^^^'l'° '^e fungiCKie
cross-resistance' phenomenon between ^V phe- '"'^'"^^' ^"'^ <"'' 'f«™lar strains are present that
nylcarbamates (such as diethofencarb) and benzi- ^'^ ^ ' ' ° '•^"''^"* ^° d.carboxim.des,

To'tnt/b'h I t ' irie^'s't^nt' 71 MATERIALS AND METHODS

cinerea and some other plant pathogens. Promis-
ing results were obtained by using this fungicide f'ungicides

baldi, 1987; Elad et al., 1988). However in the (MBC; Delsene 50 DF, E.I. DuPont de Nemours

was reported from greenhouses at one site in
Israel where cucumbers had been treated with the
mixture. From the affected crop, strains of B.
cinerea were isolated that were able to grow on
media amended with MBC, diethofencarb or a
mixtureof the two. These strains were designated
Ben'* NPC*, to indicate their resistance to both
benzimidazole and A^-phenylcarbamate (NPC)
fungicides (Katan et al., 1989), None ofthe Ben'*
ture of carbendazim + diethofencarb (Resec =
MBC + diethofencarb 25 + 25 WP, Sumitomo
Chemical Co,, Japan); and iprodione (Rovral 50
WP, Rhone-Poulene, Lyon, France), Fungicide-
amended media were prepared by adding suspen-
sions of fungicides in sterile deionized waler to
autoclaved Difco potato dextrose agar (PDA) at
45°C, Fungicide concentrations are expressed in
fig/m\ active ingredient.
 Table 1, Fungicide re Israel (winter 1989)

:ulum on medium amended w

la Faradis"
la Faradis Cucumber

Ih Faradis Cucumber -1- 2

2a Muqeble Cucumber -1- 19

30

2b Muqeble Cucumber + 19
3a Sandaia Cucumber _)_ 191
Cucumber 30
Cucumber 20
^b Sandaia Cucumber + 19
201
4 Jat Cucumber

6 Tira Cucumber _l_ 7

7 Herut Cucumber -1- 191
Cucumber -1- 19
9 Gadish Cucumber 191
0 Kfar-Hess
1 Givat-28 19
2 P. Hanna Strawberry

\verage number of colonies per plate of Botrytis-selc

Factory-prepared mixture (1:1) of MBC-fdiethofenc
5ite 1: where resistance to carbendazim-I-diethofencar etal., 1989); la and 11
eenhouses not sprayed and sprayed, respectively, witl

Survey of fungicide resistance of B, cinerea in
greenhouses
During the period January-March 1989, plates
of Botrytis-se\Ktiwe medium (BSM; Kritzman &
Netzer, 1978) with and without fungicides
(Table 1) were exposed for 1-3 h to the airborne
tomato (Lycopersicon eseulentum) and straw-
berry (Fragaria ananassa) plastic greenhouses.
Ten out ofthe 12 cucumber greenhouses surveyed
had been treated with MBC + diethofencarb,
whereas the other crops had not. After exposure,
the plates were incubated at 22X. Growth on
BSM is evident after 4-5 days as brown colo-
ration around the developing colonies (Katan,
1985). Individual colonies suspected of being
resistant to MBC-Fdiethofencarb were subcul-
tured on PDA amended with this fungicide
mixture (10 ^g/ml) and then maintained on PDA
at 19°C. Fungicide sensitivity of such isolates was
compared with that of strains of the pathogen
isolated in 1981 (VIT-2, VIT-3, GY-1405, TUT/
TIR23) (Katan, 1982) and in 1988 (EP-MIV190,
176, FAR-7, FAR-11, FAR-15) (Elad et al., 1988;
Katan et al., 1989). To determine the linear
growth rates in the presence of fungicides, myce-
lial plugs (4 mm in diameter) from the growing
margins of 3-day-old colonies on PDA were
placed at the centres of PDA plates with MBC
(5^g/ml), iprodione (2 /Jg/ml), MBC-I-dietho-
fencarb (5-1-5 fig/ml) and MBC-I-diethofencarb
-I-iprodione (5-1-5-1-2 //g/ml) and incubated at
19X. Colony diameter was measured after 3
days.
Test of pathogenicity and fungicide resistance in
vivo
Detached cotyledons of cucumber were sprayed
 Multiple fungicide-resistant Botrytis ci
with water (control), MBC (0 5 mg/ml), ipro-
dione (0 5 mg/ml) or MBC-I-diethofencarb (1
mg/ml). After 2 h the cotyledons were placed on
moist filter paper in petri dishes (15 cm diameter),
and each cotyledon was inoculated with a myce-
lial plug as in the in vitro test. The dishes were
incubated in the dark at 20"'C for 3 days and
lesion development was inspected daily.
Survey of B, cinerea populations
During January March 1989, 15 greenhouses at
12 sites were surveyed for the presence of fungi-
BSM amended with each fungicide or fungicide
mixture (Table 1) were exposed to the airborne
inoculum in each greenhouse 1-3 times during the
season. Similarly, exposed plates of unamended
BSM were used to estimate the numbers of B.
cinerea propagules that had landed on the plates
during the exposure period.
In site 1, where resistance to MBC-I-diethofen-
carb had been found for the first time in 1988
(Katan et al., 1989), the inoculum level was very
low on 6 January, when few infections were
evident on the plants. Airborne inoculum and
infection increased during February; strains
resistant to MBC-h diethofencarb, and to
MBC-H diethofencarb-f-iprodione, were found in
the greenhouses in this site regardless of whether
or not they had been treated with MBC-t-dietho-
fencarb in 1989. Strains resistant to MBC-Fdi-
ethofencarb were found on 19 January in three
out of four cucumber greenhouses in two addi-
tional sites treated with this fungicide mixture,
and a high proportion of strains with multiple
resistance to these fungicides plus iprodione was
found in these sites one month later (Table 1).
Strains with similar multiple resistance were
found during March in five additional sites
sprayed with MBC-t-diethofencarb against grey
mould, but not in cucumber, tomato and straw-
berry greenhouses in which this fungicide mixture
had not been used.
Characterization of resistant strains in vitro
Isolates suspected of being Ben" NPC* or Ben"
NPC" Dic« were transferred to individual PDA
plates and incubated at 19°C. Their colony shape
and sporulation were normal.
In previous studies with B. cinerea in Israel
(Elad et ai, 1988; Katan et al., 1989), wild-type
isolates could not grow at 0 5 /Jg/ml MBC but
43
were resistant to diethofencarb. On the other
hand, Ben" strains isolated before 1988 grew well
in the presence of MBC but could not grow at
0 3 Aig/ml diethofencarb. Consequently, neither
BenS strains (e.g. VIT-3, EP-MIV190) nor Ben**
strains isolated before 1988 (e.g. TUT/TIR23)
were able to grow in the presence of MBC + di-
ethofencarb (Table 2). Isolates resistant to
MBC-I- diethofencarb and sensitive to dicarbox-
imides, which were isolated in 1988 (FAR-11) or
1989 (FAR-24), grew well in the presence of MBC
and/or diethofencarb but were unable to grow in
the presence of iprodione, whereas Die" strains
(e.g. TUT/TIR23) grew at reduced rates on 2 ugl
ml iprodione. Isolates ofthe new phenotype Ben"
Die" NPC", with multiple resistance to MBC,
diethofencarb and iprodione (MF5-10, MF6-44,
SFA-21) grew well in the presence of MBC and/or
diethofencarb, and at reduced rates on iprodione
alone or combined with MBC-I-diethofencarb.
Mycelial growth of such strains on iprodione was
often irregular, with dense and sparse sectors, and
the growth rate was somewhat higher in the
presence of MBC-I-diethofencarb than on ipro-
dione alone (Table 2).
Fungicide resistance in vivo
The ability of B. cinerea isolates to cause disease
in host plants, with or without fungicidal treat-
cotyledons of cucumber seedlings. Mycelial plugs
of sensitive and resistant strains of the different
phenotypes were used (Table 3). Although some
variation was observed in the pathogenicity of
various isolates, strains resistant to MBC-f dieth-
ofencarb were not less aggressive than sensitive
strains on untreated cotyledons. High infection
fencarb were also obtained on cotyledons treated
with MBC with or without diethofencarb, while
Ben^ NPC"^ and Ben" NPC^ strains did not infect
cotyledons treated with MBC-h diethofencarb.
On cotyledons treated with iprodione, infection
by Die" strains was similar, or somewhat
reduced, relative to the infection of the same
strains on untreated cotyledons. No infections or
few traces were caused by Die** strains on ipro-
dione-treated cotyledons. Isolates FAR-24 and
MF6-43 caused some infections on iprodione-
treated cotyledons in spite of being considered
Dic^; therefore it was assumed that these isolates
were mixtures of Dic^ and Die" cultures (possibly
heterokaryons), or have a very low level of
resistance to dicarboximides.

(MBC), iprodione and diethofencarb on growth of Botrytis
Phenotyi
No
Isolate Ben' Die'' NPC"^
VlT-3 S S R 34
R 34
EP-MiV190 S s
TUT/TIR23 R R S 36
FAR-11 R R 38
S
FAR-24 R R 33
s
DISCUSSION
This work describes a new phenotype of multiple
fungicide resistance found among field isolates of
B. cinerea. This phenotype combines previously
described characteristics of resistance to three
groups of fungicides: the benzimidazoles (Ben"*),
the dicarboximides (Die") and the W-phenylcar-
bamates (NPC*) (Katan, 1982; Katan et al.,
1989) and is designated accordingly Beni* Die"
NPC". Resistances to benzimidazoles and dicar-
boximides have been found since the early 1980s
and are now established in many populations of
B. cinerea in Israel. Resistance to MBC + dietho-
fencarb was first recorded in 1988 in a population
of B. cinerea on protected cucumbers treated
experimentally with the fungicide mixture
MBC-I-diethofencarb. Table 1 shows that this
phenotype has survived in site 1 during the hot
summer and could incite infections in the winter
of 1989. The quick appearance of such strains and
their increase in additional sites is associated with
the application of MBC -I- diethofencarb in the
greenhouses. This finding is consistent with the
hypothesis that the Ben"* NPC* phenotype had
existed at a very low frequency within the Ben"
V. Elad etal
Radi al growth*" on PDA amended with
MBC+ MBC-I-diethofencarb-(-
MBC diethofencarb iprodione
(5 fig/ml) (2 ^g/ml) (5 + 5 fig/ml) (5 + 5 + 2/tg/ml)
0 NT
NT
40
39 35 5*
34 0 32
population of B. ciwrra (the majority of which is
Ben'* NPC=*), and that the fungicide mixture
favoured its relative increase,
Table 1 also shows that when the fungicide
mixture exerted its selective effect on a population
which had already been resistant to both benzimi-
dazoles and dicarboximides (Ben" Die"), strains
with multiple fungicide resistance of the pheno-
type Ben'* Die'' NPC* could occur. Such straitis
grew more slowly than wild-type strains on
unamended medium and more slowly than some
Ben" strains on media amended with MBC with
or without diethofencarb (Table 2). Although
their growth on media amended with iprodione
and iprodione-t-MBC-I-diethofencarb was slow
and irregular, they were able to incite grey mould
infections on cucumber eotyledons treated with
MBC, iprodione or MBC-f-diethofencarb
(Table 3).
The development of multiple fungicide resis-
tance further demonstrates the adaptive capa-
bility of B. cinerea. It emphasizes the urgent need
for new chemicals, as well as cultural means (Elad
& Volpin, 1988; Yunis era/., 1990) and biocontrol
agents (Elad, 1990), to control this plant patho-
gen.
 Multiple fungicide-resistant Botrytis cinerea
Table 3. Effect of fungieide sprays on lesion development following inoculation of detached ci

Infection of e otyledoi eated with

Phenol ype*" No treatm(;nt MBC(0 5mg/n Ipr. (0 5 n ig/ml) Resec^d mg/ml)

Isolate Ben'' Die'' NPC:' A^ B' A B A B A B

FAR-7
FAR-11
FAR-15

" Detached cotyledon

^ S, sensitive; R, resis
^' Factory-prepared m
'' Ben, benzimidazole:
= Die, dicarboximides
'"NPC,/V
r of eotyledons infected out of
)n level: tr, small lesions undern

ACKNOWLEDGEMENTS eci resistant to benziniidiizoles and dicarboxi-

We gratefully acknowledge the assistance and
cooperation of R. Ausher, Y. Sachs, G. Araf, D.
Posniak and L. Gurevitz. This study was sup-
ported in part by the Vegetable Production and
Marketing Board, Israel.
REFERENCES
Elad Y. (1990) Reasons for the delay in development of
biological control of foliar pathogens. Phytoparasit-
iea 18, 99-105.
Elad Y. & Volpin H. (1988) The involvement of
ethylene and calcium in gray mold of pelargonium,
ruscus, and rose plants. Phytoparasitiea 16, 119-131.
Elad Y., Shabi E. & Katan T. (1988) Negative cross-
resistance between benzimidazole and /^-phenylcar-
bamate fungicides and control of Botrytis cinerea on
grapes. Plant Pathology 37, 141-147.
Gulhno M.L. & Garibaldi A. (1987) Control o(Botrytis
:s with mixtures of different fungieides. Mededel-
n van de Faeulteit Landhouwwetensehappen Rijks-
•;rsiteit. Gent 52, 895-900.
T. (1982) Resistance to 3,5-dichlorophenyl-7V-
c imide ('diearboximide') fungicides in the grey
lathoge ;ted crops.
Plant Pathology 31, 1.
Katan T. (1985) Failure to detect highly resistant strains
in dicarboximide resistant field populations of Botrv-
tis einerea. EPPO Bulletin 15, 371-377.
Katan T., Elad Y. & Yunis H. (1989) Resistance to
diethofencarb (NPC) in benomyl-resistant field iso-
lates of Botrytis einerea. Plant Pathologv 38, 86-92.
Kato T., Suzuki K., Takahashi J. & Kamoshita K.
(1984) Negatively correlated cross-resistance
between benzimidazole fungicides and methyl A'-(3,5-
dichlorophenyl) carbamate. Journal of Pesticide
Seience 9, 489-495.
Kritzman G. & Netzer D. (1978) A selective medium for
isolation and identification of Botrytis spp. from soil
and onion seed. Phytoparasitica 6, 3-7.
Leroux P., Gredt M., Massenot F. & Kato T. (1985)
 vile du phcny
iicidesfor
1 Monograph No. 31, pp. 443-446.
BCPC, Farnham. Surrey. UK.
Suzuki K., Kalo T., Takahashi J. & Kamoshita K.
(1984) Mode of action of methyl A'-(3,5-dichlorophe-
Yunis H., Elad Y. & Mahrer Y. (1990) Effects of air
temperature, relative humidity and canopy wetness
on gray mold of cucumbers in unheated greenhouses.
Phvlopara.iitiea IS, 203-2\5.