RESEARCH ARTICLE

Physiological characterization of the dandelion bioherbicide, Sclerotiniaminor IMI 344141

In’aam Y. Shaheena, Mohammed H. Abu-Dieyeha$, Gavin J. Ashb and

Alan K. Watsona*

aDepartment of Plant Science, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, H9X 3V9 Canada; bE.H. Graham Centre for Agricultural Innovation (NSWDepartment of Primary Industries and Charles Sturt University), Charles Sturt University,

Locked Bag 588, Wagga Wagga, NSW, 2678, Australia

(Received 26 February 2009; returned 6 April 2009; accepted 15 October 2009)

The fungus Sclerotinia minor (IMI 344141) is being developed as a biologicalcontrol for dandelion and other broadleaf weeds in turfgrass environments. Beinga microbial pest control agent (MPCA), the S. minor strain must be characterizedto show relatedness to like organisms and to distinguish the MPCA from relatedmicroorganisms. Phenotypic variation among 30 isolates of S. minor, collectedfrom different regions and hosts, was studied on potato dextrose agar (PDA)and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates.There was high diversity (0.6) among the mycelial compatibility groups (MCG) asseven multi-member and 11 single-member groups were recognized. Isolates werecategorized into highly virulent, virulent, moderately virulent, and hypo virulentbased on 48 h post mycelial growth on detached dandelion leaves. When assessedon dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest inbiocontrol efficacy, reduction of above- and below-ground biomass, andreduction in dandelion survival. Oxalic acid production was not correlated withisolate aggressiveness or growth rate and did not vary among isolates of the sameMCG. IMI 344141 can be phenotypically distinguished from the other tested S.minor isolates by performing vegetative compatibility testing and countingsclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 producesB100 sclerotia/plate.

Keywords: phenotypic variation; mycelial compatibility; virulence; oxalic acid

Introduction

Plant pathogenic fungi regularly cause significant crop losses. However, many weed

species are also attacked by fungi which have led to investigations of fungi as

biological weed control agents. One such fungus, Sclerotinia minor Jagger, is

pathogenic to many plant species, and several studies have investigated the

pathogenicity of S. minor on dandelion (Taraxacum officinale Weber ex Wiggers)

and other broadleaf weeds in turfgrass (Ciotola, Wymore, and Watson 1991; Riddle,

*Corresponding author: Email: alan.watson@mcgill.ca$Present address: Department of Biology and Biotechnology, The Hashemite University,

PO Box 150459, Zarqa, Jordan.

ISSN 0958-3157 print/ISSN 1360-0478 online

# 2010 Taylor & Francis

DOI: 10.1080/09583150903419520

http://www.informaworld.com

Biocontrol Science and Technology,

Vol. 20, No. 1, 2010, 57�76

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Burpee, and Boland 1991; Briere, Watson, and Paulitz 1992; Schnick, Stewart-Wade,

and Boland 2002; Stewart-Wade et al. 2002; Abu-Dieyeh, Bernier, and Watson 2005;

Abu-Dieyeh and Watson 2006, 2007a,b,c). The IMI 344141 strain of S. minor is

registered in Canada as a bioherbicide for control of dandelion in turfgrass

environments (PMRA 2007).

S. minor is a soil-borne Discomycete (Sclerotineaceae) fungus characterized bysmall (0.5�2.00 mm) spherical sclerotia (Willetts and Wong 1980) that germinate by

eruptive growth of the mycelium and colonize susceptible plant tissues (Abawi and

Grogan 1979). The host range of S. minor is broad and includes 21 families, 66

genera and 94 species (Melzer, Smith, and Boland 1997; Hollowell, Shew, Cubeta,

and Wilcut 2003). S. minor is a causal agent of lettuce drop, Sclerotinia blight of

peanut, white mold of green beans, and watery soft rots of vegetables (Abawi and

Grogan 1979). In Canada, S. minor is not considered as a serious plant pathogen

except on lettuce (Melzer et al. 1997).

Individual strains of a broad spectrum fungus may vary in their ability to infect

different hosts. Therefore, the development of bioherbicide formulations based on

selected isolates requires the capability to distinguish the biocontrol agent from like

organisms (Noonan, Glare, Harvey, and Sands 2004). Accurate identification of the

microbial pest control agent (MPCA) is a key component of safety assessment and

taxonomic designation to an appropriate level to distinguish the MPCA from related

microorganisms is a regulatory requirement (PMRA 2001). The ability to

discriminate the IMI 344141 bioherbicide strain from other S. minor isolates is

also imperative for environment tracking and intellectual property and litigationprotection.

Mycelial compatibility has been used to assess the population biology of many

fungal plant pathogens. Mycelial compatibility is the ability of two strains of fungi to

fuse and form a stable heterokaryon (Leslie 1993). Strains that are compatible with

one another are frequently described as members of the same mycelial compatibility

group (MCG). Mycelial compatibility reflects genetic heterogeneity among isolates

(Viji, Uddin, O’Neill, Mischke, and Saunders 2005), reveals intrapopulation changes

(Sarma and Singh 2002), measures and categorizes intraspecific variations (Kohn,

Carbone, and Anderson 1990; Leslie 1993, 1996; Durman, Menendez, and Godeas

2003), and if combined with molecular markers, gives more precise information

about genetic diversity and relatedness among isolates of the same species (Kohn,

Stasovski, Carbone, Royer, and Anderson 1991; Noonan et al. 2004).

Pathogenic strains that are vegetatively compatible are presumed to originate

from the same clone, even if they are geographically isolated from one another

(Leslie 1993). Correlation between MCGs and other characters such as pathogeni-

city or phenotype could lead to useful diagnostics. Widely dispersed MCGs ofS. sclerotiorum showed varied aggressiveness while isolates from the same field did

not vary in aggressiveness (Kull and Pedersen 2004). Isolates of Sclerotium rolfsii

from one MCG varied in the number and size of sclerotia in culture, indicating that

phenotypic differences are not uncommon among members of a MCG (Punja and

Grogan 1983). In other studies, morphological or pathological characters were not

related to mycelial compatibility grouping (Durman et al. 2003).

The physiology of pathogenesis of plant diseases caused by Sclerotinia species has

been comprehensively reviewed and this has clarified the importance of cell wall-

degrading enzymes and oxalic acid (OA) production by the pathogen in causing

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disease (Lumsden 1979; Willetts and Wong 1980). However, pathogenesis of S. minor

specifically is not well understood. With other pathogenic Sclerotinia species, OA is

implicated in the infection process (Bateman and Beer 1965; Kritzman, Chet, and

Henis 1977), but contradicting correlations have been demonstrated between OA

production and virulence. Oxalic acid has been confirmed as a pathogenicity

determinant in S. sclerotiorum by using an OA-deficient mutant (Godoy, Steadman,

Dickman, and Daur 1990), but OA was not the sole pathogenic determinant of S.

trifoliorum (Callahan and Rowe 1991). Variation in the production of OA by isolates

of some Sclerotinia species has been reported, while few studies have examined OA

production by S. minor. One study reported correlation between aggressiveness of S.

minor isolates on peanut and mycelial growth in broth cultures, but not OA

production (Hollowell, Smith, and Shew 2001).

This study was designed to demonstrate the uniqueness of the IMI 344141

bioherbicide isolate of S. minor by comparison to other S. minor using phenological

traits, MCGs, aggressiveness, and OA production. Experiments were conducted to:

(1) detect morphological variability among S. minor isolates collected from different

regions in the world; (2) examine relatedness amongst the isolates based on mycelial

compatibility interactions; (3) compare the pathogenicity of the different isolates and

their affinity to produce oxalic acid; and (4) provide a set of descriptors to

distinguish the IMI 344141 isolate of S. minor from related microorganisms.

Materials and methods

Fungal Isolates

Thirty isolates of S. minor were obtained from various culture collections and

research colleagues (Table 1). The isolates were maintained by growing single

sclerotia, or agar discs taken from the original cultures, and growing them on potato

dextrose agar (PDA) (Difco Laboratories Inc, Detroit, MI) at 20918C in the dark.

For long-term maintenance, two sets of PDA slants were inoculated with each

isolate; one set was placed under sterile mineral oil and the other under �80928C.Sclerotia of each of the 30 isolates were produced on surface sterilized market

dandelion leaves in moist chambers. Leaves were inoculated with mycelial discs taken

from the margin of 4�5-day-old colonies grown on PDA and incubated at 20918C in

the dark. Collected sclerotia were air-dried at 20918C for 48 h, labeled, and stored in

air-tight glass vials at 490.58C.

Experiment 1. Morphological variation

Cultures of each of the 30 S. minor isolates were established by sterilizing sclerotia

(prepared above) in 70% ethanol for 40 s, 5% bleach solution for 3 min, washing

twice with sterilized distilled water, and drying on sterilized filter paper (Abu-Dieyeh

and Watson 2006). Surface sterilized sclerotia were transferred to the surface of PDA

in 9-cm diameter Petri plates and incubated at 20918C in the dark. Three-millimeter

diameter mycelial discs were taken from the margin of 4�5-day-old colonies grown

and used to inoculate PDA and oatmeal agar (OMA) (Sigma-Aldrich Canada Ltd,

Oakville, ON) plates. Plates were incubated at 20918C in the dark and examined

daily for colony diameter, mycelial density and color, sclerotial initiation and

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formation times, sclerotial arrangement, exudate presence on sclerotia, and sclerotial

size. Colony diameter was measured daily until the growth reached the edge of the 9-

cm diameter Petri plates. The experiment was replicated three times. After 20�25days of growth, the total number of sclerotia on PDA plates was counted and the

diameter of 25 sclerotia formed on both growth media per isolate per replicate was

measured; the data from the replicated plates were averaged (Sarma and Singh 2002).

Experiment 2. Mycelial compatibility groups

Each isolate was paired against itself as a control and against all other isolates on

modified Patterson’s medium (MPM) (Kohn et al. 1990). This medium contains

0.68 g KH2PO4, 0.5 g MgSO4.7H2O, 0.15 g KCl, 0.5 g yeast extract, 1 g NH4NO3,

Table 1. Designation codes, origin, host plant and mycelial compatibility group (MCG) of a

worldwide collection of Sclerotinia minor isolates.

MCG Isolate code/number Origin Host

1 IMI 344141 Quebec, Canada Lettuce (Lactuca sativa)

LRC2104 Manitoba, Canada Lettuce (Lactuca sativa)

ATCC44236 New York, USA Lettuce (Lactuca sativa)

Sm44 California, USA Lettuce (Lactuca sativa)

Sm66 Ontario, Canada Lettuce (Lactuca sativa)

2 S96-22 Suwon, Korea Unknown

S96-138 Suwon, Korea Unknown

S96-250 Suwon, Korea Unknown

S97-75 Suwon, Korea Unknown

3 R21 Virginia, USA Peanut (Arachis hypogaea)

R23 Virginia, USA Peanut (Arachis hypogaea)

4 R22 New Jersey, USA Lettuce (Lactuca sativa)

R25 New Jersey, USA Cabagge (Brassica campestris)

5 BRIP28139 New South Wales, AU Chickpea Cicer arientium

K4205 Queensland, AU Mungbean (Vigna radiata)

6 VPRI1671 Tasmania, AU Onion (Allium sativum)

VPRI1285 Victoria, AU Bathurst burr (Xanthium spinosum)

7 BPIC1681 Kifissia, Greece Lettuce (Lactuca sativa)

BPIC1949 Kifissia, Greece Lettuce (Lactuca sativa)

8 R24 New Jersey, USA Endive (Cichorium endivia)

9 JW1 Hull, UK Unknown

10 SMRF02 Virginia, USA Peanut (Arachis hypogaea)

11 TH1C New South Wales, AU Canola (Brassica napus)

12 LU286 Canterbury, NZ Lettuce (Lactuca sativa)

13 LK1 Budapest, HU Knapweed Centaurea pannonica

14 MUCL38484 Belgium Unknown

15 S94-001 Suwon, Korea Unknown

16 CBS112.17 The Netherlands Lettuce (Lactuca sativa)

17 CBS207.25 The Netherlands Sasify (Tragopogon porrifolius)

18 CBS339.39 Italy Lettuce (Lactuca sativa)

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18.4 g d-glucose, 0.2 mL of Vogel’s trace elements solution, 15 g agar (Fisher

Scientific Company, Ottawa, ON) and six drops of McCormick’s food coloring.

Each isolate was grown on MPM for 5 days prior to pairing. For pairings, mycelial

discs (3-mm diameter) taken from the edge of an actively growing colony of eachisolate were placed approximately 30 mm apart on MPM in 9-cm diameter Petri

dishes, one pairing per dish, and incubated at 20918C in the dark. Isolates were

paired in all combinations; each pairing was performed three times (three replicates).

Pairings were examined visually 7�15 days post inoculation for the presence of an

antagonistic zone. Pairings were assessed as incompatible if there was a red line in the

reaction zone between the two colonies, and as compatible when the two colonies

merged with no detectable line (Kohn et al. 1990, 1991; Sarma and Singh 2002;

Durman et al. 2003). Questionable pairings were repeated to verify results.

Experiment 3. Oxalic acid production

Cultures of the 30 S. minor isolates were established by surface sterilizing sclerotia of

each isolate as mentioned previously and plating on PDA. Plates were incubated at

20918C in the dark until mycelial growth reached two-thirds of the plate. Five

mycelial discs (6-mm diameter) taken from the margin of an actively growing colony

of each isolate were used to inoculate 100 mL potato dextrose broth (PDB, Difco).

Flasks were placed on a rotary shaker (50 rpm) at 20918C in the dark for 7 days.

Cultures were filtered through P8 filter Whatman paper (Fisher) and each filtrate

was filtered through a series of Millipore membrane filters with decreasing pore size8, 5, 1 and 0.45 mm (Millipore Corporation, Bedford, MA) using a filter holding

system (Nalgene Nunc. International, Rochester, NY) and a vacuum pump. Fifteen-

microlitre aliquots taken from the filtrate of each fungal isolate were injected into an

HPLC system utilizing an Alltech Prevail Organic Acid 5 mm Column (150�4.6 mm

ID) for organic acid analysis (Alltech Associates Inc., Deerfield, IL) and the Waters

HPLC system with a 441 Detector (Millipore Corporation, Milford, MA). The

elution solvent was 25 mM KH2PO4, pH 2.5. Organic acid detection was carried out

at 210 nm at 258C. Ten microlitres of the oxalic acid standard was chromatographedbefore the samples. Concentrations were calculated by comparing peak areas of the

samples with those of oxalic acid standard solutions. The experiment was conducted

in a completely randomized design with three replications and repeated three times.

Experiment 4. Virulence

Virulence of the S. minor isolates was determined on detached dandelion leaves and

on whole plants in the greenhouse. Mycelial discs and colonized barley grits were

used to screen the isolates on detached dandelion leaves, while the colonized barley

grits were also used on whole plants in a greenhouse to compare the virulence of the

10 most virulent isolates from the detached leaves bioassay.

Plant production

Locally collected dandelion seeds were sown onto potting soil [two-third black

pasteurized soil and one-third Pro-mix (Premier Promix, Premier Horticulture Ltee,

Riviere-du-Loup, QC) in 40�30�8 cm trays in the greenhouse (20928Cwith 15 h of

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light day1 at photon flux density minimum of 350950 mmol m�2 s�1)]. Two-week-old

seedlings with uniform vigor were individually transplanted into 15-cm diameter pots

containing the mixed potting soil (as above). Plants were grown in the greenhouse

with programmed drip irrigation of 50 mL pot�1 three times a day.

Detached leaf bioassay

Leaves were removed from 8-week-old dandelion plants grown in the greenhouse and

three dandelion leaves were placed in each of 90 glass Petri dishes lined with

Whatman No. 1 filter paper moistened with 4 mL sterile distilled water. PDA plates

of each of the 30 isolates were prepared as above. Agar plugs (2-mm diameter) were

cut from the actively growing margins of cultures of each of the 30 S. minor isolates

and placed with the mycelium-side down in the center of a detached dandelion leaf

(Briere, Watson, and Hallett 2000; Hollowell and Shew 2003). Petri dishes were

sealed with parafilm and incubated at 20928C in the dark for 48 h. Lesion diameters

were measured (lengthwise direction of the leaves) at 24 and 48 h after inoculation.

The experiment was conducted in a completely randomized design with three plate

replicates (a total of nine dandelion leaves) per isolate and conducted twice. The 48 h

post inoculation screening results were used to arbitrarily classify the S. minor

isolates into four categories: highly virulent (mean lesion diameter ]30 mm),

virulent (20�30 mm), moderately virulent (10�20 mm), and hypovirulent or avirulent

(B10 mm).Highly virulent isolates and IMI 344141 were assessed for their aggressiveness by

growing them on sterilized barley grits as described by Abu-Dieyeh and Watson

(2006) and screening them on detached dandelion leaves using the same procedures

used for mycelial discs.

Greenhouse bioassay

When potted dandelion seedlings were 3-weeks-old, 0.8 g of a commercial grass seed

mixture [30% Kentucky bluegrass (Poa pratensis), 40% creeping red fescue (Festuca

rubra L. var. rubra) and 30% turf type perennial ryegrass (Lolium perenne L.),

(C.I.L.† GolfgreenTM, Spectrum Brands Canada Ltd, Brantford, ON)] was scattered

over the surface of each pot. The grass was cut weekly with hedge shears to a height

of 10 cm, commencing 3 weeks after grass sowing. A 15�15�30, N�P2O5�K2Ofertilizer with micronutrients (PlantexTM, Plant Product Co, Brampton, ON) was

applied at 3.5 g L�1 when the dandelions were 5-weeks-old. Eight weeks after

transplanting, plants were labeled for the specific treatment, 16 plants for each

isolate (eight of which were retained as untreated controls), were distributed in a

completely randomized design on the bench, misted with water, and each was

inoculated with 0.2 g of the S. minor isolates granular formulation. Mist was applied

daily over all the pots for 1 week. Plant survival was recorded weekly for 3 weeks.

Plant re-growth (after 100% above ground damage) was recorded as decreasing

percentage damage by estimating the biomass of the new leaves. Three weeks after

the first application, surviving plants received a second application of 0.2 g of their

S. minor isolates granular formulation. Six weeks after the first application, all

dandelion plants were carefully removed from the soil, the roots were thoroughly

washed and dissected above the crown, separating above- and below-soil biomass.

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Treated and control plant materials (leaves or roots) were separately bulked for each

of the 10 isolates, placed in paper bags, oven dried at 808C for 72 h and then weighed.

The experiment was repeated.

Statistical data analysis

Bartlett’s test of SAS (SAS Institute Inc., Cary, NC 2002) was used to test the

homogeneity of variances of data from the two replicates of each experiment. In all

experiments, data from the two repeats were homogeneous and data were combined

and analyzed as one experiment. Colony diameter measurements, sclerotia count per

plate, lesion diameter on dandelion leaves and oxalic acid concentration of the

S. minor isolates were analyzed using one-way ANOVA and the mean values of the

isolates were separated using Tukey’s test at P�0.05. Sclerotia count per plate data

of two isolates, IMI 344141 and CBS339.39 were significantly less than other

isolates, but not significantly different from each other, thus they were subjected to a

paired t-test. One-way ANOVA and mean comparisons for all experiments were

performed using SigmaStat 2.03 (1992�1997).

Results

Experiment 1. Morphological variation

The studied S. minor isolates represent a worldwide sampling of S. minor populations

from more than three different hosts including the dominant host, lettuce (Table 1).

Growth characteristics were tested on PDA and OMA media. Each of the isolates

showed similar appearance of mycelial color (mainly white) and type (mainly fluffy)

on both agars. Sclerotia initiation and formation time was more rapid on OMA than

on PDA. Eight isolates required 1 day from initiation to formation on PDA, while

the 13 isolates needed 1 day on OMA. In general, the time for sclerotia completion

was 1�4 days except for isolate CBS339.39 which was the slowest in initiation and

formation, 10 and 4 days, respectively. Three isolates did not form sclerotia, even

after a month of incubation. Sclerotia formation pattern was different on the two

growth media with mostly concentric on OMA and peripheral to scattered on PDA.

Sclerotia varied in color from olive green, dark brown, grey to dominant black.

Sclerotia were spherical to irregular in shape, most with a rough surface.

The sclerotia of IMI 344141 were almost spherical, black, highly rough surfaced,

1.72�1.69 mm in diameter, and scattered over the surface of PDA. Microspores

were not detected with any of the isolates. Exudates were produced on the sclerotia of

most isolates on OMA but rarely on PDA. An earthy soil odor was clearly evident

for five isolates on OMA and for two on PDA.

Except for isolate CBS207.25, the growth rates on a particular medium were

similar among the isolates (Table 2). In general, radial growth was faster for all

isolates on PDA than on OMA, ranging from 5.3 to 17.3 mm and 5.3 to 15.1 mm

after 24 h on PDA and OMA, respectively, and 26.5 to 57 mm and 15.3 to 37.5 mm

after 48 h (Table 2). The growth rate of IMI 344141 was 13.393.22 and 48.894.25

on PDA after 24 and 48 h, respectively.

Sclerotia size was one of the most obvious quantitative characters with significant

differences among isolates in length and width dimensions. Isolate S94-001 had the

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smallest sclerotia on PDA (1.44�1 mm) and Sm66 had the smallest on OMA

(1.16�0.95 mm) (Table 3). While the largest sclerotia on both growth media were

CBS339.39, 3�2.4 mm on PDA and 2.6�2.1 mm on OMA, respectively. The very

large sclerotia suggests CBS339.39 had been misidentified and nearer to

Table 2. Growth rate of Sclerotinia minor isolates after 24 and 48 h of incubation on PDA

and OMA media. Average of four replications.

Colony diameter (mm)

PDA OMA

MCG Isolate 24 h 48 h 24 h 48 h

1 IMI344141 13.393.221 48.894.25 11.893.59 32.394.86

LRC2104 8.094.58 37.798.08 10.396.29 27.0916.02

ATCC44236 17.097.81 54.8911.64 12.695.79 33.098.76

Sm44 5.792.51 34.093.61 11.092.16 29.3910.21

Sm66 6.393.06 35.3910.79 11.394.35 36.097.39

2 S96-22 10.094.58 45.097.21 12.194.37 34.595.45

S96-138 5.392.08 32.3919.50 10.194.91 31.597.33

S96-250 13.095.20 53.8912.71 11.693.68 30.098.12

S97-75 10.794.16 45.394.62 14.893.62 34.696.05

3 R21 17.1796.75 48.0916.52 9.092.71 29.3912.23

R23 10.094.00 47.097.81 14.494.61 38.498.26

4 R22 8.6790.58 33.895.25 8.594.04 34.6913.01

R25 7.6794.04 36.396.35 12,092.94 31.099.49

5 BRIP28139 10.095.57 45.3918.77 10.996.46 30.9910.44

K4205 9.095.20 44.3912.42 11.895.85 29.9911.65

6 VPRI1671 9.093.61 39.398.15 13.392.50 28.097.26

VPRI1285 12.794.93 42.7912.74 10.693.86 29.096.78

7 BPIC1681 9.791.53 28.791.53 15.091.41 33.393.40

PBIC1949 16.096.25 44.0912.77 14.394.19 35.394.57

8 R24 16.6792.31 52.093.61 14.194.73 36.699.43

9 JW1 7.091.73 32.291.76 8.592.65 25.196.76

10 SMRF02 16.398.15 55.7913.65 15.194.37 37.598.35

11 TH1C 6.792.52 36.796.51 9.994.09 28.197.22

12 LU286 14.096.08 48.0911.27 13.892.63 34.897.41

13 LK1 7.792.31 33.093.46 7.091.58 24.9912.07

14 MUCL38484 13.295.80 38.0918.19 11.495.41 33.4910.47

15 S94-001 17.398.08 57.0916.70 12.695.04 32.597.42

16 CBS112.17 13.596.14 30.7911.24 5.394.11 15.393.86

17 CBS207.252 0.090.00 3.793.22 0.090.00 3.592.38

18 CBS339.39 9.092.00 26.597.26 9.093.92 20.093.56

1Mean9standard deviation.2All the means of this isolate are significantly different with other isolate means within the same column atthe 5% level.

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Table 3. Differences in size and number of sclerotia of Sclerotinia minor isolates.

Length/Width2

MCG1 Isolate # PDA4 OAM5Sclerotia #/Plate3

PDA

1 IMI344141 1.0590.136 e7 1.1190.20 b 92.392.08 k8

LRC2104 1.1590.28 abcde 1.1490.23 b 461.3927.01 defg

ATCC44236 1.2590.30 abcd 1.1590.25 b 622.0963.91 abc

Sm44 1.1290.17 abcde 1.1590.27 b 697.0947.00 ab

Sm66 1.2590.29 abcd 1.2390.34 b 741.3945.54 a

2 S96-22 1.1790.23 abcde 1.2090.34 b 464.0914.00 defg

S96-138 1.1590.26 abcde 1.1490.23 b 376.0999.78 gh

S96-250 1.2590.28 ab 1.2290.25 b 535.3960.52 bcdef

S97-75 1.2590.30 abc 1.1690.28b 215.7914.36 j

3 R21 1.1190.21 de 1.2090.33 b 244.0921.28 ij

R23 1.1190.18 bcde 1.1290.40 b 344.3957.01 ghi

4 R22 1.0890.16 de 1.1790.34 b 301.7964.26 hij

R25 1.1490.27 cde 1.1290.28 b 433.7951.19 fg

5 BRIP28139 1.1290.17 abcde 1.1190.18b 448.094.36 efg

K4205 1.1290.21 bcde 1.2090.27 b 376.3934.43 gh

6 VPRI1671 1.290.25abcde 1.2290.28 b 620.0946.87 abc

VPRI1285 1.1290.23 de 1.2290.36b 352.3923.18 ghi

7 BPIC1681 n.a. n.a. 0.090.00 l

BPIC1949 1.3090.47 abcd 1.3090.37 b 612.0913.23 abcd

8 R24 1.1590.23 abcde 1.2290.31 b 224.3918.58 j

9 JW1 1.0790.20 e 1.1590.28 b 692.7928.11 ab

10 SMRF02 1.2890.31 ab 1.1590.27 b 353.7965.39 ghi

11 TH1C 1.15990.24 abcde 1.1690.25 b 519.3935.23 cdef

12 LU286 1.1790.25 abcde 1.4690.42 a 450.3953.69 efg

13 LK1 1.2190.28 abcde 1.2890.59 ab 589.3925.17 abcde

14 MUCL38484 1.2990.30 a 1.2190.29 b 576.3969.47 abcdef

15 S94-001 1.1290.19 abcde 1.1390.19 b 594.3931.66 abcde

16 CBS112.17 n.a. n.a. 0.090.00 l

17 CBS207.25 n.a. n.a. 0.090.00 l

18 CBS339.39 1.2890.35 abc 1.2390.29 b 45.097.94 k

1Mycelial compatibility groups.2The data were subjected to Kruskal�Wallis one-way ANOVA on ranks. Twenty-five individual sclerotia�3 replications through time for each isolate.

3The data were subjected to square root transformation to achieve normality then analyzed by one-wayANOVA. Average of three replications through time.

4Potato dextrose agar.5Oat meal agar.6Average9Standard deviation.7Within each column, means with common letters are not significantly different at P�0.05 according toTukey’s test.

8Isolate IMI344141 was significantly different from isolate CBS339.39 at P�0.006 according to the pairedt-test (SigmaStat for Windows version 2.03 1997).

9n.a., no sclerotia formed.

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S. sclerotiorum. Length/width ratio reflects sclerotia shape which was more

homogeneous among isolates on OMA than on PDA and ranged from 1.05 to 1.3

on PDA indicating a spherical to oblong shape.

A major distinguishing feature of isolate IMI 344141 is the low number ofsclerotia (Table 3). Sclerotia production of this isolate is significantly different from

all of the S. minor isolates tested including CBS339.39 (near S. sclerotiorum) which

produced significantly less number of sclerotia than IMI 344141 according to paired

t-test (Table 3).

Experiment 2. Mycelial compatibility groups

During the MCG test, incompatible pairings were recognized by the formation in aninteraction zone where mycelia from each isolate stopped growing. Accumulation of

the red food color in the hyphal tips formed a red line between incompatible pairings

at day 7 (Figure 1), whereas compatible isolates had fused mycelia associated with

abundant sclerotia in the fusion zone. There were 435 pairings between the 30

isolates with only 21 combinations showing a compatible reaction (4.8% of all the

combinations). Consistent pairing results were obtained in the replications of each

isolate. Mycelial compatible isolates were placed in the same MCG. Eighteen

mycelial compatibility groups (MCGs), seven multi-member (consisting of two tofive isolates each) and 11 single-member (R24, JW1, TH1C, SMRF02, LU286, LK1,

MUCL38484, S94-001, CBS112.17, CBS207.25, and CBS339.39) MCGs were

identified. Each MCG was assigned a number (MCG1-MCG18) (Table 1). MCG1

included the IMI 344141 isolate and four other isolates, MCG2 had four isolates

from Korea, MCG3 to MCG7 contained two isolates each, and MCGs 8-18 had one

isolate each. All self pairings were compatible. In all incompatible reactions, sclerotia

were not formed at the interaction zone (Figure 1). All compatible reactions

occurred within the first week of incubation. Because of the slow growth of isolateCBS207.25 (Table 2), pairing plates were incubated for 2 weeks and all pairings with

this isolate were incompatibile.

Experiment 3. Oxalic acid production

Oxalic acid was detected in the culture filtrate of all the studied S. minor isolates.

HPLC analysis revealed no significant differences in OA production among the

members of each of the MCGs (Figure 2), while there were some differences betweendifferent isolates of different MCGs. Maximum OA production was measured for

isolate S96-250 belonging to MCG 2, while minimum production was from isolates

R21 and CBS207.25.

Experiment 4. Isolate virulence

After 24 h, lesion diameters on detached dandelion leaves were variable among the

isolates and no lesions had been induced by nine isolates (Table 4). After 48 h, the S.minor isolates were classified into four categories: highly virulent (mean lesion

diameter ]0 mm), virulent (20�30 mm), moderately virulent (10�20 mm), and

hypovirulent (B10 mm). Isolate IMI 344141 fell within the moderately virulent

category with 26 mm lesion diameter, but the barley based formulation demonstrated

66 I.Y. Shaheen et al.

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high virulence without significant differences from the other highly virulent isolates

(Table 5). Although isolate TH1C ranked the fourth of the highly virulent isolates

using the mycelial disc method, it was the least virulent using the barley based

formulation.

Using dandelion plants, isolate IMI 344141 was one of the four highest isolates in

exerting significant above ground damage either 5 days or 3 weeks post application

(Figure 3). However, it was significantly ranked the first among all isolates in

reducing above and below soil biomass of dandelion (Figure 4). Moreover, the high

virulence of isolate IMI 344141 resulted in the least amount of re-growth and the

Figure 1. Pairings of Sclerotinia minor isolates demonstrating representative hyphal

interactions 7 days after inoculation (except C). (A) Isolate R24�BPIC1949, incompatible,

colony reverse. (B) Colony surface. (C) Isolate ATCC44236�CBS207.25, incompatible. (D)

Isolate Sm66�ATCC44236, compatible reaction. (E) Isolate Sm66�LRC2104, compatible

reaction. (F) Self-self reaction.

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lowest survival of dandelion among isolates after the first and the second

applications (Figure 5).

Descriptors distinguishing IMI 344141

IMI 344141 can be phenotypically distinguished from the other tested S. minor

isolates by vegetative compatibility testing and counting sclerotia produced on

standard 9-cm diameter PDA plates. IMI 344141 mycelia are white and fluffy and

sclerotia initials and sclerotia are formed on PDA agar in 96 and 144 h, respectively.

The sclerotia of IMI 344141 are spherical, black in color with a very rough surface.

Sclerotia are scattered on the surface of the plates rather than peripheral. IMI

344141 produces less than 100 sclerotia/9-cm diameter PDA plate.

Discussion

Variation within a worldwide collection of 30 isolates of S. minor was studied based

on culture morphology, mycelial compatibility and virulence. Two growth media,

PDA and OMA, were used to study the different culture characteristics of the 30

isolates including growth rates, colony type, mycelial color, time needed by each

isolate to form sclerotia, mode of sclerotia formation in addition to sclerotial

morphology counts and measurement and exudate formation. Differences in the

growth rate of all isolates were recorded on both media, where it was slower on OMA

than on PDA. Isolates showed different modes of sclerotia formation on both media,

where it was mostly concentric on OMA but peripheral or scattered on PDA.

abcd

eab

cde

abcd

e

abcd

e abcd

eab

cde

abcd

eab

cde

abcd

e

a

a

ab

ff

cde

e

bcde

de

abcd

abc

abcd

abcd

e

abcd

eab

cde

de

dede

bcde

fab

cde

Sclerotinia minor isolates

Oxa

lic a

cid

conc

entra

tion

(mg

dl -1

)

0

25

50

75

100

125

150

175

200

225

250

275

S96-

138

S96-

22

SMR

F02

S94-

001

BPI

C19

49

S96-

250

LU28

6

MU

CL3

8484

S97-

75

VPR

I167

1

LK1

R24

JW1

TH1C

R21

R23

R22

R25

CB

S 11

2.17

CB

S207

.25

CB

S339

.39

BPI

C16

81

BR

IP28

139

VPR

I128

5

K42

05

IMI 3

4414

1

ATC

C44

236

Sm44

LRC

2104

Sm66

Figure 2. Oxalic acid production by different Sclerotinia minor isolates in potato dextrose

broth filtrate. Error bars refer to standard errors. Bars with common letters are not

significantly different at P�0.05 according to Tukey’s test.

68 I.Y. Shaheen et al.

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Whereas the use of the two growth media revealed these differences, it did not reveal

basic differences that we could rely on to differentiate between different isolates.

Smaller sclerotia sizes were measured for most isolates on OMA than on PDA.

Significant differences were found among isolates for sclerotia measurements on

both media where S94-001 and Sm66 had the smallest sclerotia on PDA and OMA,

respectively. CBS339.39 produced the largest sclerotia on both media. The culture

Table 4. Means and standard deviations (9 replicates) of the lesion diameters caused 24 and

48 h post inoculation of Sclerotinia minor isolates on detached dandelion leaves using 2-mm

mycelial discs.

Lesion diameter (mm)

MCG Isolate 24 h 48 h

1 IMI 344141 8.194.7 26.095.62

1 LRC2104 11.091.2 30.595.11

1 ATCC44236 4.295.1 25.996.92

1 Sm44 9.693.9 26.892.92

1 Sm66 11.49.9 28.997.72

2 S96-22 11.391.3 30.694.31

2 S96-138 12.791.7 37.196.91

2 S96-250 0.090.0 4.696.7

2 S97-75 10.792.4 30.194.71

3 R21 3.394.0 24.895.42

3 R23 12.391.0 35.393.41

4 R22 6.395.2 24.197.32

4 R25 7.294.4 18.392.43

5 K4205 11.191.2 28.695.52

5 BRIP28139 0.090.0 1.995.54

6 VPRI1671 0.090.0 13.195.63

6 VPRI1285 0.090.0 0.992.74

7 BPIC1681 0.090.0 0.090.04

7 BPIC1949 0.892.3 12.394.93

8 R24 0.090.0 0.090.04

9 JW1 4.095.0 18.098.83

10 SMRF02 12.390.7 35.094.11

11 TH1C 10.892.3 32.395.01

12 LU286 7.494.5 30.395.21

13 LK1 3.694.30 29.698.12

14 MUCL38484 9.695.7 28.597.72

15 S94-001 13.291.3 33.393.21

16 CBS 112.17 0.090.0 8.896.14

17 CBS207.25 0.090.0 0.090.04

18 CBS339.39 0.09 0.0 0.090.04

1Highly virulent ]30 mm.2Virulent (20�30 mm).3Moderately virulent (10�20 mm).4Hypo-and avirulent (B10 mm).

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Table 5. Means and standard deviations (15 replicates) of the lesion diameters caused 24 and

48 h post inoculation of the most aggressive Sclerotinia minor isolates on detached dandelion

leaves using colonized barley grits.

Lesion diameter (mm)

Isolate 24 h 48 h

IMI 344141 10.195.5 a 28.2911.4 a

R23 8.395.9 a 30.2910.0 a

LRC2104 4.894.7 ab 22.995.7 ab

SMRF02 8.494.7 a 27.5911.4 ab

TH1C 1.292.67 b 17.398.2 b

S94-001 6.295.77 ab 25.497.7 ab

S96-22 6.595.3 ab 21.9910.7 ab

S96-138 10.192.7 a 28.395.5 a

S97-75 6.794. 7 ab 28.194.7 a

LU286 8.494.7 a 27.5911.4 ab

Within each column, means with similar letters are not significantly different at P�0.05 according toTukey’s test.

Sclerotina minor isolatesIM

I3441

41

S96-18

3

S97-75

S94-00

1

S96-22

SMRF02TH1C LR

CLU

286

R23

Abo

ve-g

roun

d da

mag

e (%

)

0

20

40

60

80

100

120 5 days post application 3 weeks post application

aab

abcab

abc

a

c

ab

bcabc

m

n

o

mn mnmn

non nn

Figure 3. Above ground damage to dandelion caused by different Sclerotinia minor isolates.

Error bars represent the standard errors of the means (average of 16 plant replicates). Within

each post application time, bars with a common letter are not significantly different at P�0.05

according to Tukey’s test.

70 I.Y. Shaheen et al.

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characteristics and large dimensions of the sclerotia of this isolate suggest that

CBS339.39 was misclassified and is likely closer to S. sclerotiorum. Spherical to

oblong sclerotial shape was a reflection of length/width ratio measurements, which

was more homogeneous on OMA than on PDA. Woodard and Simpson (1993)

reported a range of 1000�3000 sclerotia per plate on PDA for S. minor isolates from

peanut. Our range of sclerotia production was lower than this even for those isolates

isolated from peanut plants (R21, R23, SMRF02). Most isolates produced 200�700sclerotia per PDA plate, whereas two isolates: IMI 344141 and CBS339.39 produced

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Bio

mas

s (g

pla

nt -1

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

IMI3

4414

1

S96

-183

S97

-75

S94

-001

S96

-22

SM

RF0

2

TH1C

LU28

6

R23

LRC

cont

rol

30

2.5

2.0

1.5

1.0

0.5

Above-groundRoots

a

b

c

bb

bb

b

b

bb

a

b

bbc

cd

bbcbc bc bc

Figure 4. Effects of different Sclerotinia minor isolates on the above- and below-ground

biomass of dandelion after two consecutive spot applications of 0.2 g/plant of S. minor

colonized barley granules. Error bars represent the standard errors of the means (average of 16

plant replicates). Within each group, bars with a common letter are not significantly different

at P�0.05 according to Tukey’s test.

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significantly fewer sclerotia per plate on PDA than all sclerotia-producing isolates.

Non-sclerotia producing isolates were also part of this study.

Diversity in fungal populations can be measured by MCGs (Leslie 1993).

Mycelial compatibility in different Sclerotinia species, other than S. minor, has been

comprehensively studied. The present study demonstrates for the first time the

occurrence of MCG diversity within S. minor. Results from this study showed that,

this worldwide collection of S. minor isolates is represented by 18 MCGs, seven of

them as multi-member groups. Of the tested isolates, 36.6% were unique isolates that

could not be assigned to any of the eight multi-member MCGs indicating numerous

single-member MCGs may exist. MCG Diversity is the ratio between the number of

MCGs and the total number of isolates (Viji et al. 2005). Our results demonstrate

high diversity (0.6, highest diversity is 1) among the tested isolates of S. minor which

agree with the findings for the closely related species S. sclerotiorum (Kohn et al.

1991). This high diversity among MCGs could be correlated with the wide host range

reported for S. minor (Melzer et al. 1997; Hollowell et al. 2003).

The recovery of an MCG of S. minor in diverse geographical areas (MCG1) or

hosts (MCG4, MCG5 and MCG6) could be attributed to spread by agricultural

practices and other human activities through contaminated soil, crop material, and

equipment (Harlton, Levesque, and Punja 1995; Punja and Sun 2001; Cilliers,

Pretorius, andVanWyk 2002; Durman et al. 2003; Atallah, Larget, Chen, and Johnson

2004). The occurrence of certainMCGs likeMCG3andMCG7 in a specific geographic

region oron the same host species has been reported forSclerotium rolfsii (Nalim, Starr,

Woodard, Segner, and Keller 1995; Punja and Sun 2001; Cilliers et al. 2002).

Virulence assays could help in subdividing populations of fungi into different

groups and isolates within a MCG could have similar virulence (Kull and Pedersen

2004; Viji et al. 2005). In our study, however, results from the virulence tests did not

show a distinct pattern of relationship between virulence and MCG except for three

Sclerotinia minor isolatesIM

I3441

41

S96-18

3

S97-75

S94-00

1

S96-22

SMRF02TH1C LR

CLU

286

R23

Dan

delio

n su

rviv

al (%

)

0

20

40

60

80

100

IMI34

4141

S96-18

3

S97-75

S94-00

1

S96-22

SMRF02TH1C LR

CLU

286

R230

20

40

60

80

100

Partial damageRe-growth after 100% above-ground damage

b

a aa

a

a

a aa

a a

aa

a

a a

b

bc

c

a

A B

Figure 5. Effects of different Sclerotinia minor isolates on re-growth from roots and

dandelion survival. (A) Three weeks after the first application of 0.2 g plant�1 of S. minor

barley based formulation; (B) 3 weeks after the second application of the same rate. Mean

values represent averages of 16 plant replicates. Within each graph, bars with a common letter

are not significantly different at P�0.05 according to Tukey’s test.

72 I.Y. Shaheen et al.

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isolates, S96-22, S96-138 and S97-75, from MCG2 and the isolates of MCG1 where

the lesion diameter ranged between 26 and 30.5 mm. Positive correlation between

virulence in vitro and mycelial growth was not always applicable, since many of the

studied isolates with high growth rate on PDA (Table 2) were hypovirulent and thiscontradicts earlier reports where positive correlation was observed between mycelial

growth and aggressiveness in vitro for isolates of S. sclerotiorum (Boland and Hall

1992; Durman et al. 2003).

In our study, virulence tests on detached dandelion leaves were performed using

two types of inocula, mycelial discs and the barley based formulation. In the mycelial

discs study, measurements were taken 24 and 48 h after inoculation and relative

virulence of the different isolates was ranked according to lesion size after 48 h. IMI

344141 has strong bioherbicidal activity against dandelion (Abu-Dieyeh et al. 2005;Abu-Dieyeh and Watson 2006) and appeared in the moderate virulent group of

isolates. However, when the barley-based formulations of S. minor were tested

on detached dandelion leaves, IMI 344141 was one of the most virulent isolates

(Table 5). When the barley based formulations of the 10 most virulent isolates were

applied onto dandelion plants, IMI 344141 caused the greatest biocontrol effect in

reducing above and below ground biomass (Figures 3�5). These results suggest

caution in relying on detached leaves in virulence assessment of S. minor.

Earlier studies attempted to positively correlate virulence with levels of oxalicacid produced by the fungi Cryphonectria parasitica (Vinnini et al. 1993), Sclerotium

rolfsii (Bateman and Beer 1965; Kritzman et al. 1977), and Sclerotinia sclerotiorum

(Marciano, Magro, and Favaron 1989; Godoy et al. 1990; Boland and Hall 1992;

Zhou and Boland 1999). Conflicting data regarding differences in oxalic acid

production by hypovirulent and virulent fungal isolates have been reported (Havir

and Anagnostakis 1983; Bennett and Hindal 1989). In our study, there were no

significant differences in production of OA among isolates of the same MCG and

there was no correlation between OA production and virulence or growth rate onsolid media since many of the high OA producing isolates had slow growth and were

hypovirulent.

IMI 344141 was clearly distinguished from all other S. minor isolates tested based

on the number of sclerotia produced on PDA. IMI 344141 produced fewer than 100

sclerotiaper platewhile otherS.minor isolates producedmore than 200 andupwards of

700 sclerotia per plate. In conclusion, groups based on morphological or pathological

characters could not be easily related to mycelial compatibility groups and without

molecular marker genotyping, MCGs alone do not suffice to completely characterizepopulations. To this end, molecular characterization of IMI 344141 is ongoing.

Acknowledgements

The authors are grateful to Miron Teshler for his help in various aspects of the project.Financial support from the Natural Sciences and Engineering Research Council of Canada(NSERC) Idea to Innovation (I2I) grant and Sarritor Inc. are gratefully acknowledged. Thefollowing graciously loaned S. minor isolates: G. Abawi, Cornell University, Geneva, USA; G.Boland, University of Guelph; J. Whipps, Warwick Horticulture Research International, UK;H. Huang, Agriculture and Agri-food Canada, Brandon; P. Phipps, Virginia Tech; S. O’Neill,Queensland Dept. Plant Industry, Brisbane; Tamrika Hind-Lanoiselet, Wagga WaggaAgricultural Institute, NSW; S. Morley, Herbarium VPRI, Victorian Department of PrimaryIndustries, Knoxfield, Victoria, Australia; K. Elena, Benaki Phytopathological Institute,Athens, Greece; Alison Stewart, Lincoln University, Christchurch, New Zealand; Levente

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Kiss, Hungarian Plant Protection Institute, Budapest; P. Charue, Belgian CoordinatedCollections of Microorganisms, Louvain; J. Tatnell and M. Fuhlbohm, Australian Depart-ment of Plant Industry, Kingaroy, QSLD; W.G. Kim, National Institute of AgriculturalScience and Technology, Suwon, Republic of Korea; F. Snippe-Claus, Centraalbureau voorSchimmelcultures, Utrecht, The Netherlands.

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