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Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
41
Characterization of Rhizoctonia solani Isolates Associated
with Patch Diseases on Turfgrass
Stacy R. Blazier
1
and Kenneth E. Conway
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater,
OK 74078-3032
Cultural characteristics and pathogenicity of Rhizoctonia solani isolates obtained
from brown patch on creeping bentgrass, Agrostis pulustris Huds. and large patch on
zoysiagrass, Zoysia japonica Steud, were evaluated and compared with known R. solani
anastomosis groups: AG-2-2III-B, AG-2-2IV, AG-1-IA, AG-4, and AG-5. Bentgrass and
zoysiagrass isolates were obtained from infected grass leaf sheaths along disease patch
margins. The bentgrass and zoysiagrass isolates differed culturally from one another.
The bentgrass isolate and the AG-2-2IIIB tester both showed irregular clusters of myce-
lia (not sclerotia), concentric zonation, dark brown main hyphae, and sparse aerial hy-
phae on potato dextrose agar after two weeks of incubation at 22°C, 12h/12h light/dark.
These two isolates caused high levels of disease on creeping bentgrass cv. Crenshaw in
in vitro pathogenicity tests. The zoysiagrass isolate most closely matched R. solani AG-
2-2IV in both cultural characteristics and pathogenicity on creeping bentgrass cv.
Crenshaw. The zoysiagrass isolate and the AG-2-2IV tester both had abundant aerial
hyphal growth, dark brown main hyphae, and no sclerotial formation or zonation on
potato dextrose agar after two weeks of incubation. Optimum temperature for growth of
both isolates was 25°C but unlike the bentgrass isolate and the AG-2-2IIIB tester, the
zoysiagrass isolate and the AG-2-2IV tester did not grow at 35°C. The zoysiagrass isolate
and the AG-2-2 IV tester caused low levels of disease on creeping bentgrass cv. Crenshaw
in in vitro pathogenicity tests. Results indicate that cultural characteristics and host
range of the bentgrass isolate and those of the zoysiagrass isolate are different. Isolates
representing R. solani AG-2-2IIIB and AG-2-2IV were tested for sensitivity to azoxystrobin
in in vitro tests. Sensitivity to azoxystrobin (effective concentration causing 50% growth
inhibition [EC
50
]) was determined by radial growth on potato dextrose agar amended
with 0, 1, 3.2, 10, 31.2, 100, 316, and 1000 mg a.i. azoxystrobin /L after three days incuba-
tion at 22°C. EC
50
values for AG-2-2 IIIB isolates averaged approximately 193 mg a.i.
azoxystrobin/L while those for AG-2-2IV isolates averaged approximately <1 mg a.i.
azoxystrobin/L. Results suggest there is some variability in fungicide sensitivity be-
tween and within R. solani AGs and that R. solani AG-2-2IIIB isolates may be less sensi-
tive to azoxystrobin fungicide than AG-2-2IV isolates. © 2004 Oklahoma Academy of
Science
1
Former Graduate Assistant, current address: 125 Hill
Farm Lane, Homer, LA 71040
INTRODUCTION
Rhizoctonia solani Kuhn is a worldwide, eco-
logically diverse soilborne fungus belong-
ing to Order Ceratobasidiales of the
Basidiomycota and is the mycelial or imper-
fect state of Thanatephorus cucumeris (A. B.
Frank) Donk. Relationships within the spe-
cies Rhzoctonia solani are very complex and
confusing. Identification is based on anas-
tomoses groups (Ag) among isolates and
cultural characteristics. Important taxo-
nomic characteristics of the species include:
(1) the absence of asexual spores, or conidia;
(2) the absence of clamp connections, or
structures involved in genetic recombina-
tion; (3) the absence of rhizomorphs; (4)
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
42
small (< 1mm), round, dark brown sclerotia
that may or may not be present; (5) multi-
nucleate hyphal cells; (6) pigmented myce-
lia (shades of brown); (7) right-angled hy-
phal branching; (8) septum formation in
hyphal branches near points of hyphal ori-
gin; (9) presence of a dolipore septum; and
(10) an optimum growth temperature of 20
to 30
o
C (Baker 1970, Parmeter and Whitney
1970, Brown and McCarter 1976, Anderson
1982).
The host range of R. solani is extensive.
The pathogen is capable of causing seed-
ling damping-off, root rot, collar rot, stem
canker, crown rot, bud and fruit rots, and
foliage blight on a variety of susceptible
agriculturally important crops ( Baker 1970,
Anderson 1982) like soybean (Glycine max
(L.) Merr.; Liu and Sinclair 1991), cotton
(Gossypium hirsutum L.; Brown and
McCarter 1976), canola (Brassica campestris
L.; Yitbarek et al 1987), wheat (Triticum
aestivum L.; Wiseman et al 1995), beet (Beta
vulgaris L.; Carling et al 1987), potato
(Solanum tuberosum L. subsp. tuberosum;
Escande and Echandi 1991), and rosemary
(Rosemarinus officinalis L.; Conway, et al
1997). Rhizoctonia solani also infects a num-
ber of turfgrass species (Couch 1995). The
fungus was first identified as the causal
agent of a disease known as Rhizoctonia
blight (brown patch) on creeping bentgrass
(Agrostis palustris Huds.) in 1913 (Burpee
and Martin 1992, Couch 1995) and has since
become regarded as one of the most destruc-
tive diseases of both warm- and cool-sea-
son turfgrasses including zoysiagrasses
(Zoysia spp. Willd; Burpee and Martin 1992,
Couch 1995, Aoyagi et al 1998), tall fescue
(Festuca arundinacea Schreb.; Burpee and
Martin 1992, Couch 1995, Aoyagi et al 1998),
Kentucky bluegrass (Poa pratensis L.; Couch
1995, Aoyagi et al 1998), centipedegrass
(Eremochloa ophiuroides (Munro) Hack.;
Couch 1995, Aoyagi et al 1998), and creep-
ing bentgrass (Burpee and Martin 1992,
Couch 1995, Aoyagi et al 1998). Rhizoctonia
blight on cool-season grasses such as
bentgrass is called “brown patch” and on
warm-season grasses like zoysiagraass, the
disease is referred to as “large patch”
(Aoyagi et al 1998, Hyakumachi et al 1998)
because of slight differences in symptoma-
tology, time of year of disease outbreaks, and
R. solani isolate cultural morphology (Couch
1995, Hyakumachi et al 1998).
Variability in disease symptoms, host
range, and geographical location of R. solani
isolates suggests that there are several
strains of the species (Burpee and Martin
1992). As of 1994, 12 different strains of the
fungus (AG-1 through AG-11 and AG-BI)
(Carling et al 1994) have been recognized
based on affinities for hyphal fusion (anas-
tomosis), a genetic feature that results in
exchange of nuclei and the combining of
different genotypes (Kataria et al 1991,
Burpee and Martin 1992). Anastomosis
groups are categorized based on their myce-
lial compatibilities for hyphal fusion. Anas-
tomosis occurs between fungal isolates of
the same AG but not between isolates of
different AG’s. Each AG therefore seems to
be genetically independent from all others
(Parmeter et al 1969, Ogoshi 1985, Burpee
and Martin 1992).
Anastomosis groups appear to be fairly
host plant specific. For instance, AG-3 oc-
curs commonly on Solanaceae and AG-4 is
regularly associated with Pinaceae,
Chenopodiaceae, Cruciferae, Leguminosae,
Malvaceae, and Solanaceae (Butler 1993).
Four anastomosis groups of R. solani, AG-1
(specifically, subgroup IA on cool-season
turf in Japan), AG-2, AG-4, and AG-5, have
been isolated from turfgrasses (Burpee and
Martin 1992, Aoyagi et al 1998). Subgroup
2 of AG-2 has been consistently associated
with Rhizoctonia blight of turfgrasses
(Burpee and Martin 1992, Green et al 1993,
Zhang and Dernoeden 1995). Reports indi-
cate that brown patch on cool-season turf is
typically caused by intraspecific group IIIB
of subgroup AG-2-2, while large patch on
warm-season turf is incited by intraspecific
group IV of subgroup AG-2-2 (Burpee and
Martin 1992, Green et al 1993, Zhang and
Dernoeden 1995; however, Aoyagi, et al
S.R. BLAZIER and K.E. CONWAY
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
43
(1998) established that this isolate should be
included in a new group, AG-2-2LP for
large patch on Zoysiagrass. Type IIIB is
usually associated with infections of foliar
portions of family Poaceae, while type IV
primarily causes root rots of the
Chenopodiaceae (Burpee and Martin 1992).
Several fungicides have been labeled for
control of Rhizoctonia blight including
flutolanil (Prostar, AGREVO Corporation,
Wilmington, DE), propiconazole [Banner,
Ciba-Geigy Corporation, (Novartis) Greens-
boro, NC], fenarimol (Rubigan, Dow-Elanco
Specialty Products, Indianapolis, IN),
iprodione (Chipco 26109, Rhone-Poulenc
AG Company, Research Triangle Park, NC),
chlorothalonil (Daconil 2787, ISK Biotech
Corporation, Mentor, OH), quintozene
(Terraclor, Uniroyal Chemical Co.,
Middlebury, CT), mancozeb (Fore, Rohm
and Haas Co., Philadelphia, PA) (Couch
1995), and azoxystrobin (Heritage 50WDG,
Zeneca Agrochemicals, Jealott’s Hill Re-
search Station, Bracknell, Berkshire, RG42
6ET, UK). Azoxystrobin is a new beta-
methoxyacrylate fungicide that has been
used for control of several ascomycete, ba-
sidiomycete, and oomycete fungal diseases
on such crops as cereals, cucurbits, veg-
etables, fruits, peanuts, ornamentals, rice,
potatoes, and turf. Azoxystrobin is a deriva-
tive of the chemically similar strobilurins, a
class of naturally occurring fungicides pro-
duced by Strobilurus tenacellus, a wood-de-
caying fungus of the mushroom family
Tricholomataceae (Dernoeden 1998,
Aspinall and Worthington 1999). The fun-
gicide has broad spectrum activity with
protectant and acropetal systemic capabili-
ties, meaning the chemical can be taken up
by plant xylem and then move upward in
the transpiration stream. The chemical is
also effective for controlling established in-
fections and can be absorbed by both roots
and leaves. Azoxystrobin interferes with
cellular respiration in sensitive fungal
pathogens by inhibiting transport of mito-
chondrial electrons (Aspinall and
Worthington 1999).
It is not known whether these fungi-
cides are effective against all R. solani AGs
or just a select few. Kataria et al (1991) tested
different fungicides against various isolates
of several anastomosis groups and found
variability in fungicide sensitivity between
and within AGs. Knowledge of which AGs
are involved in a given Rhizoctonia blight
outbreak and their sensitivities to different
fungicides may help to facilitate selection of
the most appropriate fungicide for manage-
ment of the disease in any particular area or
situation.
Because zoysiagrass is often planted
around the shoulders of bentgrass greens
this study was conducted to classify R. solani
isolates from brown patch on creeping
bentgrass and large patch on zoysiagrass
into appropriate AGs based on observations
of cultural and pathogenicity characteristics
and comparisons with those of known anas-
tomosis testers; and to evaluate the AGs
commonly associated with Rhizoctonia
blight for sensitivity to azoxystrobin in vitro.
MATERIALS AND METHODS
Collection and Isolation. Grass leaf sheaths
and blades with symptoms of brown patch
and large patch were collected from creep-
ing bentgrass and zoysiagrass, respectively,
at the Horticulture Turfgrass Research Cen-
ter in Stillwater, Oklahoma, in 1997.
Samples were taken from the extreme mar-
gins of patch areas with forceps and trans-
ported to the laboratory in polyethylene
bags. Sections of necrotic, straw-colored tis-
sue were removed from infected plant ma-
terial and were surface sterilized with 10%
Clorox (The Clorox Company, Oakland, CA)
for 1 min, plated onto potato dextrose agar
(PDA, Difco Laboratories, Detroit, MI)
amended with 0.3 g/L streptomycin sulfate
(Sigma Chemical Co., St. Louis, MO) con-
tained in petri dishes, and incubated at 22°C
(12-h light/12-h dark regime). After 48 h of
incubation, hyphal tips from each isolate
were transferred to fresh PDA petri dishes
(100 x 15 mm). Following another 48 h of
PATCH DISEASES ON TURFGRASS
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
44
incubation, hyphal tips were transferred to
PDA slants and stored in a fungal culture
collection at 11°C. The bentgrass isolate des-
ignated as #345, and the one from
zoysiagrass as #414. Rhizoctonia solani iso-
lates #96, #300, #309 wer e obtained from
stock slant cultures from Dr. Kenneth E.
Conway’s laboratory (Oklahoma State Uni-
versity, NRC Rm 326, Department of Ento-
mology and Plant Pathology, Stillwater, OK,
74078) fungal culture collection. These iso-
lates were originally obtained from R. J.
Cook (United States Department of Agricul-
ture, Agricultural Research Service Pullman,
WA) and were chosen for the experiment
because they are representatives of the fol-
lowing AGs: AG-4, AG-1-IA, and AG-5, re-
spectively. Also chosen for the experiment
were R. solani isolates #410, a cool season
isolate from zoysiagrass and #411, isolated
from warm season zoysiagrass and in Kan-
sas and presumably representing anasto-
mosis intraspecific groups AG-2-2IV, and
AG-2-2IIIB, respectively. These two isolates
were obtained from N. A. Tisserat (Kansas
State University, Manhattan, KS). Accord-
ing to Dr. Tisserat (pers. comm.), isolate #410
was not associated with warm-season patch
disease and isolate #411 was isolated from
zoysiagrass in the Kansas City area and was
associated with cool weather (fall and
spring) injury to Zoysiagrass. Mycelial frag-
ments were removed from the slant cultures,
plated onto PDA, and were incubated as
above for use in subsequent experiments.
Cultural characteristics. R. solani iso-
lates #345 and #414 were identified based
on comparisons of cultural characteristics,
hyphal anastomosis, and number of nuclei
per hyphal cell with those of the known AG
testers R. solani #96, R. solani #300, R. solani
#309, R. solani #410, and R. solani #411. Five
PDA cultures per isolate were observed for
colony color, sclerotial formation, growth
zonation, and aerial mycelium after incuba-
tion for two weeks at 22°C, 12-h light/12-h
dark and the experiment was repeated once.
To confirm the multinucleate condition,
mycelia from each isolate were removed
from PDA cultures and teased apart on a
clean glass slide. Hyphae were stained with
acridine orange (Sigma Chemical, St. Louis,
MO; Dhringra and Sinclair 1985) and ob-
served using epifluorescence under an ul-
traviolet microscope. Following a modified
procedure described by Aoyagi et al (1998),
we observed 15 hyphal cells per isolate for
the multinucleate condition in two separate
experiments.
Hyphal anastomosis. Hyphal anasto-
mosis reactions were observed by remov-
ing mycelial plugs (0.75 cm in diameter) of
isolates #345 and #414 from actively grow-
ing week old PDA cultures and pairing them
with mycelial plugs of tester isolates of
known AG having the same cultural char-
acteristics as isolates #345 and #414. Tests
were conducted on 2% reverse osmosis (RO)
water agar in 100 x 15 mm petri dishes fol-
lowing modified procedures of Parmeter et
al (1969), and Zhang and Dernoeden (1995).
Petri dishes were incubated at 22°C until
hyphae from paired isolates began to over-
lap (two or three days). Overlapping hy-
phae were then stained with lacto-fuchsin
red (Carmichael 1955, Escande and Ecandi
1991) and were observed for two types of
anastomosis reactions by using light micros-
copy at 400X magnification. Perfect anas-
tomosis occurs between hyphae from the
same isolate or a genetically identical iso-
late (clone) and is characterized by complete
fusion of cell walls and cytoplasm. Imper-
fect anastomosis is the result of cell wall fu-
sion but exchanged cytoplasm does not re-
main viable as with perfect anastomosis
(Yokoyama et al 1985a, 1984b, Wilkinson
1988, Aoyaki et al 1998).
Ten water agar plates per pairing were
observed for 25 points of contact each. Pair-
ings of mycelial plugs from the same iso-
late were designated as control reactions. A
pair of separate isolates was considered ge-
netically identical if more than 80% of fu-
sion contacts were perfect anastomoses
(Aoyagi et al 1998).
Temperature-growth experiment.
Mycelial plugs (0.75 cm in diameter) were
S.R. BLAZIER and K.E. CONWAY
°
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
45
cut from actively growing 2-d-old PDA cul-
tures of isolates #345 (brown patch), #414
(large patch), #411 (AG-2-2IIIB), and #410
(AG-2-2IV) and plated onto new PDA petri
dishes. Five replicate petri dishes for each
isolate were incubated in the dark at 21, 25,
26, 30, and 35°C. Two perpendicular colony
diameters were measured on the bottom of
each plate after 24-h incubation. Agar plug
diameters were subtracted from every mea-
surement. The two colony diameters for
each plate were averaged, and a mean
growth rate was calculated from the five
replicate plates for each temperature. The
test was conducted twice with similar re-
sults.
Pathogenicity tests using Conetainers.
All R. solani isolates were tested for patho-
genicity to creeping bentgrass cv. Crenshaw.
Inoculum was produced by adding one 100
x 15 mm PDA culture, chopped into ap-
proximately 1 cm
2
pieces, to 250-ml Erlen-
meyer flasks containing 20 g oat seed and 3
ml RO water that had been autoclaved
(121°C, 1.05 kg/cm
2
, 20 min) on each of three
consecutive days. Flasks containing oat seed
were incubated at 22°C (12-h light/12-h
dark regime) and after 14 da the colonized
seed was removed and dried overnight un-
der a laminar-flow hood.
Creeping bentgrass seed was obtained
from Lofts Great Western Seed Company
(Albany, OR). Pathogenicity of R. solani iso-
lates on creeping bentgrass was tested in
plastic Conetainers (Stuewe & Sons, Inc.,
Corvallis, OR) 3 cm in diameter and 21 cm
deep following a modified procedure pre-
viously described by Wilkinson (1988).
Conetainers were filled with 100 ml of au-
toclaved vermiculite (W. R. Grace & Co.,
Cambridge, MA) and seeded with 0.25 g
bentgrass seed that had been surface steril-
ized with 10% NaOCl for 1 min. Seeds were
covered with a thin layer of vermiculite,
watered every other day with a Peter’s 20-
20-20 solution (W. R. Grace & Co.,
Fogelsville, PA), and maintained in a growth
chamber (12-h light/12-h dark, 20 to 22°C).
Conetainers were covered with plastic wrap
to maintain 100% relative humidity.
Two weeks after planting, the bentgrass
was cut to a height of 1 cm with sterile scis-
sors. One infested oat seed was then intro-
duced aseptically to the vermiculite surface
of each conetainer except for the controls,
which were inoculated with noninfested oat
seed. Inoculated bentgrass was then placed
in a growth chamber (15-h light/9-h dark,
20 to 25°C) and kept moist (100% relative
humidity) with plastic covering. Inoculated
bentgrass was watered every other day with
Peter’s 20-20-20 solution. Disease was rated
2 wk after inoculation using the disease in-
dex described by Aoyagi et al (1998), where
0 = healthy, 1 = 1 to 25% diseased, 2 = 26 to
50% diseased, 3 = 51 to 75% diseased, and 4
= 76 to 100% diseased. Disease severity was
calculated as
disease index x no. of inoculated
grass samples in each index
maximum index x total no.
of inoculated grass samples
All treatments consisted of four repli-
cates and tests were conducted two times
with similar results.
Fungicide variability tests. Aliquots of
a stock solution of azoxystrobin dissolved
in molten 3/4 strength PDA were added to
subsequent molten 3/4 strength PDA to
obtain final concentrations of 1, 3.2, 10, 31.2,
100, 316, and 1000 mg a.i. azoxystrobin per
L medium (ppm). These concentrations re-
sulted in equal spacing on a log
10
scale
(Keinath and Zitter 1998). Three-quarter
strength PDA plates without fungicide were
used as controls. Mycelial plugs 0.75 cm in
diameter were cut from actively growing
cultures of the fungal isolates and placed
inverted either onto control or fungicide-
amended plates, each containing 20 ml of
agar medium. Eight replicate plates were
used for each concentration. After 3 da of
growth at 22°C (12-h light/12-h dark re-
gime), two perpendicular colony diameters
Σ
x 100
][
PATCH DISEASES ON TURFGRASS
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
46
were measured on the bottom of each plate.
Agar plug diameters were subtracted from
every measurement. The two colony diam-
eters for each plate were averaged and a
mean diameter was calculated from the
eight replicate plates. Percent radial growth
inhibition was calculated as
mean dia. on unamended
PDA – mean dia
on fungicide – amended PDA
mean dia on unamended PDA
Azoxystrobin dose response curves
were constructed for the R. solani isolates by
plotting probit-transformed (Zadoks and
Schein 1979) percent radial growth inhibi-
tion against log-transformed fungicide con-
centration. The concentration of
azoxystrobin causing 50% growth inhibition
compared to growth on unamended PDA
(EC
50
) was estimated for each isolate by in-
terpolation from the fitted regression line
(second-degree polynomial) using SAS re-
gression. The activity of azoxystrobin fun-
gicide was considered to be strong if the EC
50
was <10 mg a.i./L, moderate if the EC
50
was
11 - 100 mg a.i./L, weak if the EC
50
was 101-
1000 mg a.i./L, and ineffective if the EC
50
was >1000 mg a.i./L. The experiment was
repeated once with similar results.
Statistical analysis. Data from the
pathogenicity experiments were subjected
to analysis of variance using a general lin-
ear model (GLM), and mean separation was
determined with Duncan’s multiple range
test (P≤0.05) (SAS, version 6.10, SAS Insti-
tute Inc., Cary, NC). Fungicide variability
data were analyzed using SAS regression.
RESULTS
Cultural characteristics. R. solani isolate
#345 obtained from brown patch on creep-
ing bentgrass in Stillwater, Oklahoma was
most similar to the AG-2-2IIIB tester isolate
#411. Both isolates were buff in color early
in the growth development stage but turned
to a dark brown color within two weeks.
Isolates #345 and #411 also had irregular
clusters of mycelia (not sclerotia), zonation
or concentric rings, and sparse aerial hyphae
on PDA after two weeks of incubation (Table
1). Rhizoctonia solani isolate #414 obtained
X 100
][
Table 1. Cultural characteristics of Rhizoctonia solani isolates commonly associated with
turfgrasses
Rhizoctonia Anastomosis Colony Nuclear Aerial
isolate group Color
1
condition
2
mycelium Sclerotia Zonation
#96 (T)
4
AG-4 B-DB
3
>2 Absent Present No
#300 (T) AG-1-IA C >2 Absent Present No
#309 (T) AG-5 B >2 Absent Present No
#345 (BG) AG-2- B-DB >2 Absent Absent Yes
2IIIB
#410 (ZG) AG-2- DB >2 Present Absent No
2IV
#411 (BG) AG-2- B-DB >2 Absent Absent Yes
2IIIB
#414 (ZG) AG-2- DB >2 Present Absent No
2IV
1
Cultures grown on PDA at 22°C for two weeks.
2
Multinucleate condition is a distinguishing characterisitic of R. solani.
3
Colony color designations: C = cream, B = buff, DB = dark brown.
4
BG = bentgrass isolate, ZG = zoysiagrass isolate, T = tester culture.
S.R. BLAZIER and K.E. CONWAY
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
47
from large patch on zoysiagrass most closely
resembled the AG-2-2IV tester isolate #410.
Both isolates were dark brown early in
growth and remained that color after two
wk. These two isolates exhibited abundant
aerial mycelia and neither had sclerotial for-
mation or zonation patterns (Table 1).
Hyphal anastomosis. Known anasto-
mosis testers #410 and #411 were chosen for
hyphal anastomosis tests with unknown AG
isolates #345 and #414, respectively, based
on identical characteristics in culture. When
isolates were paired with self as control re-
actions, 100% perfect anastomosis was ob-
served (Fig. 1). It was determined that R.
solani isolates #345 (from creeping
bentgrass) and #414 (from zoysiagrass) are
not identical strains (perfect fusion fre-
quency of only 8%) but belong to separate
anastomosis intraspecific groups AG-2-2
IIIB and AG-2-2 IV, respectively. Perfect fu-
sion was observed among 94.4% of hyphal
fusions between isolates #345 and #411 (AG-
2-2 IIIB). When #345 was paired with the
representative AG- 2-2 IV tester #410, mean
perfect anastomosis frequency of 3.33% was
obtained. Pairings between isolates #414
and #410 (AG-2-2 IV) resulted in a mean
perfect fusion frequency of 84.6%. Pairings
between isolate #414 and the representative
AG-2-2 IIIB tester #411 resulted in a mean
perfect fusion frequency of only 4.4%.
Temperature-growth of R. solani iso-
lates. Rhizoctonia solani isolates #345 and
#411 grew at all five temperatures tested .
The optimum temperature of these two iso-
lates was 25°C, with mean colony diameters
of 1.52 cm and 1.67 cm, respectively. This
evidence lends support to the earlier con-
clusion made by observations of cultural
Figure. 1. Pathogenicity of Rhizoctonia solani turfgrass isolates on creeping bentgrass
cv. Crenshaw grown in conetainers. Disease severity = S (disease index x the number of
grass samples in each index)/(maximum index x the total number of grass samples) x 100
(Aoyagi et al 1998). Disease index of brown patch was rated two weeks after incubation
at 20 to 25°C using a scale of 0 to 4, where 0 = no symptoms and 4 = dead grass. Isolate
#96 = AG-4; #300 = AG-1-IA; #309 = AG-5, #345 = AG-2-2IIIB; #410 = AG-2-2IV ; #411 =
AG-2-2IIIB; #414 = AG-2-2IV. Column values having the same letter(s) do not differ
significantly (P≤0.05) according to Duncan’s multiple range test.
#96 #300 #309 #345 #410 #41 1 #414 control
Isolate
b
bc
b
a
cd
a
cd
d
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Disease severity (%)
PATCH DISEASES ON TURFGRASS
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
48
characteristics and anastomosis reactions
that isolate #345, like #411, belongs to AG-
2-2IIIB. Isolates #414 and #410 also had
growth rate optima at 25°C, with mean
colony diameters of 0.94 cm and 1.30 cm,
respectively. This evidence further con-
firmed conclusions from earlier experiments
that both isolate #414 and #410 were repre-
sentatives of AG-2-2IV.
Pathogenicity tests using Conetainers.
Rhizoctonia solani isolates #345 (bentgrass
isolate) and #411 (warm season zoysia iso-
late) caused the highest levels of disease on
creeping bentgrass compared to all of the
other isolates (Fig. 1). Initial leaf symptoms
observed were small, tan lesions that en-
larged and became surrounded by reddish
brown margins over time. Eventually grass
leaves became necrotic and brown in color.
These symptoms were similar to symptoms
of brown patch on creeping bentgrass un-
der field conditions. Zoysiagrass isolates
#410 and #414 were the least aggressive
pathogens to bentgrass. Moderate levels of
disease were produced by the tester isolates
#96, #300, and #309. All uninoculated con-
trol bentgrass remained healthy. Koch’s
postulates were tested and R. solani was iso-
lated from all treatments except the control.
Fungicide variability tests. The four R.
solani isolates from AG-2-2 IIIB and AG-2-
2IV grew at all seven azoxystrobin concen-
trations after 3 da (Fig. 2). Isolates #345 and
#411 (both AG-2-2 IIIB) had similar re-
sponses to azoxystrobin. These isolates
were slightly less sensitive to the fungicide
than isolates #410 and #414 (AG-2-2IV). At
1 mg a.i. azoxystrobin/L, isolate #345
growth was inhibited by only 16% (probit =
4.01) and isolate #411 was inhibited by 40%
(probit = 4.75). Isolates #410 and #414 had
similar responses to azoxystrobin. Isolate
#410 growth was inhibited by 51% (probit
= 5.03) while isolate #414 growth was in-
hibited by 61% (probit = 5.28). At 1000 mg
a.i. azoxystrobin/L, isolates #345 and #411
Figure 2. Dose-response curves for four isolates of Rhizoctonia solani to azoxystrobin
fungicide. Percent inhibition (relative colony diameter) = (diameter on unamended
medium – diameter on azoxystrobin – amended medium)/(diameter on unamended me-
dium) X 100. R. solani isolates are as follows: #345 = AG-2-2IIIB; #411 = AG-2-2IIIB;
#410 = AG -2-2IV; and #414 = AG-2-2IV. The 50% effective concentrations were approxi-
mately 355 mg a.i. azoxystrobin/L and 31.2 mg a.i. azoxystrobin/L for isolates #345 and
#411, respectively, and <1 mg a.i. azoxystrobin/L for isolates #410 and #414.
0 0.5 1 1.5 2 2.5 3
Azoxystrobin concentration (log
10
[mg/L])
#410 (R
2
= 0.7336)
#345 (R
2
= 0.7975)
#411 (R
2
= 0.9336)
#414 (R
2
= 0.9065)
Probit-transformed percent inhibition
6
5
4
S.R. BLAZIER and K.E. CONWAY
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
49
(AG-2-2IIIB) again demonstrated slightly
lower sensitivity than the AG–2-2IV isolates
(#410 and #414). Isolates #345 and #411
were inhibited by 66% (probit=5.41) and
70% (probit = 5.52), respectively. Isolates
#410 and #414 wer e inhibited by 82% (probit
= 5.92) and 84% (probit = 5.99) at 1000 mg
a.i. azoxystrobin/L, respectively. The fun-
gicide concentration in the recommended
label rate is approximately 1500 mg a.i.
azoxystrobin/L. Our results indicate that
the four R. solani isolates are sensitive to fun-
gicide at 1000 mg a.i. azoxystrobin/L, all
showing >60% growth inhibition; therefore,
in field situations, the label rate should be
effective for inhibiting of the growth of these
four isolates.
The azoxystrobin concentrations that
reduced radial growth of isolates by 50%
(EC
50
) were determined to be approximately
355 mg a.i. azoxystrobin/L and 31.2 mg a.i.
azoxystrobin/L for isolates #345 and #411
(both AG-2-2 IIIB), respectively, and <1 mg
a.i. azoxystrobin/L for isolates #410 and
#414 (both AG-2-2 IV). There is some vari-
ability in fungicide sensitivity between
strains of R. solani and between isolates of
AG-2-2 IIIB. In this case, AG-2-2 IIIB iso-
lates from brown patch were less sensitive
to azoxystrobin than AG-2-2IV isolates from
large patch.
DISCUSSION
Rhizoctonia blight on cool- and warm-sea-
son turfgrasses may be caused by two sepa-
rate intraspecific groups of R. solani, specifi-
cally AG-2-2 IIIB and AG-2-2 IV, respectively
(Burpee and Martin 1992, Zhang and
Dernoeden 1995, Aoyaki et al 1998,
Hyakumachi et al 1998). However, three
other strains of R. solani (AG-1-IA, AG-4, and
AG-5) have also been associated with
Rhizoctonia blight infections but with less
consistency than strains AG-2-2IIIB and AG-
2-2IV (Anderson 1982, Burpee and Martin
1992). We wanted to confirm AGs of R. solani
isolates, #345 (BG) and #414 (Z), fr om brown
patch on creeping bentgrass and large patch
on zoysiagrass in Oklahoma, respectively,
by comparing their cultural and pathoge-
nicity characterisitics with those of known
anastomosis turfgrass isolates.
Zhang and Dernoeden (1995) suggested
that anastomosis classification by the tradi-
tional means of microscopic observation of
hyphal pairings is tedious and time-con-
suming and that simple observations of cul-
tural characteristics such as colony color,
presence or absence of sclerotia, presence or
absence of zonation patterns, and type of
mycelial growth are usually reliable enough
to tentatively classify isolates into anasto-
mosis groups. In this study, we found that
observations of cultural characteristics of
our isolates and comparisons of those char-
acteristics with isolates of known anastomo-
sis grouping were quite dependable in clas-
sifying our isolates (#345 and #414) into
their respective AGs. Of the five known
anastomosis tester turfgrass isolates used
(AG-2-2IIIB, AG-2-2IV, AG-1-IA, AG-4 and
AG-5), isolate #345 from brown patch most
closely matched the AG-2-2IIIB (isolate
#411) tester in cultural characteristics while
isolate #414 from large patch most closely
matched the AG-2-2IV (isolate #410) tester.
To further confirm our AGs, we chose to
observe hyphal anastomosis reactions be-
tween pairings of the brown patch isolate
(#345) and the large patch isolate (#414)
with the tester isolates AG-2-2 IIIB (#411)
and AG-2-2IV (#410), respectively, using
light microscopy.
In this study, imperfect fusion was not
observed in positive control pairings be-
tween identical isolates and was observed
only infrequently between different isolates
of the same anastomosis grouping. Follow-
ing the relationship defined by Aoyagi et al,
(1998), we considered the relationship be-
tween isolates as clonal (i.e., identical AG)
if the frequency of perfect fusion was greater
than 80%. In pairings of brown patch iso-
late #345 with the AG-2-2IIIB tester (#411)
and of large patch isolate #414 with the AG-
2-2IV tester (#410), we observed >80% per-
fect fusion frequency, lending support to our
PATCH DISEASES ON TURFGRASS
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
50
earlier conclusions of AG of isolates #345
and #414 based on cultural characteristics
alone.
Butler (1993) stated that AGs appear to
be plant host specific. We wanted to exam-
ine whether there were any differences in
pathogenicity to creeping bentgrass cv.
Crenshaw between the R. solani turfgrass
isolates. Our results showed that pathoge-
nicity varies with AG. We found that iso-
late #345 (AG-2-2IIIB) and isolate #411 (AG-
2-2IIIB tester) were most pathogenic on
creeping bentgrass cv. Crenshaw while iso-
lates #414 (AG-2-2IV) and #410 (AG-2-2IV
tester) were least pathogenic to the grass.
We observed moderate pathogenicity by iso-
lates #96 (AG-4 tester), #300 (AG-1-IA
tester), and #309 (AG-5 tester). Even though
AG-4, AG-1-IA, and AG-5 may be capable
of causing average levels of disease, AG-2-
2 IIIB isolates appear to be more pathogenic
on creeping bentgrass cv. Crenshaw.
Kataria et al (1991) have documented
and demonstrated that there is variability
in fungicide sensitivity within and between
AGs because of differences in molecular and
biochemical characteristics. It has been sug-
gested that knowledge of fungicide sensi-
tivity levels between and within AGs is use-
ful in selecting appropriate fungicides for
reliable and efficient control of R. solani dis-
eases (Kataria et al 1991, Zhang and
Dernoeden 1995). An analysis of AG sensi-
tivity to fungicides allows us to draw firm
conclusions about the consistency or vari-
ability of performance of a fungicide both
within and between AGs. We wanted to
determine if such variations in sensitivity
to azoxystrobin, a common fungicide used
to control Rhizoctonia blight on cool- and
warm-season turfgrasses, were evident be-
tween strains AG-2-2IIIB from brown patch
and strains AG-2-2IV from large patch and
within isolates of strains AG-2-2IIIB and AG-
2-2IV. We found that fungal isolates #410
and #414 belonging to AG-2-2IV (large
patch) were more sensitive to azoxystrobin
fungicide than fungal isolates #345 and #411
belonging to AG-2-2IIIB (brown patch). The
AG-2-2IV isolates (#410 and #414) demon-
strated consistent azoxystrobin sensitivity,
both having EC
50
values of <1 mg a.i.
azoxystrobin/L. There was, however, vari-
ability in fungicide sensitivity between iso-
lates #345 and #411 representing AG-2-2IIIB.
Isolate #345 was less sensitive (EC
50
= 355
mg a.i. azoxystrobin/L) than isolate #411
(EC
50
=3 1.2 mg a.i. azoxystrobin/L). Our
results indicate that azoxystrobin fungicide
may be more effective in controlling Rhizoc-
tonia blight (large patch) on warm-season
turfgrasses than in controlling Rhizoctonia
blight (brown patch) on cool-season
turfgrasses because isolates representing
AG-2-2IV were more sensitive to
azoxystrobin than isolates representing AG-
2-2IIIB. However, our results from isolates
of two major turfgrass anastomosis groups
only approximate azoxystrobin sensitivity
levels of representative R. solani popula-
tions; therefore, it may not be safe to draw
accurate conclusions about the specificity of
these AGs to azoxystrobin fungicide. We
can only speculate that there is variability
in azoxystrobin sensivity within and be-
tween entire AGs. Additional in vitro test-
ing with greater numbers of isolates for each
anastomosis group, and in vivo tests on dis-
eased turfgrass in growth chambers would
be necessary to confirm our findings.
ACKNOWLEDGMENTS
Approved for publication by the Director,
Oklahoma Agricultural Experiment Station.
Mention of a trademark, proprietary prod-
uct or vendor does not constitute a guaran-
tee or warranty of the product by Oklahoma
State University nor imply their approval
to the exclusion of other products or ven-
dors that may be suitable. The authors
thank Lofts Great Western Seed Company
(Albany, OR) for their generous donation of
bentgrass seed used in this study. Cultures
AG-2-2III and AG-2-2IV were provided by
Dr. Ned Tisserat, Department of Plant Pa-
thology, Kansas State University, Manhat-
tan, KS. This manuscript is a portion of a
S.R. BLAZIER and K.E. CONWAY
Proc. Okla. Acad. Sci. 84: pp 41-51 (2004)
51
thesis by the first author submitted in par-
tial fulfillment of the requirements for the
M.S. degree at Oklahoma State University.
Portions of this research were supported by
Hatch appropriations, OKLO 2187, pro-
vided to the second author by the Division
of Agricultural Sciences and Natural Re-
sources, Oklahoma State University,
Stillwater, Oklahoma 74078, and from grant
funds from CREES #97-34103-5036.
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Received: September 22, 2004; Accepted: December 15, 2004
PATCH DISEASES ON TURFGRASS
