Australasian Plant Pathol.
DOI 10.1007/s13313-014-0277-8
Tunisian Rhizoctonia solani AG3 strains affect potato shoot
macronutrients content, infect faba bean plants and show
in vitro resistance to azoxystrobin
Naceur Djébali & Salem Elkahoui & Wael Taamalli &
Kamel Hessini & Belhassen Tarhouni & Moncef Mrabet
Received: 21 January 2014 / Accepted: 27 January 2014
# Australasian Plant Pathology Society Inc. 2014
Abstract Rhizoctonia solani reduces quality and yield of po-
tato crops in the south of Tunisia and has become an impediment
for potatoes export. The lack of studies on the pathogen
prompted us to characterize the growth capacity at different
temperatures, the aggressiveness, the host specificity and the
sensitivity to the fungicides fludioxonil, pencycuron and
azoxystrobin of local R. solani strains. Morphological character-
ization and sequencing of the rDNA ITS gene of the fungal
strains showed that they belong to R. solani anastomosis group
3 (AG3). All strains showed a maximum growth at 25 °C with a
preference to cold (15–20 °C) in comparison to high tempera-
tures (30–36 °C). All R. solani strains decreased potato shoot dry
weight in infected versus control plants. The nitrogen and potas-
sium levels were higher in the shoot of infected potato plants in
comparison to the control; however the opposite behaviour was
noticed for phosphorus concentration, indicating an imbalance in
the nutritional statue of infected potato plants. The effect of
R. solani on potato macronutrients uptake is discussed. All
R. solani strains were able to infect faba bean roots with the
strain RS5.2 reduced significantly plant growth and nodulation.
Therefore, faba bean may provide a suitable alternative host for
R. solani AG3. The local R. solani strains were highly sensitive
to fludioxonil (EC50: 0.007 μg a.i. ml−1) and pencycuron (EC50:
0.006 μg a.i. ml−1); but resistant to azoxystrobin with an EC50
exceeding 19 μg a.i. ml−1. This highlights the risk of resistance to
quinone outside inhibitor fungicides (QoI) in R. solani popula-
tions, although up to now no decrease in field performance was
noticed. It is recommended to delay build up of QoI resistance by
N. Djébali (*) : S. Elkahoui : W. Taamalli : K. Hessini : M. Mrabet
Laboratory of Molecular Physiology of Plants, Centre of
Biotechnology of Borj Cedria, B. P. 901, Hammam-Lif 2050, Tunisia
e-mail: dnaceur@yahoo.fr
B. Tarhouni
Technical Centre of Potato and Artichoke, Route Jedaida,
Saïda 2031, Tunisia
an integrated approach, combining optimised fungicide use with
the choice of R. solani resistant cultivars and the application of
farming practices.
Keywords Black scurf of potato . Fungicide resistance . In
vitro growth . Specificity . Solanum tuberosum . Vicia faba
Introduction
Rhizoctonia solani Kühn, (teleomorph Thanatephorus
cucumeris (A. B. Frank) Donk) is a soil borne Basidiomycete
occurring world-wide, with complex biology and causes dis-
ease on a broad array of host plant species (Agrios 1988). It
causes different disease symptoms like root, crown and stem
rot and damping-off on host plants. Because of the lack of
conidia and the scarcity of the sexual spores, R. solani exists
as vegetative hyphae and sclerotia in nature and the identifi-
cation of the fungus is based on characteristics of vegetative
hyphae as with other Rhizoctonia spp. Great variations among
the isolates in terms of myclial colour, number of sclerotia,
size of aerial mycelium, growth rate, saprophytic behavior,
enzyme production and pathogenicity were found
(Hyakumachi et al. 1988). Rhizoctonia solani is called a
species complex because it contains many related but genet-
ically isolated subspecific groups. Many criteria have been
used to delineate subgroups of the R. solani species. Earlier
methods of subgrouping included differences in colony mor-
phology, host range virulence, whereas recent methods in-
clude biochemical and molecular techniques (Carling et al.
1994; Sharon et al. 2006). The grouping of R. solani isolates
was mostly accomplished by evaluation of the anastomosis
reactions of the vegetative hyphae of the individual fungal
strains (Carling 1996). Nevertheless, the rDNA internal tran-
scribed spacer (ITS) sequence analysis is currently the most
appropriate method for classification of Rhizoctonia spp.
 N. Djébali et al.

Generally, the clusters of the isolate sequences of multinucle- Table 1 Identification of the studied Rhizoctonia solani strains and their
sites of origins
ate Rhizoctonia were supportive of the anastomosis groups
(AGs) and subgroups based on hyphal fusion anastomosis Strain Region of Main environmental
(Sharon et al. 2006). Thirteen AGs of R. solani have been code isolation factors of sampling sites
described by Carling et al. 2002. Isolates of 11 AGs have been
RS1.1 Mannouba Soil texture: Clay
associated with potato. Of these, AG3 is acknowledged by
(North of Tunisia) Climate: Sub-humid
most to be the principal cause of Rhizoctonia disease of potato
RS1.2 Temperature mean: 18.8 °C
(El Bakali and Martín 2006). The R. solani AG3 group was
Temperature max.: 33.5 °C
also isolated from several solanaceae crops such as tomato,
RS1.2 Annual rainfall: 616 mm
tobacco and egg plants (Carling et al. 2002). The investiga-
Relative humidity mean: 74.3 %
tions of the relation between AG groups and host plant spec-
ificity demonstrated that isolates from one or a few AGs are RS2.1 Sidi Bouzid Soil texture: Sandy
generally present on one specific plant species, however some (Centre of Tunisia) Climate: Arid superior
studies showed that R. solani isolates were able to infect plants RS2.2 Temperature mean: 19.7 °C
from families other than the one from which they were orig- Temperature max.: 45.1 °C
inally isolated (Keijer et al. 1997). For example, isolates from RS3.2 Annual rainfall: 272 mm
AG3 group that were isolated from nonsolanaceous plants Relative humidity mean: 60.6 %
could cause disease in potato (Richter and Schneider 1953). RS5.1 Gafsa Soil texture: Sandy
In the last decade, the southern region of Tunisia has become (South of Tunisia) Climate: Arid inferior
an important area for potato production because of its suitable Temperature mean: 20.6 °C
climate for the production of this crop from January to March, RS5.2 Temperature max.: 45.3 °C
during which the export of potatoes to European countries is Annual rainfall: 165 mm
exempt from taxes. Since 2005, severe R. solani black scurf Relative humidity mean: 54.0 %
infections on potato tubers were found in this region which
reduces potato quality and yield and has became an impediment
for export of potato (Daami-Remadi et al. 2008; Djébali and strains from faba bean was done from symptomatic roots and
Belhassen 2010). Nevertheless, there is no study in Tunisia nodules of inoculated plants at 2 months post inoculation
describing the aggressiveness of R. solani strains isolated from following the same protocol described above. To study the
potato and their host specificity toward plant species cultivated in effect of temperature on R. solani in vitro growth, PDA plates
rotation with potato (especially faba bean). In addition in the were inoculated with agar plugs (9 mm in Ø), cut form 7-days-
attempt to have control measures against this pathogen, several old fungal culture, with the mycelium facing down and incu-
fungicides were introduced and approved in Tunisia for potato bated at 15, 20, 25, 30 and 36 °C in darkness. The fungal
seed treatment against black scurf. Therefore, the aim of this growth was determined at 2 days post sub-culture (dps) by
study was to investigate the growth capacity at different temper- measuring the diameter (mm) of the fungal colony (DFC) at
atures, the aggressiveness, the host specificity and the fungicide two perpendicular directions with the original mycelial plug
sensitivity of local R. solani strains. diameter subtracted. The speed of mycelia growth (SMG) was
determined at 2 dps according to the following formula:

DFC at 2 dps−DFC at 1 dps

Materials and methods
Fungal isolation and culture
The original Rhizoctonia inocula were obtained from potato
tuber-borne sclerotia from Mannouba, Sidi Bouzid and Gafsa
regions (Table 1). The sclerotia removed from tuber were
surface disinfected in ethanol 95° for 1 min, then washed with
sterile distilled water and dried on sterile filter paper.
Disinfected sclerotia were divided in two pieces and left to
culture on Potato Dextrose Agar (PDA Pronadisa) medium
for 2 days at 25 °C under 16 h of florescent light and 8 h of
obscurity. Eight strains (Table 1) were purified by the hyphal tip
method (Zhang et al. 2007). The fungal isolates were stored on
PDA slants at 4 °C in the dark. The re-isolation of the R. solani
SMGðmm=dayÞ ¼
2 dps−1 dps
Molecular identification of fungal strains
Based on the morphological differences and the geographical
origin, four R. solani strains (RS1.3, RS3.2, RS5.1 and RS5.2)
were selected for the molecular identification. The fungal
DNA extraction and the amplification of the internal tran-
scribed spacers (ITS) of the ribosomal DNA (rDNA) region
were performed according to the method of Mahuku (2004).
The ITS1 and ITS4 primers (White et al. 1990) were used for
the amplification of the ITS including the ITS1, 5.8S and ITS2
regions. The amplified DNA fragments were purified from
agarose gels using Wizard SV Gel and PCR-clean up system
Aggressiveness and fungicide sensitivity of R. solani
from Promega. The Applied Biosystems® 3130 Genetic
Analyser was used for the sequencing of the amplified frag-
ments using the same primers. The sequences were assembled
using the CAP program available on the NCBI website http//
www.ncbi.nlm.njh.gov/blast. The nucleotide sequences data
were submitted to GenBank and the accession numbers were
obtained.
Inoculum preparation and inoculation tests
The R. solani inoculum was prepared by sub-culturing 25
fungal agar-discs (9 mm in Ø) from 7 days-old culture in
500 mL liquid Potato Dextrose Browth medium for 10 days
at 25 °C in darkness without shaking. The obtained mycelium
was crushed by an electrical mixer and directly used for plant
inoculation. Potato and faba bean plants were inoculated at
three leaves stage by pouring 100 mL of the fungal inoculum
at the base of the stem.
Plant material and culture
The cultivar Nicola of potato and a local cultivar of faba bean
(Vicia faba L.) were used in this study. The potato seeds were
disinfected by immersing in 3 % sodium hypochlorite for
30 min and then washed several times in water. Disinfected
tuber seeds were kept in darkness to germinate until sprouts
length was about 1 to 2 cm. Faba bean seeds were surface
sterilized by immersion in 70 % alcohol for 30 s and then in
0.2 % HgCl 2 for 2 min and extensively washed with
autoclaved distilled water. Imbibed seeds were transferred on
humidified autoclaved Perlite and incubated at 25 °C in dark-
ness until radicle emergence. Potato and faba bean germinated
seeds were planted in autoclaved (30 min at 121 °C) sandy
soil-compost mixture (2:1) contained in 2 Kg pots. Physical
and chemical characteristics of the used soil are given in
Table 2. Plants were irrigated twice a week. Potato and faba
bean plants were harvested at 3 and 2 months post inoculation
Table 2 Main physical
and chemical character- Element Concentration
istics of the soil used for
potato and faba bean Clay (%) 8 percent of inhibition related to the control of mycelia growth
cultures Silt (%) 7 versus the log10 transformation of the seven concentrations of
Sand (%) 85
C.E (μs/cm) 512
pH 8.0
N (g/kg) 1.37
Na (meq/100 g) 1.62
K (meq/100 g) 1.85
Ca (meq/100 g) 5.67
Mg (meq/100 g) 1.37
P2O5 (ass ppm) 109
with R. solani, respectively. The pathogenicity and specificity
tests of the R. solani strains were repeated twice with five
replicates for each treatment in a randomized complete bloc
design.
Determination of nitrogen, potassium and phosphorus
concentrations
Plant dry weight was determined after samples were dried to a
constant weight at 65 °C. Dried material was ground and
reduced to a fine powder for determination of inorganic ions.
Organic nitrogen (N) was assayed with the Kjeldahl method.
The potassium cation (K) and the inorganic phosphorus anion
(Pi) were extracted with 30 mL of 0.1 M nitric acid added to
30 mg of plant dry powder. The K was assayed by flame
emission photometry (flame spectrophotometer IL 151) as
described by Hessini et al. (2005) and the inorganic phospho-
rus was determined by colorimetry as described by Fleury and
Leclerc (1943).
Fungicides sensitivity assay
The effect of three commercial fungicides (Table 3) on the
in vitro growth of four R. solani strains representing the three
localities was studied. The fungicides were added at different
concentrations (microgram of active ingredient per milliliter:
μg a.i. ml−1) to autoclaved PDA medium and then distributed
in 9 cm Petri dishes. Plates were inoculated with plugs of agar
(5 mm in Ø) of 7-days-old R. solani culture with the mycelium
facing down and incubated at 25 °C in darkness for
3 days. For each plate, the diameter of the fungal
colony (DFC) was determined in two perpendicular
directions with the original mycelial plug diameter (9 mm)
subtracted. The percentage of growth inhibition (GI) was
calculated according to the following formula:
 
DFC in presense of fungicide
GIð%Þ ¼ 100−  100 :
DFC of the control
For each strain, a linear regression of the probit of the
each fungicide was obtained. The 50 % effective concentra-
tion (EC50), which was the fungicide concentration that re-
sulted in 50 % mycelial growth inhibition, was calculated with
the regression equation for each strain (Zhang et al. 2007). In
azoxystrobin sensitivity assay, the salicylhydroxamic acid
(SHAM), which act as an inhibitor of alternative oxydase,
was not added to the medium since previous data showed that
azoxystrobin was effective alone in inhibiting in vitro R. solani
mycelial growth and the addition of SHAM showed little
synergistic effects with it (Jin et al. 2009). This experiment
 N. Djébali et al.

(concentration of a.i. in g l−1)

rate for potato seed treatment
was repeated twice and arranged in a randomized complete
Manufacture recommended block design with three replicates per treatment.

(0.25 g a.i. l−1)

(1.5 g a.i. l−1)

(2.5 g a.i. l−1)

Microscopic observations

1000 ml/t

250 ml/t
600 ml/t

For bright field microscopy (Olympus BX41, Japan), fresh

R. solani mycelium was mounted on glass slides and observed
without staining. For electron microscopy, agar plugs contain-
Formulation (concentration

ing intact fungal mycelium were mounted on metal stubs and

observed without staining in a Scanning Electron Microscope
Suspendable liquid
Suspendable liquid

Suspendable liquid

(Hitachi S 450, Singapore) at 15 KV.

of a.i. in %)

Statistical analysis
(25 %)

(25 %)

(10 %)

Statistical analysis was performed using Statistica software

version 5.1 (www.statsoft.com) and a statistics package
Trade name (manufacture)

(Microsoft Excel 2007). In all experiments, One-Way

ANOVAs, specifically Fisher’s least significant difference
Monceren® 250 FS

Maxim® 100 FS

(LSD) test with a P value 0.05, were used to analyze signif-

icant differences between each treatment group. The Correla-
(Syngenta)

(Syngenta)
Amistar®

tions between measured parameters were estimated by com-

(Bayer)

puting Pearson’s correlation coefficient (r) using XLSTAT

software v 7.5 (Addinsoft, USA).
Interferes with transport
electron transferring
Inhibits mitochondrial

mechanism in cells
Inhibits cell division
Mode of action

Results

Morphologic and molecular characterization of the local

R. solani strains

The fungal strains were isolated from potato tubers showing

N-[(4-chlorophenyl)-methyl]-N-cyclopentyl-

typical black sclerotia of R. solani at the surface (Fig. 1a). The

pyrimidin-4-yl]oxyphenyl]-3-methoxy

eight isolated fungal strains developed initially a white colony

4-(2,2-difluoro-1,3-benzdioxol-4-yl)-1
methyl (Z)-2-[2-[6-(2-cyanophenoxy)

on PDA medium which progressively turns to brown with the

formation of dark brown to black solid sclerotia with irregular
shape within 7 days all over the mat and not only around the
Table 3 Some characteristics of the studied fungicides

h-pyrrole-3-carbonitrile
N′-phenylurea (C.A.)

site of inoculation (Fig. 1b). In observation under microscope

the fungal strains showed colorless vegetative mycelium
(about 8.2 μm in diameter) with typical R. solani right-angle
prop-2-enoate
Chemical name

branching of main hyphae, with constriction (about 3.5 μm in

diameter) at the point of branching (Fig. 1c and d).
To confirm the result of the morphological identification
the R. solani strains RS1.3, RS3.2, RS5.1 and RS5.2 were
used for the sequencing of the rDNA ITS gene. The blast of
Active ingredient (a.i.)

the ITS sequences using the Genbank data base showed that
the fungal strains exhibited 100 % of identity to Rhizoctonia
Azoxystrobin

solani species AG3 group. The sequences were deposited in

Pencycuron

Fludioxonil

the GenBank website under the accessions numbers:

KF892037 (RS1.3), KF892038 (RS3.2), KF892039 (RS5.1)
and KF152934 (RS5.2).
Aggressiveness and fungicide sensitivity of R. solani

Fig. 1 Morphology of
Rhizoctonia solani sclerotia
(yellow arrows) on infected
potato tuber (a). One month old
R. solani culture (PDA medium)
showing the production of
sclerotia (red arrows) (b).
Colourless mycelia observed
under light (c) and electronic
scanning (d) microscopes (white
arrow: indicates constriction of
the mycelium at the brunching
point). Bars = 1 cm in (a) and (b)
and 20 μm in (c) and (d)

Effect of temperature on R. solani mycelia growth
All R. solani strains showed a maximum of growth at 25 °C on
PDA medium in darkness (Fig. 2). At this optimal temperature
the studied R. solani strains did not relatively show differences
in their growth. Better growth differentiation between R. solani
strains was observed at 20 °C in comparison to the other
studied temperatures (15, 25, 30 and 36 °C). In general, the
R. solani strains showed better growth in temperatures bellow
25 °C in comparison to those superior to this temperature. The
strains RS5.1 and RS5.2 showed better tolerance to temperature
variation between 20 °C and 30 °C in comparison to the other
strains. In fact, they showed the least variation in their growth in
comparison to the other strains when varying temperature be-
tween 20 and 30 °C. The strains RS1.2, RS1.3, RS2.2, RS2.3
did not show any growth at 36 °C after 7 days of culture
(Fig. 2). In order to verify if this temperature was lethal or not
for these strains, their culture plates were transferred to 25 °C.
All tested strains showed re-growth after transferring from 36 to
25 °C, but their speed of growth was decreased in comparison
to their respective control (25 °C–25 °C) (Fig. 3). The two
strains RS5.1 and RS5.2 showed the higher speed of mycelia
re-growth after temperature recovery in comparison to the other
strains (Fig. 3).

Fig. 2 Effect of temperature on

Rhizoctonia solani mycelia
growth 2 days after sub-culturing
on PDA medium in darkness.
Bars represent standard errors
 N. Djébali et al.

Fig. 3 Effect of temperature

recovery on the speed of
Rhizoctonia solani mycelia re-
growth at 2 days on PDA medium
in darkness. Bars represent
standard errors

Effect of R. solani on potato mineral nutrition and yield
Typical disease symptoms include cankers on underground
stems (Fig. 4a) and stolons and sclerotia formation on progeny
tubers (typical black scurf sclerotia) and at the base of stem
were observed at 3 months post infection of potato plants (cv.
Nicola) (Fig. 4b,c). The formation of tuber born sclerotia was
associated in some cases with the development of malformed
(misshapen) tubers and an alteration in the target size and the
number of tubers (Fig. 4d). Some infected plants showed the
formation of purple tubers at leaf axils (Fig. 4f), which was
associated with severe rotting at base of the stem.
All studied strains decreased potato shoot dry weight in infect-
ed versus control plants (Table 4). The dosage of nitrogen (N),
potassium (K) and phosphorus (P) in the vegetative part of potato
plants showed that N and K were significantly higher in R. solani
infected plants in comparison to the control; however the opposite
behaviour was noticed for the P element. The infection of potato
plants with the eight R. solani strains caused the formation of stem
cankers at the base with different percentage and level of

Fig. 4 Symptoms of Rhizoctonia solani infection and sclerotia production on the basis of stem (a, b) and progeny potato tubers (c) of the cultivar Nicola
3 months post inoculation. Malformed progeny tubers (d), non infected tubers (e), purple tubers at leaf axils (f). Bars = 1 cm
Aggressiveness and fungicide sensitivity of R. solani

57.8 (±12.2) ab
60.1 (±11.5) ab

70.8 (±11.9) ab
50.4 (±10.3) b
64.4 (±7.7) ab
58.3 (±8.8) ab
Infection with
infection. The strains RS3.2 and RS5.2 caused superior percent-

83.3 (±8.3) a

0.0 (±0.0) c
67.7 (8.3) ab
sclerotia (%)
age and level of cankers on stem and percentage of tuber infection
with sclerotia in comparison to the other strains. Although, only
two R. solani strains (RS1.1 and RS5.2) caused significant de-
crease in tuber fresh weight per plant, the other strains did not
Fresh weight per

124.8 (±12.7) bc
139.0 (±4.8) abc
139.5 (±6.3) abc
152.8 (±7.7) abc

148.5 (±5.0) abc

145.0 (±5.6) abc
reduced significantly the plant production (Table 4).

164.3 (±6.7) ab

170.0 (±3.8) a
112.5 (±9.0) c
The correlation between the measured parameters showed
that the disease severity expressed by the percentage of stem
plant (g)

with cankers, the level of cankers on stem and the percentage

of progeny tubers with sclerotia, was not associated with tuber
6.3 (±0.5) bc fresh weight reduction per plant (Table 5). However, the
8.5 (±0.5) ab
8.3 (±0.4) ab

6.3 (±0.4) bc
Number per

9.3 (±0.2) a
5.3 (±0.4) c

9.5 (±0.3) a

8.8 (±0.4) a
9.5 (±0.4) a

percentage of stem with cankers was positively associated to

shoot N (r=0.69) and K (r=0.72) contents and the level of
Tuber

plant

cankers on stem was positively associated to shoot N (r=0.82)

content and negatively associated to shoot P content (r=−0.71).
The percentage of progeny tubers infected with sclerotia was
Level of cankers

positively correlated to shoot N (r=0.74) and K (r=0.86)

1.8 (±0.2) ab
1.3 (±0.4) ab

1.5 (±0.3) ab
1.3 (±0.2) ab
1.3 (±0.3) ab
0.8 (±0.2) b

1.0 (±0.2) b

0.0 (±0.0) c
2.5 (±0.1) a

contents and negatively correlated to shoot dry weight

on stems

(r=−0.78) and shoot P content (r=−0.81). The percentage of

progeny tubers infected with sclerotia was positively correlated
to the percentage of stem with cankers (r=0.82) and the level of
58.3 (±12.5) ab

cankers on stem (r=0.81) (Table 5). Potato shoot dry weight

41.6 (±10.4) b

41.6 (±10.4) b
33.3 (±11.7) b
31.2 (±11.8) b

48.3 (±8.8) ab

82.5 (±5.9) ab
100.0 (±0.0) a

0.0 (±0.0) c

was negatively associated to shoot K content (r=−0.80) and

cankers (%)
Stem with

positively associated to P shoot content (r=0.79) (Table 5).

Effect of R. solani on faba bean growth, blooming

and nodulation
4.0 (±0.7) a

3.5 (±0.6) a
3.5 (±0.3) a
3.5 (±0.5) a
2.3 (±0.2) a
2.8 (±0.1) a

3.0 (±0.5) a

3.3 (±0.1) a
4.5 (±0.3) a
per plant
Number
Table 4 Effect of Rhizoctonia solani strains on potato growth, shoot mineral content and yield

Means in the same column followed by the same letter are not different at p≤0.05 (LSD test)

In order to evaluate the specificity of infection of the 8

Stem

R. solani strains, they were inoculated on roots of a local faba

bean cultivar usually used in crop rotation with potato in
1.1 (±0.3) ab
1.5 (±0.1) ab
1.7 (±0.2) ab
1.3 (±0.3) ab

1.7 (±0.4) ab
0.4 (±0.0) b
0.8 (±0.2) b
0.9 (±0.3) b

Tunisia. The results showed that the majority of the studied

2.6 (±0.4) a
Phosphorus
(mg/g DW)

R. solani strains (7/8) had a minor effect on the measured

parameters on faba bean inoculated plants (Table 6). Only the
strain RS5.2 caused significant decrease in 5 out of 7 mea-
sured parameters on faba bean inoculated plants (Table 6). In
21.3 (±2.8) ab
21.7 (±3.5) ab
24.8 (±2.7) ab
21.6 (±3.7) ab

23.9 (±4.7) ab

21.1 (±2.5) ab
15.5 (±1.7) b

28.1 (±5.3) a

7.7 (±0.3) c

fact, this R. solani strain caused a decrease in faba bean stem,

(mg/g DW)
Potassium

leaf and flower numbers, shoot dry weight and nodule number
per plant. To satisfy Koch’s postulates, fragments of faba bean
roots and nodules showing symptoms of browning were sur-
face sterilised, cut in two pieces and placed on PDA medium.
15.0 (±0.6) ab
15.1 (±0.2) ab
15.6 (±0.3) ab

15.8 (±0.6) ab

15.5 (±0.3) ab
13.5 (±0.4) b
13.5 (±0.3) b

16.3 (±0.3) a
10.9 (±0.5) c

All studied R. solani strains were successfully re-isolated from

(mg/g DW)
Nitrogen

root parts at an average frequency of 21.9 %. No fungal

colonies were observed on any of the non-inoculated control
root and nodule fragments.
Dry weight per

Effect of fungicides on R. solani mycelia growth

2.8 (±0.3) b

3.0 (±0.3) b
2.4 (±0.2) b
3.1 (±0.3) b
3.4 (±0.1) b
3.2 (±0.1) b
2.3 (±0.3) b

3.4 (±0.1) b

4.8 (±0.1) a
plant (g)
Shoot

In order to have measures of control against R. solani attack in

potato crops in Tunisia, we tested the sensitivity of this fungal
pathogen to three commercial fungicides, containing different
Control
Fungal

RS3.2
RS1.1
RS1.2

RS5.2
RS1.3
RS2.2
RS2.3

RS5.1

active ingredients with different mode of actions: fludioxonil,

strain

pencycuron and azoxystrobin, recommended for the control

 N. Djébali et al.

Infection with
sclerotia (%)
of potato black scurf disease (Table 3). The results showed
that the three fungicides inhibited the growth of the tested
R. solani strains in vitro, but at different levels. All tested

1
R. solani strains showed a typical S-shaped dose–response
relationship between growth inhibition and fungicide concen-
Fresh weight per

tration of Fludioxonil and Pencycuron (Fig. 5) with a low

EC50 values about 0.007 μg a.i. ml−1 and 0.006 μg a.i. ml−1,

−0.57 ns
plant (g)

respectively (Table 7). The complete growth inhibition of the

four R. solani strains occurred at 3 μg a.i. ml−1 and 0.1 μg a.i.

1
ml−1 for pencycuron and fludioxonil, respectively. The four
Number per

R. solani strains presented in general the same behaviour of

−0.43 ns
0.82**
Tuber

sensitivity to these two fungicides. The R. solani strains

plant

1 showed some dose response to azoxystrobin, but EC50 values

could not be accurately estimated because growth was not
Level of cankers

inhibited by more than 50 %, except for RS2.2 strain, even on

the highest concentration tested (30 μg a.i. ml−1) (Fig. 5). The
−0.32 ns
−0.44 ns
0.81**
on stem

EC50 values of these strains clearly exceeded 30 μg a.i. ml−1

1

and were considered to be resistant to azoxystrobin (Table 7).

cankers (%)
Stem with

−0.45 ns
−0.45 ns
0.86**

0.82**

Discussion
1

Black scurf caused by R. solani is one of the most damaging

Number per

diseases of potato worldwide. In Tunisia, this pathogen caused

ns

ns
ns
ns

ns

severe attacks on potato crops especially in the south region

−0.46
−0.30
0.27
0.20

0.05
Stem

plant

which is suitable for potato production destined for export to

European countries. The morphological and molecular char-
Phosphorus
(mg/g DW)

acterization showed that the isolated strains from potato tuber

0.16 ns
−0.66 ns

0.58 ns

−0.81**
0.78*
−0.71*

born sclerotia belong to R. solani AG3, which is the most

1

prevalent and damaging AG in potato worldwide, probably

due to global trading of tubers as suggested by Tsror (2010).
(mg/g DW)
Potassium

0.55 ns
−0.29 ns

−0.30 ns
−0.40 ns

To the authors’ knowledge, this is the first report of AG3

0.86**
−0.75*

0.72*

infecting potato in Tunisia. The studied R. solani strains

Table 5 Pearson’s correlation coefficient (r) between measured parameters

showed a maximum of in vitro growth on PDA medium at

25 °C, confirming the results of Ritchie et al. (2009). The local
(mg/g DW)

0.61 ns
−0.55 ns

−0.18 ns
−0.26 ns
0.06 ns
Nitrogen

0.82**

R. solani strains showed better growth in cool (15–20 °C)

0.69*

0.74*

versus hot (30–36 °C) temperatures, indicating that planting

1

potato when the soil is cool can increase the attacks of plants
by the pathogen as indicated by El Bakali and Martín (2006).
Dry weight per

It appears form our data that the R. solani strains isolated from
−0.59 ns
−0.55 ns
0.41 ns
0.66 ns
−0.62 ns

0.04 ns
plant (g)

−0.80**

the Gafsa region in the South (RS5.1 and RS5.2) are more
−0.78*
0.79*
Shoot

adapted to high and large amplitude variations of tempera-

1

tures, likely indicating an adaptation of these strains to the

**highly significant (0.01≥p>0.001)
Infection with sclerotia (%)

desert climate of the Gafsa region. This hypothesis will be

Fresh weight per plant (g)
Level of cankers on stem
Dry weight per plant (g)

Phosphorus (mg/g DW)

deeply investigated by increasing the R. solani collection from

Stem with cankers (%)
Potassium (mg/g DW)
Nitrogen (mg/g DW)

different potato cropping areas in Tunisia.

*significant (0.05≥p>0.01)
ns not significant (p>0.05)
Number per plant
Number per plant

The inoculation of the potato cultivar Nicola with each of the

eight R. solani strains caused cankers on underground stems
Parameters

and sclerotia formation on progeny tubers (typical black scurf

sclerotia). Disease severity was not associated with yield reduc-
tion; however, formation of tuber-borne sclerotia downgrades
Tuber
Shoot

tuber quality, causing development of misshapen tubers and an

Stem

alteration in the target size of tubers (El Bakali and Martín

Aggressiveness and fungicide sensitivity of R. solani

Table 6 Effect of Rhizoctonia solani strains on faba bean growth, blooming and nodulation

Fungal strain Stem number Leaf number Shoot dry Time for appearance Flower number Root dry Nodule number
per plant per plant weight (g) of the 1st flower at full blooming weight (g) per plant

RS1.1 4.3 (±1.0) ab 24.5 (±6.6) ab 6.5 (±1.4) ab 46.5 (±3.0) a 16.0 (±8.0) c 3.1 (±2.0) a 68.8 (±36.0) ab
RS1.2 2.5 (±0.6) b 23.5 (±11.6) ab 5.5 (±1.9) ab 43.0 (±6.6) a 16.5 (±5.4) bc 2.7 (±1.4) a 62.5 (±31.2) ab
RS1.3 3.8 (±1.0) ab 23.3 (±5.0) ab 6.6 (±2.3) ab 41.5 (±5.7) a 26.8 (±2.9) bc 1.7 (±1.1) a 30.0 (±0.0) b
RS2.2 3.8 (±2.5) ab 22.0 (±8.9) ab 6.7 (±2.2) ab 44.0 (±3.5) a 16.3 (±12.1) bc 2.1 (±2.0) a 56.3 (±22.5) ab
RS2.3 3.5 (±1.7) ab 22.0 (±5.8) ab 4.9 (±1.2) ab 48.0 (±0.0) a 15.3 (±1.5) c 2.3 (±1.3) a 47.5 (±35.0) ab
RS3.2 3.5 (±1.3) ab 27.3 (±8.7) ab 6.5 (±2.6) ab 39.8 (±9.0) a 25.0 (±17.7) bc 2.8 (±2.4) a 48.8 (±18.9) ab
RS5.1 2.8 (±1.5) ab 19.5 (±5.8) b 4.0 (±1.0) b 39.0 (±10.0) a 30.8 (±6.7) ab 1.4 (±0.7) a 47.5 (±35.0) ab
RS5.2 2.0 (±0.0) b 18.0 (±3.3) b 4.5 (±1.4) b 41.3 (±7.0) a 13.6 (±9.6) c 1.3 (±0.7) a 41.3 (±7.5) b
Control 5.0 (±0.8) a 33.3 (±10.7) a 7.5 (±1.1) a 41.0 (±8.1) a 42.3 (±7.9) a 2.1 (±1.1) a 82.5 (±35.0) a

Means in the same column followed by the same letter are not different at p≤0.05 (LSD test)

2006). However, diseases symptoms on stem and progeny
tubers were associated to an increase in N and K and to a
decrease in P contents in potato shoots, indicating that R. solani
creates an imbalance in the nutritional statute of the potato
plants. Under disease pressure plants evolve numerous defence
mechanisms including the synthesis of several nitrogen com-
pounds such as alkaloids, proteins (defensive) and enzymes (ex.
Chitinases) to fight against the pathogen. This synthesis of
nitrogen compounds requires the uptake of N from soil, mainly
nitrate (NO3−) as potato plants are nitrophilic, and uptake of K
as an essential compound for enzyme activity and leaf protein
synthesis. Also, it is well known also that N uptake is positively
mediated by K levels in the soil solution (Hagin et al. 1990).
Conversely, the increase in NO3− uptake compared to ammo-
nium (NH4+) likely causes alkalinisation of the rhizosphere soil
which result in a decreased availability and uptake of soluble
nutrients such as inorganic phosphorus (Pi) (Ruan et al. 2000;
Hessini et al. 2009) added to that its low availability due to slow
diffusion and high fixation in soils (Shen et al. 2011). So these
statements probably explain, at least in part, the increase of N
and K and the decrease of P in R. solani infected potato plants.
In accordance, Filippi and Prabhu (1998) showed that rice
panicle N content was positively correlated with panicle blast
(causal agent Pyricularia grisea) severity and Graham (1983)
showed that low P levels tend to increase disease incidence in
particular for fungal diseases such as powdery mildew and
Pythium root rot. Thus, to avoid such an imbalance in the
nutritional statues of plants in R. solani infected soils we should
increase the input of phosphate fertilizers making P more
available for plants. In addition, we observed for some plants
the formation of aerial tubers associated with severe rotting on
the stem base. The pathogen probably destroy the vascular
tissues starting by the phloem because it is at the periphery of
the stele causing the interruption of the photosynthates flux
from leaves to underground parts of the plants and conducing
to the formation of the areal tubers which may explain again the
increase in N and K levels in the shoot of infected potato plants.
In pathogenicity tests, the used R. solani strains showed a better
difference in their aggressiveness when assessing the severity of
the disease on stem (percentage of stem with cankers) in
comparison to tubers (percentage of tubers with sclerotia) of
the potato cultivar Nicola, even if there is a positive correlation
between the two parameters.
The majority of the studied potato R. solani AG3 strains
(AG3-PT) had a small effect on faba bean growth, blooming
and nodulation, nevertheless they were able to infect roots and
nodules of this legume species. Therefore, faba bean may
provide a suitable alternative host for R. solani and potentially
increase the risk of disease in subsequent potato crops. To our
knowledge, this is the first report of AG3-PT infecting faba
bean in Tunisia. Alternative hosts can support the long sur-
vival of R. solani in the fields where potato crops are grown.
Indeed, Tsror (2010) reported that R. solani AG-3 has been
isolated from the roots of barley, flax and sugar beet. In
pathogenicity trials, many other crops were susceptible to
AG-3 isolates, including different plant families such as
Fabaceae (bean, lucerne, clover, sweet clover), Poaceae
(wheat, maize, oats), Solanaceae (tobacco, tomato),
Asteraceae (lettuce, sunflower) Bassicaceae (radish, cauli-
flower), Apiaceae (carrot) and Amaryllidaceae (onion)
(Carling et al. 1986). In addition, R. solani AG-3 has been
isolated from the roots and stems of many weeds widespread
in potato fields (El Bakali et al. 2000). The large spectrum of
alternative hosts that can be either R. solani infected with
symptoms or latently infected has to be considered in the
integrated management of the disease e.g. by crop rotation
and sanitation.
The fungicide sensitivity assays showed that the local
R. solani strains were highly sensitive at low concentrations
to pencycuron (EC50: 0.006 μg a.i. ml−1) and fludioxonil
molecules (EC50: 0.007 μg a.i. ml−1). Fludioxonil proved
maximum efficacy in totally reducing R. solani in vitro growth
at 0.1 μg a.i. ml−1. So, to fight against R. solani it is recom-
mended to use fludioxonil and alternatively pencycuron for
 N. Djébali et al.

Fig. 5 Percentage of inhibition of

mycelia growth of four
Rhizoctonia solani strains after
3 days of culture on PDA medium
supplemented with three
fungicides fludioxonil (a),
pencycuron (b) and azoxystrobin
(c). Bars represent standard errors

potato seed treatment. Fludioxonil provide effective control of fludioxonil can be used for protection against seedborne and
Rhizoctonia stem canker and black scurf of potato in Canada soilborne fungi that cause seed decay, damping-off and seed-
(Bains et al. 2002). According to Zang et al. (2001), ling blight such as Fusarium spp. and Rhizoctonia spp.
Strobilurin fungicides including azoxystrobin inhibited mi-
tochondrial electron transferring by binding to the quinone
Table 7 The effective concentrations (EC50) of the fungicides that
inhibit 50 % of the growth of the Rhizoctonia solani strains
outside center of cytochrome bc1 complex and interfered with
ATP synthesis (Esser et al. 2004). Jin et al. (2009) showed that
Fludioxonil Pencycuron Azoxystrobin azoxystrobin was effective alone in inhibiting the in vitro my-
EC50 (μg a.i. ml−1)
celial growth of R. solani with EC50 value 0.008 μg a.i. ml−1.
RS1.2 0.006 0.005 > 30.000 However, our study indicated that the four studied Tunisian
RS2.2 0.007 0.004 19.894 R. solani strains showed high level of resistance to
RS3.2 0.005 0.014 > 30.000
azoxyxtrobin with EC50 value exceeding 19 μg a.i. ml−1.
RS5.2 0.010 0.004 > 30.000
Several plant pathogens, such as Magnaporthe grisea (Kim
et al. 2003), Septoria tritici (Ziogas et al. 1999), Botrytis cinerea
a.i. active ingredient (Tamura et al. 1999) and Venturia inaequalis (Zheng et al.
Aggressiveness and fungicide sensitivity of R. solani
2000), express resistance to strobilurin fungicides by the induc-
tion of alternative oxidase pathway, indicating that mitochon-
drial electron transfer with the alternative oxidase pathway
activity and thus, ATP synthesis is sustained by circumvention
of the cytochrome b site of QoI action (Joseph-Horne and
Hollomon 2000). Our data highlights the risk of resistance to
quinone outside inhibitor fungicides (QoI) also in R. solani
populations. Although up to now azoxystrobin was effective
in reducing black scurf of potato and no decrease in field
performance was noticed in Tunisia (Djébali and Belhassen
2010). It has been shown that alternative oxidase pathway,
which played an important role in vitro, did not decrease the
efficacy in the field application of QoIs (Kim et al. 2003). It is
assumed that plant antioxidants such as flavone presented in the
host quenched the active oxygen species from reaching the
level sufficient for inducing alternative oxidase pathway during
QoI action (Olaya and Köller 1999). Nevertheless, it is recom-
mended to delay build up of QoI resistance by an integrated
approach, combining optimised fungicides use (Ma and Uddin
2009) with the choice of R. solani resistant cultivars and the
application of farming practices (Djébali and Belhassen 2010).
In conclusion this study constitutes the first report of
R. solani AG3 infecting potato and faba bean in Tunisia. We
showed also that R. solani induce a nutritional imbalance in
infected potato plants which mainly affect phosphorus uptake.
Finally, the in vitro fungicide sensitivity assays showed that
the Tunisian R. solani strains were highly sensitive to
fludioxonil and pencycuron at low concentrations; but resis-
tant to azoxystrobin.
Acknowledgments Financial support of this work was obtained from
the Tunisian Ministry of High Education and Scientific Research and the
grant of the CTPTA-CBBC bilateral project 2009–2012.
References
Agrios GN (1988) Plant pathology. Academic, London
Bains PS, Bennypaul HS, Lynch DR, Kawchuk LM, Schaupmeyer CA
(2002) Rhizoctonia disease of potatoes (Rhizoctonia solani): fungi-
cidal efficacy and cultivar susceptibility. Am J Potato Res 79:99–106
Carling DE (1996) Grouping in Rhizoctonia solani by hyphal anastomo-
sis. In: Sneh B, Jabaji- Hare S, Neate S, Dijst G (eds) Rhizoctonia
species: taxonomy, molecular biology, ecology, pathology and dis-
ease control. Kluwer Academic Publishers, Dordrecht, pp 37–47
Carling DE, Leiner RH, Kebler KM (1986) Characterisation of Rhizoctonia
solani and binucleate Rhizoctonia-like fungi collected from Alaskan
soils with varied crop histories. Can J Plant Pathol 8:305–310
Carling DE, Rothrock CS, MacNish GC, Sweetingham MW, Brainard
KA, Winters SW (1994) Characterization of anastomosis group 11
(AG-i 1) of Rhizoctonia solani. Phytopathology 84:1387–1392
Carling DE, Baird RE, Gitaitis RD, Brainard KA, Kuninaga S (2002)
Characterization of AG13 a newly reported Anastomosis Group of
Rhizoctonia solani. Phytopathology 92:893–899
Daami-Remadi M, Zammouri S, El Mahjoub M (2008) Effect of the level
of seed tuber infection by Rhizoctonia solani at planting on potato
growth and disease severity. African J Plant Sci Biotechnol 2:34–38
Djébali N, Belhassen T (2010) Field study of the relative susceptibility of
eleven potato (Solanum tuberosum L.) varieties and the efficacy of two
fungicides against Rhizoctonia solani attack. Crop Prot 29:998–1002
El Bakali AM, Martín MP (2006) Black scurf of potato. Mycologist 20:
130–132
El Bakali MA, Martin MP, García FF, Moret BA, Nadal PM (2000) First
report of Rhizoctonia solani AG-3 on potato in Catalonia (NE
Spain). Plant Dis 84:806
Esser L, Quinn B, Li YF, Zhang MQ, Elberry M, Yu LD, Yu CA, Xia D
(2004) Crystallographic studies of quinol oxidation site inhibitors: a
modified classification of inhibitors for the cytochrome bc1 com-
plex. J Mol Biol 341:281–302
Filippi MC, Prabhu AS (1998) Relationship between panicle blast sever-
ity and mineral nutrient content of plant tissue in upland rice. J Plant
Nutr 21:1577–1578
Fleury P, Leclerc M (1943) Misson colorimetric nitro-vanado-molybdate
method for phosphorus determination. Its interest in biochemistry. B
Soc Chim Biol 25:201–205
Graham RD (1983) Effects of nutrient stress on susceptibility of plants to
disease with particular reference to the trace elements. Adv Bot Res
10:221–276
Hagin J, Olsen SR, Shaviv A (1990) Review of interaction of
ammonium-nitrate and potassium nutrition of crops. J Plant Nutr
13:1211–1226
Hessini K, Lachâal M, Soltani A (2005) Physiological response to sodi-
um chloride of wild Swiss Chard. J Plant Nutr 28:877–888
Hessini K, Lachaâl M, Cruz C, Soltani A (2009) Role of ammonium to
limit nitrate accumulation and to increase water economy in wild
Swiss Chard. J Plant Nutr 32:821–836
Hyakumachi M, Mushika TY, Ogosi Y, Toda T, Kageyama K, Tsuge T
(1988) Characterization of new cultural type of R. solani AG-2-2
isolated from warm season turf grass and its genetic differentiation
from other cultural type. Plant Pathol 47:1–9
Jin L, Chen Y, Chen C, Wang J, Zhou M (2009) Activity of azoxystrobin
and SHAM to four phytopathogens. Agric Sci China 8:835–842
Joseph-Horne T, Hollomon DW (2000) Functional diversity within the
mitochondrial electron transport chain of plant pathogenic fungi.
Pest Manag Sci 56:24–30
Keijer J, Korsman MG, Dullemans AM, Houterman PM, De Bree J, Van
Silfhout CH (1997) In vitro analysis of host plant specificity in
Rhizoctonia solani. Plant Pathol 46:659–669
Kim YS, Dixon EW, Vincelli P, Farman ML (2003) Field resistance to
strobilurin (QoI) fungicides in Pyricìlaria grisea caused by muta-
tions in the mitochondrial cytochrome b gene. Phytopathology 93:
891–900
Ma B, Uddin W (2009) Fitness and competitive ability of an
azoxystrobin-resistant G143A mutant of Magnaporthe oryzae from
perennial ryegrass. Plant Dis 93:1044–1049
Mahuku GS (2004) A simple extraction method suitable for PCR based
analysis of plant, fungal, and bacterial DNA. Plant Mol Biol Report
22:71–81
Olaya G, Köller W (1999) Baseline sensitivities of Venturia inaequalis
populations to the strobilirin fungicide kresoximmethyl. Plant Dis
83:274–278
Richter H, Schneider R (1953) Untersuchungen zur morphologischen
und biologischen Differenzierung von Rhizoctonia solani K.
Phytopathol Z 20:167–226
Ritchie F, Bain RA, McQuilken MP (2009) Effects of nutrient status,
temperature and pH on mycelial growth, sclerotial production and
germination of Rhizoctonia solani from potato. J Plant Pathol 91:
589–596
Ruan J, Zhang F, Wong MH (2000) Effect of nitrogen form and phos-
phorus source on the growth, nutrient uptake and rhizosphere soil
property of Camellia sinensis L. Plant Soil 223:63–71
Sharon M, Kuninaga S, Hyakumachi M, Sneh B (2006) The
advancing identification and classification of Rhizoctonia

spp. using molecular and biotechnological methods compared
with the classical anastomosis grouping. Mycoscience 47:
299–316
Shen J, Yuan L, Zhang J, Li H, Bai Z, Chen X, Zhang W, Zhang
F (2011) Phosphorus dynamics: from soil to plant. Plant
Physiol 156:997–1005
Tamura H, Mizutani A, Yukioka H, Miki N, Ohba K, Masuko M (1999)
Effect of the methoxyiminoacetamide fungicide, SSF129, on respi-
ratory activity in Botrytis cinerea. Pestic Sci 55:681–686
Tsror LJ (2010) Biology, epidemiology and management of Rhizoctonia
solani on Potato. J Phytopathol 158:649–658
White TJ, Brunsn SL, Taylorn JW (1990) Amplification and
sequencing of fungal ribosomal RNA genes for phyloge-
netics. In: Innis MA, Gefand DH, Sninsky JJ, White TJ
N. Djébali et al.
(eds) PCR protocols: a guide to methods and applications.
Academic Press, Inc., New York
Zang LE, Shetty KK, Watrin CG, Forster B (2001) Fludioxonil, a low use
rate seed treatment for the control of Fusarium on corn and potatoes.
Crop Prot 76:257–262
Zhang CQ, Yuan SK, Sun HY, Qi ZQ, Zhou MG, Zhu GN (2007)
Sensitivity of Botrytis cinerea from vegetable greenhouses to
boscalid. Plant Pathol 5:646–653
Zheng D, Olaya G, Köller W (2000) Characterization of laboratory
mutants of Venturia inaequalis resistant to the strobilurinrelated
fungicide kresoxim-methyl. Curr Genet 38:148–155
Ziogas BN, Baldwin BC, Young JE (1999) Alternative respiration: a
biochemical mechanism of resistance to azoxystrobin (ICIA5504)
in Septoria tritici. Pestic Sci 50:28–34