J. Northeast For. Univ. VoL 5, No. 4, Dec. 1994

RESISTANCE OF SCLEROTINIA SCLEROTIORUM TO

CARBENDAZIM

]I . Factors influencing ECs0of earbendazim to S. sclerotiorum and the virulence of a

S. sclerotiorum strain tolerant to the chemical

Yang Qian (]~i~)

Collage of Forest Resource and Environment, Northeast Forestry University.

ABSTRACT This study covers the observation of the factors which influence the resistance de-

velopment of S. sclerotiorum to carbendazim, the comparison betweeen normal course of infec-

tion and infection of hosts by a strain of S. sclerotiorum when resistance to carbendazim has been de-

veloped.

Key words: Resistance, Carbendazim, Sclerotinia sclerotiorum, Virulence

INTRODUCTION

Although the resistance development of

S. sclerotiorum to carbendazim is influ-

enced by chemical and the fungus itself

mainly,some other factors on

methodoloogy aspect may be also involved

in the resistance development. If it is true

and the factors are not aware, they will not

be controlled properly, then they will shift

the-results signigicantly. On the other

hand, when a fungus like S. sclerotiorum

developed resistance to fungicide, whether

the resistance will influence the virulence of

the fungus, which is related to the disease

control. However, none of the study on

these aspects above have been done so far.

Therefore, the factors influencing ECs00of

carbendazim to S. sclerotiorum were ob-

served, and the normal course of infection

and infection of hosts by a strain of the

fungus tolerant to the chemical were com-

pared in this study.

MATERIALS AND METHODS

The isolate of S. sclerotiorum examined

in this study was isolate YQ-Y. Both

sclerotia and mycelium from these isolates

were tested with carbendazim at different

concentrations. The formulation of

carbendazim was #Bavistin # (50% WP).

Water suspensions of 'lBavistin u at differ-

ent concentration were mixed with PDA.

The sclerotia and mycelium were then in-

oculated onto this medium. After inocula-

tion, the fungus on the medium was re-

corded 2-7 days after inoculation with

sclerotia, and 18-60 hours after inocula-

tion with mycelium.

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Inhibition of growth (IG) was em-

ployed to evaluate the effects of treating

time and mycelium age on the resistance

development of the isolate of the fungus to

carbendazim.

Normal mycelium and the mycelium

from sclerotia formed on 3/~g

ml-lcarbendazim in PDA medium about 7

weeks were prepared by culturing on PDA

at 22~C for 3-5 days. When the mycelia

covered nearly all the media in the plates,

they were inoculated on detached stems of

oilseed rape, soybeam and linseed. The

lesion length on the stems was recorded ev-

ery 24 hours after inoculation.

RESULTS

Factors influencing ECsoof thr Chemi-

cal to the Fungus During observation

of the ECs0of carbendazim for isolate

YQ-Y of S. sclerotiorum, it was found that

various factors could influence the

EC50values. These factors mainly included

age of the mycelium and time of observa-

Table 1

tion of the mycelium growth after inocula-

tion.

The age of mycelium when it could

give normal results was quite flexible, but

usually ranged from 2 to 4 days (though

sometimes even longer). When the

mycelium started to thicken at the edge of

the culture, it was too old to use in an

ECs0test, because at this stage, the

mycelium has started to form sclerotia and

tends to stop outward hyphal growth. Thus

tests carried out when the mycelium is ag-

ing are usually irregularr. Therefore, in or-

der to estimate the ECs0value accurately,

the mycelium should be tested before the

older growth stage. The best time is imme-

diately the advanced edge of the mycelium

has almost reached the margin of the PDA

plate or slightly earlier.

Making observations at different

times after inoculation of the mycelium on

the media containing different concentra-

tions of carbendazim tended to result in

variations in the relative colony growth

and hence ECs0values (Table 1).

Inhibition of mycelial growth of S. sclerotiorum growing on media containing carbendazim, ob-

served at different concentration ( p g / m l ) of carbendazim, obseraved at different times after inocu-

lation

Time of the

observation

Inhibition to growth (%) by differenteoneentration of carbendazim (/zg / ml)

0.04 0.13 0.4

24h 29.9 48.8 87.1

36h 32.8 40.5 79.4

48h 28.9 35.2 68.2

The results of tests on both mycelium

and sclerotia (Table 1 and 2) showed that

with mycelium, inhibition of growth start-

ed to decrease after 24h exposure to low

level of the chemical, in particular to the

concentrations of 0.13 and 0.4 /~g ml -i.

From the results with both mycelium and

sclerotia, it appears that the time of obser-

vation influences the apparent inhibition to

growth of the fungus; the later the observa-

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tion was made, the lower the inhibitioh was likely to appear.

Table 2 Inhibition to myeelial growth from sclerotia of S. scterotiorum treated with different concentrations

(Pg / ml) of carbendazim observed at different times after inoculation

Time of the

observation

Inhibition to growth (%) by different concentrations of carbendazim ~ g / ml)

0.03 0.3 3

3 days 22.1 95.0 100.0

4 days 11.5 93.3 99.4

5 days 4.7 86.1 97.2

Comparison between Normal Course of

Infection and Infection o f Hosts by a Strain

of S. sclerotiorum Tolerant to

Carbendazim In order to find out if

there were any differences in pathogenicity

of the fungus to the hosts between the

normal mycelium and that from sclerotia

formed on 3 /zg ml-lcarbendazim in PDA

medium about 7 weeks before this experi-

ment, the different mycelia were inoculated

on detached stems of oilseed rape, soybean

and linseed. The results (Table 3) showed

that the normal cycelium had higher viru-

lence to oilseed rape and soybean than

cycelium from the sclerotia which had sur-

vived on the mekium containing 3 pg

ml-~carbendazim. The mycelia from the

two different sources did not show very

much difference in level of pathogenicity

when infecting linseed stems. The results

from oilseed rape and soybean seemed to

suggest that the chemical treatment could

make the pathogen less virulent to these

two host crops in the stem infections.

Table 3 The means of lesion length on stems of oilseed rape (OSR), soybean and linseed caused by original

wild type mycelium and the same mycelium trained to be tolerant to carbendazim

Lesion length on stems(mm)

Crops Inoculation with two mycelial cultures

Wild type Tolerant

OSR Mean 47.2 29.0

Soybean Mean 27.0 15.3

Linseed Mean 60.0 59.8

_ LSD 3.4

LSD: least significant difference ( p < 0.05 df = 30)

DISCUSSION

Factors influencing ECs0of the chemi-

cal to the fungus mainly included age of the

mycelim and time of observation of the

mycelium growth after inoculation. During

the ECs0test, if the factor of age of the

mycelium is not controlled properly, it will

influence the ECs0values significantly, and

mislead the conclusion in the fungus

resistant or sensitive to carbendazim.

Therefore, it is extramly important to

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know the effects of age of the mycelium,

and to control it properly before the test.

Time of observation of the mycelium

growth after inoculation also influence the

ECs0values significantly, which appeared

that the later the observation was made,

the lower the inhibition was likely to ap-

pear. Although it is difficult to explain the

reasons so far, one of the possible reasons

is that the growth curves of the fungus on

PDA and on carbendazim were different.

The fungus grew relatively faster at earlier

time on PDA than on carbendazim because

it has long lag phase on carbendazim.

It was observed in the comparison be-

tween the normal course of infection and

the infection of hosts by strain of S.

sclerotiorum tolerant to carbendazim that

the mycelium which was tolerant to

carbendazim grew slower during the infec-

tion of some of its hosts than the normal

mycelium of the isolate, which is similar

with the observation on Botrytis cinereaby

Liu Bo et al.. Therefore, although exposure

to 0.3 /zg ml-lcarbendazim may have in-

creased the tolerance of the isolate, it also

appears to have decreased the virulence of

the fungus in some degree. By contrast, no

significant difference was observed in the

comparison between the two infections of

linseed. Although the reason for this is dif-

ficult to determine, the responses of

fifferent crops to the two different mycelia

of the fungus may occur in different ways.

Alternatively, even mycelium tolerant to

carbendazim may already be sufficiantly

virulent to a more susceptible crop like lin-

seed.

REFERENCES

1. Delen, N. & M. Yildiz. 1982. Fungicide re-

sistance of some fungal pathogens isolated

from greenhouses in Turkey. Journal of

Turkish Phytopatthology 11, (1-2)33-40

2. Yang Qian ! 99 I. Interaction of some crops

with Sclerotinia sclerotiorum(Lib.) de Bary.

PhD Thesis, Reading University. Chapt 8

Pathogen Response to a Fungicide.

133-144

3. Liu Bo, Ye Zhongyin, Liu Jingfen and

Zhou Mingguo 1992. Study on the proper-

ties of the resistant strains to procymidone

in Botrytis cinerea. Acta Phytojphylacica

Sinica 19, (4) 297-302

4. Yang Qian 1991. Interaction of Some

Crops with Sclerotinia sclerotiorum (Lib.)

de Bary. PhD Thesis, Reading University,

Chapt 3 Susceptibilities of diffferent crops

to the disease, 46-62

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