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1314 Plant Disease / Vol. 94 No. 11
Characterization of Rhizoctonia spp. Isolates Associated with Damping-Off Disease
in Cotton and Tobacco Seedlings in Greece
C. Bacharis, A. Gouziotis, and P. Kalogeropoulou, Aristotelian University of Thessaloniki, Faculty of Agriculture,
Plant Pathology Laboratory, Thessaloniki, Greece; O. Koutita, Plant Breeding Department of Hellenic Sugar Indus-
try S.A., Thessaloniki, Greece; and K. Tzavella-Klonari and G. S. Karaoglanidis, Aristotelian University of Thes-
saloniki, Faculty of Agriculture, Plant Pathology Laboratory, Thessaloniki, Greece
Rhizoctonia spp. are soilborne patho-
gens attacking many of the agricultural and
horticultural crops throughout the world
and causing damping-off, root rot, foot rot,
stem rot, fruit rot, and foliar blight in more
than 150 plant species (33). In Greece,
Rhizoctonia seedling blight is widespread
throughout the country, causing significant
losses due to pre- and post-emergence
damping-off in field crops such as cotton,
tobacco, canola, and sugar beet. Isolates
within the genus Rhizoctonia vary greatly
in their colony morphology, growth char-
acteristics, and virulence on several hosts
(15,33).
Although a preliminary discrimination
can be achieved by classifying Rhizoctonia
spp. into uninucleate, binucleate, or multi-
nucleate groups based on the number of
nuclei in vegetative hyphal cells, the most
widely used method of classification of
Rhizoctonia spp. is based on grouping the
isolates into anastomosis groups (AGs;
48). Hyphae of isolates will only anasto-
mose each other if they are within the
same AG.. Classification in AGs is valid
for both multinucleate (Thanatephorus)
and binucleate (Ceratobasidium) taxa
within the Rhizoctonia spp. complex. Four-
teen AGs (AG-1 to AG-13) and one sub-
group of bridging isolates AG2-BI have
been described (8,9) in Rhizoctonia solani
Kühn, the anamorph of Thanatephorus
cucumeris (Frank) Donk. The latter iso-
lates have the ability to anastomose with
isolates of several AGs. Seven of the 14
AGs (AG-1, AG-2, AG-3, AG-4, AG-6,
AG-8, and AG-9) have been further subdi-
vided into subgroups based on several
characteristics such as frequency of anas-
tomoses, fatty acid and isozyme profiles,
thiamine requirement, colony morphology,
and host range of the isolates (33,37,48).
Binucleate Rhizoctonia spp. are currently
divided into 19 AGs, designated as AG-A
through AG-S (38).
Rhizoctonia spp. are major threats in
cotton and tobacco production worldwide,
causing damping-off on young seedlings
of both hosts. R. solani also causes target
spot of tobacco, an important foliar disease
(22,47). Despite the significance of the
cotton crop in several regions of the world
and the high economic importance of
Rhizoctonia spp. attacks on this crop, only
a few reports have characterized Rhizocto-
nia spp. isolated from this host. According
to these reports R. solani AG-4 is the pre-
dominant AG (6,26,28). In addition, iso-
lates of R. solani AG-7 (2), AG-13 (8), and
a binucleate Rhizoctonia sp. (26) have
been recovered from diseased cotton seed-
lings. Similarly, in tobacco, the predomi-
nant AG associated with damping-off is the
AG-4 (16,32), while isolates of AG-1 to
AG-5 have also been recovered from to-
bacco (27,32). Isolates of AG-1, AG-2,
AG-4, and AG-5 are associated with damp-
ing-off symptoms while those of AG-3 are
associated with foliar symptoms (target
spot) (10,17,23,27).
Characterization of Rhizoctonia popula-
tions within a cropping region can help in
the development of environmentally
friendly and sustainable crop management
systems. Factors affecting the dynamics,
existence, and distribution of AGs in the
soil include crop rotation, tillage (13,36),
climate, and soil characteristics (14,54).
Although the occurrence of Rhizoctonia
spp. is well documented in Greece, there
are no studies related to the occurrence of
AGs and subgroups of R. solani or binu-
cleate Rhizoctonia spp. Therefore, this
study was initiated to characterize the AGs
of isolates of Rhizoctonia spp. obtained
from cotton and tobacco seedlings grown
on cultivated fields in central and northern
Greece. We examined those isolates based
on (i) nuclear staining, (ii) observation of
hyphal anastomosis with tester isolates,
(iii) sequence analysis of internal tran-
scribed spacer (ITS) region on ribosomal
(r)DNA, (iv) mycelial growth in different
temperatures, and (v) pathogenicity tests
on five crops, including tobacco and cot-
ton.
MATERIALS AND METHODS
Pathogen isolation. A survey was con-
ducted in the main cotton- and tobacco-
cultivating regions of Greece in spring
2007 to obtain isolates of Rhizoctonia spp.
(Fig. 1). In each region, 3 to 15 fields were
inspected for seedlings showing symptoms
of damping-off. Diseased cotton and to-
bacco seedlings were arbitrarily selected at
several sites in each field and transported
ABSTRACT
Bacharis, C., Gouziotis, A., Kalogeropoulou, P., Koutita, O., Tzavella-Klonari, K., an
d
Karaoglanidis, G. S. 2010. Characterization of Rhizoctonia spp. isolates associated with damp-
ing-off disease in cotton and tobacco seedlings in Greece. Plant Dis. 94:1314-1322.
Isolates of Rhizoctonia spp. were obtained during the spring of 2007 from diseased cotton and
tobacco seedlings showing damping-off symptoms. The sampled fields were located in the main
cotton- and tobacco-cultivating regions of central and northern Greece. Among the 79 isolates
obtained from cotton plants, 17 were binucleate and 62 were multinucleate whereas, among the
89 isolates obtained from tobacco plants, 87 were multinucleate and only 2 were binucleate.
Characterization of anastomosis groups (AGs) was performed with hyphal anastomosis reactions
using tester isolates of known AG groups. Anastomosis reactions in cotton mutlinucleate isolates
showed that 54 of them belonged in Rhizoctonia solani AG-4, 6 in AG-7, 1 in AG-2, and 1 in
AG-3. In the 87 tobacco multinucleate isolates, anastomosis reactions showed that 70 of them
belonged in R. solani AG-2, 16 in AG-4, and 1 in AG-5. In addition, molecular characterization
was carried out using the specific ribosomal DNA internal transcribed spacer region, in a ran-
domly selected number of isolates. In cotton, the most prevalent AG was AG-4, with 18 isolates
to the subgroup HG-I, 1 isolate to the subgroup HG-II, and 7 isolates to the subgroup HG-III,
followed by AG-7 (7 isolates), AG-2-1 (1 isolate), and AG-3 (1 isolate). In tobacco, the most
prevalent group was AG-2-1 (70 isolates), followed by AG-4 (6 isolates to the subgroup HG-I
and 5 isolates to the subgroup HG-III) and a single isolate belonging to AG-5. Phylogenetic
analysis showed that the isolates were distinctly separated based on their AG types. Pathogenic-
ity and aggressiveness of the isolates to several hosts was determined. AG-4 isolates from either
cotton or tobacco were the most aggressive on the hosts tested, while AG-2-1 isolates were o
f
moderate aggressiveness and were not pathogenic on barley.
Corresponding author: G. S. Karaoglanidis
E-mail: gkarao@agro.auth.gr
Accepted for publication 28 June 2010.
doi:10.1094 / PDIS-12-09-0847
© 2010 The American Phytopathological Society
Plant Disease / November 2010 1315
to the laboratory in individual polyethylene
bags to prevent cross contamination.
The surface of symptomatic roots and
hypocotyls was disinfected with a 10%
sodium hypochlorite solution for 1 min
and washed three times with sterilized
distilled water. Pieces from the diseased
roots and hypocotyls were placed on 1.5%
water agar, acidified with lactic acid (10%,
pH 4.5) and incubated at 25°C in the dark
(Libeler incubators). Two petri dishes,
each containing three to five segments,
were prepared from each plant. After 2 to 3
days, hyphae grown from segments were
observed microscopically for morphologi-
cal characteristics of Rhizoctonia spp. and
hyphal tips from Rhizoctonia spp.-like
colonies were transferred on potato dex-
trose agar (PDA; Ovoid, Unipart Ltd.,
Basingstoke, England). From each petri
dish, only one isolate was kept in pure
culture. The cultures were incubated at
25°C in the dark for 3 days and then were
stored at 10°C until use. In total, 79 iso-
lates of Rhizoctonia spp. were obtained
from 34 cotton fields (COT isolates) and
89 isolates from 23 tobacco fields (TOB
isolates) (Table 1).
Nuclear staining. Nuclei of each isolate
were stained for confirmation of binucleate
or multinucleate status based on the meth-
odology described by Bandon (4). Single
5-mm-diameter disks of 2-day-old cultures
grown on PDA were transferred on clean,
sterilized glass slides covered by 2% water
agar. The slide cultures were incubated in a
moist chamber at 25°C in the dark. After 2
to3 days, the slides were removed from the
moist chamber and one drop of safari O
and one drop of 3% KOH were added. The
number of nuclei per cell was counted
from 20 cells in each isolate under ×400
magnification. Measurements were taken
two times.
Hyphal anastomosis. The determina-
tion of the AGs was performed using tester
isolates of known AG provided by Dr. R.
Grouch (Institute of Vegetable and Orna-
mental Crops, Gerbera, Germany) and Dr.
J. H. M. Schneider (Institute of Sugar Beet
Research, Bergen op Zoom, Netherlands)
(Table 2).
Anastomosis reactions were observed
using the glass-slide technique, following
the method described by Carling (7). A 5-
mm mycelial plug of an actively growing
culture of each isolate and a 5-mm myce-
lial plug of each tester isolate was placed 1
to 2 cm apart on water-agar-coated, sterile
glass slides. The glass slides were placed
in a moist chamber and incubated at 25°C
in the dark until the growing hyphae came
into contact (2 to 3 days). The region of
the overlapping hyphae was stained with
one drop of safari O and one drop of 3%
KOH and examined microscopically at
×400 and ×1,000 to determine anastomosis
reactions. The categories of hyphal anas-
tomosis reactions were scored as follows
(11): C0, no interaction; C1, hyphal con-
tact; C2, hyphal fusion with killing reac-
tion; and C3, fusion with no cell death.
Isolates were placed into AGs based on the
presence of at least five C2 reactions with
each of the tester isolates per microscopic
field. The anastomosis determination pro-
cedure with the tester isolates was con-
ducted two times.
DNA extraction. To determine se-
quence similarity of the rDNA-ITS region,
36 COT isolates and 34 TOB isolates were
used. The isolates were selected to repre-
sent all the groups identified in the anas-
tomosis reaction tests. Each isolate was
grown in potato dextrose broth (Sigma-
Aldrich, St. Louis), filtrated, dried, lyophi-
lized, ground to a fine powder using micro
pestles (Eppendorf International, Weasel-
ing, Germany), and stored at –20°C until
use. DNA was extracted using Quay Pure
gene Core Kit A (Siegen GmbH, Hilden,
Germany) according to the manufacturer’s
protocol. The concentration of the ex-
tracted DNA was measured by UV spec-
trophotometer (Gene Quant2; Pharmacia
Biotech, Cambridge, UK).
DNA amplification and sequencing.
ITS1 and ITS2 regions, including the ribo-
somal 5.8S RNA gene, were amplified
using the universal ITS-1 (5-TCCGTA
GGTGAACCTGCGG-3) and ITS-4 (5-
TCCTCCGCTTATTGATATGC-3) primers
that anneal to the flanking 18S and 28S
rRNA genes (53). The PCR amplification
reactions were performed in a 50-µl mix-
ture containing 5 µl of polymerase chain
reaction (PCR) buffer (1×) (Dynasty II Hot
Start Reaction Buffer, Fanzines, Espoo,
Finland), 2 µl of dNTPs (200 µM), 0.5 µl
of each primer (0.5 µM) (Lark Technolo-
gies Inc., Essex, UK), 0.5 µl (1 U per 50
µl) of Taq DNA polymerase (Dynasty II
Hot Start; Fanzines), 5 µl of template DNA
(50 ng), and 36.5 µl of sterile highly puri-
Fig. 1. Map of Greece showing tobacco and cotton production areas where seedlings were collected
for isolation of Rhizoctonia spp. 1 = Larissa, 2 = Pieria, 3 = Imathia, 4 = Pella, 5 = Kilkis, 6 = Thessa-
loniki, 7 = Serres, 8 = Drama, 9 = Xanthi, and 10 = Rodopi.
Tab l e 1. Geographic origin and number o
f
Rhizoctonia spp. isolates obtained from cotton
and tobacco fields of central and northern
Greece
Region, crop No. of
fields No. of
Isolates
Cotton isolates
Serres 6 12
Drama 2 2
Thessaloniki 1 1
Pella 5 14
Imathia 12 23
Larissa 8 27
Total 34 79
Tobacco isolates
Pieria 1 2
Xanthi 2 13
Kilkis 1 5
Serres 15 44
Rodopi 4 25
Total 23 89
1316 Plant Disease / Vol. 94 No. 11
fied H2O. In all PCR sets, a negative con-
trol without DNA was included.
PCR mixtures were covered with a drop
of mineral oil to prevent evaporation and
incubated in a PTC 200 thermal cycler (MJ
Research, Waltham, MA) using the follow-
ing conditions: DNA denaturization for 5
min at 94°C and 30 cycles of DNA dena-
turization for 30 s at 94°C, primer anneal-
ing for 30 s at 57°C, and extension of DNA
for 1 min at 72°C. DNA amplification was
terminated by primer extension for 5 min
at 72°C and final incubation at 4°C. After
the end of each reaction, set aliquots of the
reaction mixture (5 µl) were analyzed on
2% agarose gel in Tris-borate EDTA
buffer, supplemented with ethidium bro-
mide (0.1 µg ml–1) by electrophoresis, and
viewed under UV light.
PCR products of each isolate were puri-
fied using the Qiaquick PCR Purification
Kit (Siegen GmbH). The purified products
were subjected to sequencing in both di-
rections using the universal primers ITS-1
and ITS-4. The sequencing was carried out
by Lark Technologies Inc.
Sequence data and phylogenetic
analysis. Sequences were aligned using
the computer software package Clustal W
2.0.9 (25,51) and BioEdit 7.0.9.0 (18). The
alignment of both directions in each isolate
was checked visually. The obtained se-
quences of all isolates were compared with
sequences in the National Center for Bio-
technology Information (NCBI) data base
using BlastN 2.2.18 (55). Sequence simi-
larity between the ITS1-5.8S-ITS2 se-
quences of isolates obtained in this study
and those of isolates already deposited in
GenBank was checked using Clustal W
2.0.9. Phylogenetic analyses were per-
formed using neighbor-joining method
(40) based on the alignment using ITS1-
5.8S-ITS2 sequences of the isolates used
in this study and isolates of known AG.
The computational analysis to generate the
phylogenetic tree was performed using
Mega 4.0 (50). The distances in the ITS-
5.8S rDNA region were determined by
Kimura’s two-parameter model (21), omit-
ting all sites with gaps. A bootstrap analy-
sis of 1,000 replications was carried out.
Sequences of R. cerealis (GenBank acces-
sion no. AJ302009) and binucleate Rhizoc-
tonia AG-F (accession nos. AB219144 and
DQ102433) were used as outgroups for
rooting the phylogenetic trees constructed
from isolates obtained from cotton and
tobacco, respectively.
Hyphal growth response at different
temperatures. Hyphal growth rate of 86
isolates was measured at six different tem-
peratures (10, 15, 20, 25, 30, and 35°C) on
PDA. Isolates were selected to include all
the AGs and subgroups revealed in the
study. Mycelial plugs (5 mm in diameter)
from 3-day-old cultures were transferred to
the center of 9-cm petri dishes. The cul-
tures (two replicate cultures per isolate and
temperature) were incubated in the dark.
The experiments for all the isolate–
temperature combinations were carried
simultaneously in different growth cham-
bers. Two perpendicular colony diameters
were measured on the bottom of each plate
at 24-h intervals until the colony had
reached the edge of the petri dish. Agar
plug diameters were subtracted from every
measurement. The average daily hyphal
growth rate per isolate was calculated per
temperature. The experiment was per-
formed two times.
Pathogenicity and aggressiveness. In
all, 21 COT isolates and 20 TOB isolates
were selected to represent the AGs and
subgroups revealed in the study. The
pathogenicity and aggressiveness of the
selected isolates was tested on cotton (cv.
Flora), tobacco (cv. Xanthi), barley (cv.
Mutsu), radish (cv. Saxa 2), and sugar beet
(cv. Creta) using a method modified from
Carling et al. (9). Each isolate used as
inoculum was incubated on PDA plate for
2 to 3 days. A 5-mm agar plug with myce-
lium actively grown on PDA was trans-
ferred on the center of 9-cm petri dish
Tab l e 2. Tester isolates of Rhizoctonia solani used to determine anastomosis groups (AGs) of isolates
of the pathogen collected from cotton and tobacco grown in Greece
AG Isolate code Host Country Source
1-1A CS-KA Rice Japan Schneider
1-1A PRG-97-1 Rice Japan Grosch
1-1B B-19 Sugar beet Japan Schneider
1-1C BV-17 Sugar beet Japan Schneider
2-1 Ps-4 Bean Japan Schneider
2-1 SH-3 Soil Japan Grosch
2-2IIIB C-96 Bent grass Japan Schneider
2-2IV RI-64 Sugar beet Japan Schneider
3 ST-11-6 Potato Japan Schneider
3 B-15 … Israel Grosch
4-HGI AH-1 Peanut Japan Schneider
5 GM-10 Soybean Japan Schneider
5 SH-4 … Japan Grosch
6 6R-1 … Israel Grosch
7 1-529 … Japan Grosch
8 08-01 Wheat United States Grosch
9 09-01 … … Grosch
10 10-03 Alfalfa Australia Grosch
12 12-01 Plant residues Australia Grosch
13 13-01 Cotton United States Grosch
Tab l e 3. Nuclear status of Rhizoctonia spp. isolates and anastomosis grouping (AG) of R. solani obtained from diseased cotton and tobacco seedling plants
from northern and central regions of Greece
Number of isolatesy Anastomosis group (AG)z
Region, crop Total Binucleate Rhizoctonia solani AG-2 AG-3 AG-4 AG-5 AG-7
Cotton isolates
Serres 12 1 (8.3) 11 (91.7) 0 1 (9.0) 10 (91.0) 0 0
Drama 2 0 2 (100.0) 0 0 2 (100.0) 0 0
Thessaloniki 1 0 1 (100.0) 0 0 1 (100.0) 0 0
Pella 14 4 (28.5) 10 (71.5) 0 0 6 (60.0) 0 4 (40.0)
Imathia 23 9 (39.1) 14 (60.9) 0 0 13 (92.8) 0 1 (7.2)
Larissa 27 3 (11.1) 24 (88.9) 1 (4.2) 0 22 (91.6) 0 1 (4.2)
Total 79 17 (21.5) 62 (78.5) 1 (1.6) 1 (1.6) 54 (87.1) 0 6 (9.7)
Tobacco isolates
Pieria 2 1 (50) 1 (50) 0 0 0 1 (100.0) 0
Xanthi 13 0 13 (100.0) 13 (100.0) 0 0 0 0
Kilkis 5 0 5 (100.0) 5 (100.0) 0 0 0 0
Serres 44 1 (2.2) 43 (97.8) 36 (83.7) 0 7 (16.3) 0 0
Rodopi 25 0 25 (100.0) 16 (64.0) 0 9 (36.0) 0 0
Total 89 2 (2.2) 87 (97.8) 70 (80.5) 0 16 (18.4) 1 (1.1) 0
y Number in parenthesis is the percentage of the total number of isolates.
z Number in parenthesis is the percentage of the of the total number of isolates per AG.
Plant Disease / November 2010 1317
containing 2% water agar. Sterile PDA
plugs were used as controls. After 2 days
of incubation, 10 seeds of each host were
placed at equal distances in a circular pat-
tern along the outer edges of the petri
dishes. Prior to use, seed were surface
sterilized in 10% NaOCl for 1 to 2 min.
Petri dishes were incubated at 21°C in the
dark. Three days after the placement of the
seed into the water agar medium, damage
was assessed on the developing cotton,
barley, radish, and sugar beet seedlings,
where 0 = no damage; 1 = minor discol-
oration of the hypocotyl; 2 = discoloration
plus small (<1-mm-diameter) necrotic
lesions on the stem, hypocotyl, or root; 3 =
discoloration plus large (>1-mm-diameter)
necrotic lesions on the stem, hypocotyl, or
root; and 4 = seedling dead (9). The dis-
ease index scale was applied to all hosts
except tobacco. Due to the small size of
tobacco seedlings, pathogenicity on this
host was determined by counting the num-
ber of dead 3-day-old seedlings. For each
isolate, three replicate petri dishes were
used and the experiment was carried out
two times.
Data analysis. Data of isolates` myce-
lial growth rate and aggressiveness on
several hosts were subjected to analysis.
Due to uneven number of isolates within
groups and subgroups, nonparametric
analysis was performed. Overall differ-
ences in mycelial growth rates at the tem-
peratures tested and the aggressiveness
data of each AG or subgroup were tested
with the Kruskal-Wallis test and, in case of
a significant result, pairwise comparisons
were performed by means of a series of
Mann-Whitney tests. Due to the relatively
high number of pairwise comparisons, the
significance level per comparison was
adjusted according to Bonferroni’s method
(P = 0.002). All the statistical analyses
were performed using SPSS (SPSS Inc.,
Chicago).
RESULTS
Isolation and identification. In total,
168 isolates of Rhizoctonia spp. were ob-
tained from diseased cotton and tobacco
seedling plants. The isolates were collected
from 57 fields distributed across the 10
most important cotton- and tobacco-
cultivating regions of Greece (Table 1).
Nuclear staining using safari O revealed
that multinucleate isolates were dominant
among isolates obtained from cotton
(79.5%) and tobacco (97.8%) (Table 3).
The remaining isolates were binucleate.
Anastomosis grouping. All multinucle-
ate isolates were paired with tester isolates
of R. solani AGs AG-1 to AG-13. Positive
anastomosis reactions were observed with
the tester isolates of AG-2, -3, -4, -5, and
-7 (Table 3). Among 62 COT isolates, 54
isolates gave a C2 reaction with the AG-4
tester isolate, 6 isolates with AG-7, and
one isolate each with AG-2 and AG-3.
Similarly, among the 87 multinucleate
Tab l e 4. Hyphal anastomosis reactions and analysis of the ribosomal (r)DNA internal transcribed
spacer region of Rhizoctonia solani and two binucleate Rhizoctonia spp. isolated from cotton and
tobacco seedlings in Greece
Isolate name Anastomosis groupingy rDNA sequencing Accession no.z
Cotton isolates
P20B AG-7 AG-7 FJ480891
P2B3 AG-4 AG-4-HG-I FJ480867
P4B3 AG-7 AG-7 FJ480892
P7B2 AG-7 AG-7 FJ480893
AMP4B AG-4 AG-4-HG-III FJ480881
L2B7 AG-4 AG-4-HG-I FJ480874
L3B1 AG-7 AG-7 FJ480894
L3B10 AG-4 AG-4-HG-I FJ480865
L4B2 AG-4 AG-4-HG-I FJ480868
L4B9 AG-4 AG-4-HG-I FJ480864
L6B8 AG-2 AG-2-1 FJ480890
L7B1 Binucleate AG-F FJ480897
L7B5 AG-4 AG-4-HG-III FJ480882
L7B10 AG-4 AG-4-HG-I FJ480866
L8B2 AG-4 AG-4-HG-I FJ480875
L10B7 AG-4 AG-4-HG-I FJ480873
SB2B AG-4 AG-4-HG-III FJ480883
SG1B AG-4 AG-4-HG-I FJ480876
SD2B AG-4 AG-4-HG-I FJ480869
SZ6B AG-3 AG-3 FJ480889
SH3B AG-4 AG-4-HG-I FJ480863
D2B21 AG-4 AG4-HG-III FJ480884
D3B28 AG-4 AG-4-HG-II FJ480880
TH1 AG-4 AG-4-HG-III FJ480886
A8B AG-4 AG-4-HG-III FJ480887
G9B AG-4 AG-4-HG-I FJ480871
D1B AG-4 AG-4-HG-I FJ480870
D3B AG-4 AG-4-HG-I FJ480862
H3B AG-4 AG-4-HG-I FJ480872
T4B AG-4 AG-4-HG-III FJ480885
I9B AG-4 AG-4-HG-I FJ480879
K1B AG-4 AG-4-HG-I FJ480878
K9B AG-7 AG-7 FJ480895
M9B AG-4 AG-4-HG-I FJ480877
B1B6 Binucleate AG-F FJ480896
H20B AG-4 AG-4-HG-III FJ480888
Tobacco isolates
SAK2 AG-4 AG-4 HG-III FJ480930
SAK3 AG-4 AG-4 HG-III FJ480927
SAK5 AG-2 AG 2-1 FJ480920
SAK7 AG-2 AG 2-1 FJ480902
SBK7 AG-2 AG 2-1 FJ480903
SCK1 AG-2 AG 2-1 FJ480921
SCK2 AG-2 AG 2-1 FJ480904
SCK7 AG-4 AG-4 HG-I FJ480922
SDK3 AG-4 AG-4 HG-III FJ480928
SDK4 AG-2 AG 2-1 FJ480901
SEK1 AG-2 AG 2-1 FJ480900
SZK3 AG-2 AG 2-1 FJ480905
SZK5 AG-2 AG 2-1 FJ480906
SZK8 AG-4 AG-4 HG-III FJ480929
SHK4 AG-2 AG 2-1 FJ480907
SHK8 AG-4 AG-4 HG-I FJ480923
SIK1 AG-2 AG 2-1 FJ480908
SKK2 AG-2 AG 2-1 FJ480899
SKK3 AG-2 AG 2-1 FJ480898
SMK1 AG-2 AG 2-1 FJ480909
SOK1 AG-2 AG 2-1 FJ480910
KA5K AG-2 AG 2-1 FJ480911
KB9K AG-2 AG 2-1 FJ480913
KB10K AG-2 AG 2-1 FJ480912
KC7K AG-4 AG-4 HG-I FJ480925
KC10K AG-4 AG-4 HG-I FJ480924
KD5K AG-4 AG-4 HG-I FJ480926
KD7K AG-2 AG 2-1 FJ480914
XK2 AG-2 AG 2-1 FJ480915
XK9 AG-2 AG 2-1 FJ480916
KK4 AG-2 AG 2-1 FJ480917
DOI4K AG-2 AG 2-1 FJ480918
DOI7K AG-2 AG 2-1 FJ480919
LIT3K AG-5 AG-5 FJ480931
y Anastomosis groups (AGs) based on hyphal anastomosis reactions with tester isolates.
z Accession number of sequences obtained from the National Center for Biotechnology Information.
1318 Plant Disease / Vol. 94 No. 11
TOB isolates, 70 isolates gave a C2 reac-
tion with the AG-2 tester isolate, 16 iso-
lates with AG-4, and one isolate with AG-5
(Table 3).
ITS and phylogenetic analysis. Most
of the isolates tested yielded a 700-bp
product, whereas the binucleate isolates
and the isolates belonging in AG-7 yielded
a 680-bp product. Sequence analysis of the
PCR products and comparison with al-
ready existing sequences in NCBI showed
that, among the 36 COT isolates tested, 18
belonged to AG-4-HG-I, 1 to AG-4-HG-II,
8 to AG-4-HG-III, 1 belonged to AG-2-1, 1
to AG-3, 5 to AG-7, and 2 binucleate iso-
lates to AG-F (Table 4). Similarly, among
the 34 TOB isolates tested, 24 belonged to
AG-2-1, 5 to AG-4-HG-I, 4 to AG-4-HG-
III, and 1 to AG-5 (Table 4). For both
hosts, molecular data were in accordance
with the classification of the isolates based
on the results of the hyphal anastomosis.
The alignment of the obtained se-
quences showed that the 5.8S rDNA gene
sequence was completely conserved in all
the isolates tested independently of AG
classification (data not shown; sequences
are available upon request). The phyloge-
netic tree of COT isolates was rooted using
a R. cerealis sequence (GenBank accession
no. AJ302009) while the phylogenetic tree
of the TOB isolates was rooted using two
binucleate Rhizoctonia spp. isolates (ac-
cession nos. AB219144 and DQ102433).
The phylogenetic trees showed that the
isolates tested separated distinctly based
on their AG and that each AG clustered
together. The phylogenetic tree for the
cotton isolates was mainly separated into
two clades (Fig. 2). One clade was com-
posed of isolates AG-2-1 and AG-3, and
another contained AG-4, AG-7, and binu-
cleate AG-F. Each HG in AG-4 was sepa-
rated in the clade composed of AG-4 iso-
lates. Similarly, the phylogenetic tree for
the tobacco isolates was also separated
into two clades (Fig. 3). One clade was
composed only of AG-2-1 isolates and
another was composed of AG-4 and AG-5
isolates. Both phylogenetic trees also
revealed that isolates obtained from dif-
ferent regions could not be separated into
distinct clades on the basis of their geo-
graphic origin.
Hyphal growth response at different
temperatures. As a group, binucleate
isolates had an optimum growth tempera-
ture of 30°C. They showed only a slight
growth at 10°C but grew well at 35°C
(Table 5). On average, the AG-2-1 isolates
grew more slowly than isolates of other
AGs. The AG-3 isolate had an optimum
growth temperature of 25°C but showed
minimal growth at 10 and 35°C. The AG-4
and AG-7 isolates had an optimum growth
at 30°C, grew well at 35°C, and showed
minimal growth at 10°C. The AG-4 sub-
groups did not vary significantly in growth
rate, with the exception of the AG-4-HG-II
isolate, which had a reduced growth rate at
30°C and grew only slightly at 35°C. The
AG-5 isolate had the ability to grow at
both 10 and 35°C and had an optimum
temperature of 25 to 30°C (Table 5).
Pathogenicity and aggressiveness. On
cotton plants, the isolate AG-2-1 and the
binucleate isolates obtained from cotton
were less aggressive, with disease index
Fig. 2. Phylogenetic tree constructed by the neighbor-
j
oining method based on the sequences of the
internal transcribed spacer (ITS)1-5.8S-ITS2 ribosomal DNA regions of Rhizoctonia spp. isolated from
cotton seedlings. Distances were determined by Kimura’s two-parameter model, omitting all sites with
gaps. A Rhizoctonia cereale sequence (AJ302009) was used as an outgroup. Numbers next to the
branches represent the percentage of congruent clusters in 1,000 bootstrap trials when the values were
more than 70%. Numbers in parenthesis are the accession numbers of sequences obtained from the
National Center for Biotechnology Information.
Plant Disease / November 2010 1319
values of 1.0 and 1.4, respectively, com-
pared with other COT isolates belonging to
several AGs (Table 6). Even lower was the
disease index value of 0.3 caused on cotton
by the AG-5 isolate obtained from tobacco.
The most aggressive isolates on cotton
were the AG-4 isolates, independently of
their host of origin, and the AG-7 isolates
obtained from cotton.
Similarly, on radish and sugar beet, the
AG-2-1 and the binucleate isolates were
the least aggressive, with disease index
values of 0.8 to 2.8 (Table 6). The remain-
ing isolates tested were equally aggressive
on both hosts.
Barley was the least susceptible host.
Binucleate, AG-2-1, AG-3, and AG-5 iso-
lates caused no symptoms on barley plants.
AG-7 and AG-4 isolates caused damage on
barley seedlings with disease index values
ranging from 0.0 to 1.4 (Table 6).
On tobacco, the most aggressive isolates
were AG-4-HG-I, AG-4-HG-III, and AG-7
causing symptoms on 46 to 76% of the
seedlings (Table 6). Interestingly, the AG-
4-HG-II isolate from cotton caused no
symptoms on tobacco seedlings. The binu-
Fig. 3. Phylogenetic tree constructed by the neighbor-joining method based on the sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 ribosomal
DNA regions of Rhizoctonia solani isolated from tobacco seedlings. Distances were determined by Kimura’s two-parameter model, omitting all sites with
gaps. Two binucleate Rhizoctonia sequences were used to rooted the tree. Numbers alongside branches represent the percentage of congruent clusters in
1,000 bootstrap trials when the values were more than 70%. Numbers in parenthesis are the accession numbers of sequences obtained from the National
Center for Biotechnology Information.
1320 Plant Disease / Vol. 94 No. 11
cleate and AG-3 isolates from cotton were
less aggressive on tobacco, causing symp-
toms on 3 and 20% of the seedlings, respec-
tively. The AG-2-1 isolates, independently
of their host of origin, were moderately
aggressive, causing symptoms on 35 to 40%
of tobacco seedlings (Table 6).
DISCUSSION
The results of the study indicated that
damping-off disease of cotton and tobacco
seedlings in Greece caused by Rhizoctonia
spp. is mainly due to R. solani. Variability
in the number and frequency of different
AGs was observed on both hosts.
The relatively high proportion of binu-
cleate Rhizoctonia spp. (21.5% of isolates)
isolated from cotton in this study was also
found previously in Spain (26). In reports
from other hosts such as sugar beet (20),
soybean (12), or cauliflower (35), the pro-
portion of binucleate isolates was low.
Molecular data presented in the current
report showed that binucleate Rhizoctonia
isolates from cotton were of AG-F. To the
best of our knowledge, this is the first re-
port of AG-F isolates attacking cotton
worldwide.
AG-4 predominated among the cotton
isolates of R. solani, followed by AG-7,
which agrees with previous reports from
other countries (2,3,6,26,28). Isolates of
AG-4 were obtained from all the sampled
cotton fields in all the sampling regions. In
this study, samples were taken from lati-
tudes ranging from 39 to 41°N and distri-
bution of AG-4 isolates varied with lati-
tude, as previously observed in the United
States (19). Isolation of AG-2 and AG-3 R.
solani from cotton is reported, to the best
of our knowledge, for first time.
AG-2-1 was the predominant AG iso-
lated from tobacco, followed by AG-4.
Previous reports suggested that damping-
off of tobacco seedlings is most closely
associated with AG-4 (16,32). The effect
of the temperature and differences in the
soil texture, along with possible effects of
the differences in agronomic practices
among different countries, may explain the
observed prevalence of AG 2-1 isolates in
tobacco seedlings in Greece in contrast to
reports of AG-4 isolate prevalence in other
countries. In addition, one AG-5 isolate
was recovered. To the best of our knowl-
edge, infection of tobacco seedlings by
AG-5 R. solani has been previously re-
ported only in Italy (31).
The levels of variability in ITS1 and
ITS2 for the isolates examined were simi-
lar to those observed in previous studies
(5,23,24,41,45). Construction of phyloge-
netic trees showed that the three different
AGs (AG-2, AG-4, and AG-5) recovered
from tobacco isolates were located in three
different clades on the phylogenetic tree,
suggesting low genetic relatedness among
them. Phylogenetic analysis of the cotton
isolates showed that the AG-2 and AG-3
isolates were located on the same clade,
suggesting high genetic similarity, as pre-
viously reported (45). This may explain the
morphological similarity of these isolates.
Similarly, the binucleate isolates of AG-7
and the R. solani subgroups of AG-4 and
the AG-7 isolates were located on the same
clade. This genetic relatedness of binucle-
ate and AG-4 isolates has been observed in
another recent study (44). Such results are
informative for evaluating the evolutionary
relationships among the several AGs of R.
solani and contribute to the better under-
standing of the biology the species com-
plex.
Tab le 5 . Radial growth rate (mm/24 h) on potato dextrose agar of Rhizoctonia fungi by anastomosis group (AG) and subgroup that were isolated from cotton
and tobacco seedlings in northern and central regions of Greece
Temperature (°C)z
AG No. of isolates 10 15 20 25 30 35
Cotton isolates
Binucleate 16 3.4 a 10.9 a 16.4 a 21.4 a 28.8 a 21.0 ab
AG-2-1 1 2.3 a 5.2 a 19.5 ab 22.0 ab 19.0 a 3.7 a
AG-3 1 4.3 a 12.6 ab 11.6 a 18.3 a 15.8 a 8.0 a
AG-4-HG-I 18 4.6 a 17.0 b 26.0 b 31.0 b 33.3 a 19.2 a
AG-4-HG-II 1 8.7 a 16.0 ab 23.7 ab 24.0 ab 24.2 a 7.3 a
AG-4-HG-III 8 6.4 a 17.1 b 24.1 ab 28.1 b 31.0 a 23.9 b
AG-7 5 4.9 a 15.9 ab 23.3 ab 24.9 ab 33.8 a 26.3 c
Tobacco isolates
Binucleate 2 1.8 a 9.4 a 19.0 a 22.5 ab 28.2 ab 24.2 b
AG-2-1 24 5.3 a 11.3 a 18.5 a 19.0 a 16.2 a 4.3 a
AG-4-HG-I 5 3.2 a 16.6 a 25.1 a 30.2 b 33.4 b 16.9 b
AG-4-HG-III 4 3.6 a 16.9 a 24.6 a 28.6 ab 31.0 b 23.7 b
AG-5 1 7.8 a 14.7 a 25.0 a 25.5 ab 26.5 ab 14.1 b
z Least square mean values per temperature followed by the same letter are significantly different at P = 0.002 according to the nonparametric tests o
f
Kruskal-Wallis and Mann-Whitney.
Tab l e 6. Disease severity on cotton, radish, sugar beet, and barley seedling plants and disease incidence on tobacco seedling plants caused by binucleate
Rhizoctonia spp. and Rhizoctonia solani isolates belonging to different anastomosis groups (AGs) and subgroupsz
AG No. of isolates Cotton Radish Sugar beet Barley Tobacco
Cotton isolates
Binucleate 2 1.4 a 2.5 b 2.2 a 0.0 a 3.8 a
AG-2-1 1 1.0 a 0.8 a 2.8 a 0.0 a 37.5 b
AG-3 1 3.3 c 3.6 cd 3.4 b 0.0 a 20.0 ab
AG-4-HG-I 8 3.3 c 3.5 cd 3.8 c 0.8 c 76.3 c
AG-4-HG-II 1 2.7 b 3.7 cd 4.0 c 1.1 cd 2.5 a
AG-4-HG-III 5 2.4 b 3.5 c 3.8 c 1.4 d 46.0 b
AG-7 3 3.5 c 3.8 d 3.9 c 0.3 b 53.3 b
Tobacco isolates
Binucleate 2 1.4 a 2.4 ab 2.8 ab 0.0 a 35.0 a
AG-2-1 10 1.3 a 1.6 a 2.6 a 0.0 a 31.3 a
AG-4-HG-I 4 2.4 a 3.5 ab 4.0 b 0.7 ab 68.1 b
AG-4-HG-III 2 3.0 a 3.9 b 4.0 ab 1.2 b 52.5 ab
AG-5 1 0.3 a 3.2 ab 4.0 ab 0.0 a 30.0 a
z Disease severity was measured using a disease index scale ranging from 0 (no damage) to 4 (seedling dead), and disease incidence was measured as per-
centage of dead seedling plants. Least square means followed by different letter within the columns are not significantly different according to the nonpara-
metric tests of Kruskal-Wallis and Mann-Whitney at P = 0.002.
Plant Disease / November 2010 1321
As reported in previous studies (39,46),
AG-4 and AG-7 isolates had higher growth
rates than the remaining AGs and sub-
groups at temperatures of 10 to 30°C. The
AG-7 isolates showed higher growth rates
at 30 and 35°C compared with other iso-
lates, as previously reported (2). The simi-
larity of our results to those from countries
with diverse climates suggests that most
isolates and subgroups of R. solani do not
exhibit temperature adaptation across geo-
graphic areas (19). However, the lower
mycelial growth rate of the AG-2-1 isolates
compared with that of AG-4, and the lower
temperature optima for these isolates,
agree with previous reports (34,43,46).
AG-2-1 isolates are known to prevail in
colder conditions than AG-4 isolates
(48,49). In the current study, the AG-2-1
isolates were obtained mainly from to-
bacco fields located in cool, semi-
mountain regions where tobacco is culti-
vated in Greece.
Results of pathogenicity studies were
similar to previous reports (1,2). AG-2-1
isolates were less aggressive than AG-4
and AG-7 isolates and did not cause any
symptoms on barley. R. solani AG-2-1 is a
heterogeneous subgroup in terms of both
genetic variability and virulence on several
hosts (9,35,42,49,54). Although AG-5
isolates are considered to be weak patho-
gens (30), in the current study the AG-5
isolate was highly aggressive against rad-
ish and sugar beet and was moderately
aggressive on tobacco. Nicoletti and Lahoz
(31) also found that AG-5 was not aggres-
sive on tobacco seedlings. Although
pathogenicity to different hosts has been
used in the past to classify AGs and sub-
groups of R. solani (9), it seems that
pathogenicity is not a reliable characteris-
tic of these groups (29,33,52).
In Northern Greece, tobacco and cotton
are rotated with susceptible hosts such as
sugar beet, tomato, and maize, suggesting
that crop rotation may be insufficient for
the successful management of diseases
caused by R. solani and binucleate Rhizoc-
tonia spp. Small grains that are less sus-
ceptible to the AGs recovered in the study
could be included in the rotation schemes.
Wheat and barley are the two main small-
grain species cultivated in northern
Greece. The predominant AGs found in
this study should be tested for pathogenic-
ity on the main wheat and barley cultivars
cultivated in the region to determine their
suitability as rotation crops. Furthermore,
based on the findings of the current report,
the efficacy of fungicides applied either as
seed treatments in cotton or as soil-drench
in tobacco should be investigated.
ACKNOWLEDGMENTS
C. Bacharis was financially supported by On-
assis Foundation. We thank R. Grosch and J. H.
M. Schneider for their kind offer of Rhizoctonia
solani tester isolates used in the study and G..
Menexes for his aid in the statistical analysis of
the results.
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