MACROPHOMINA PHASEOLINA ISOLATES FROM SUGAR BEET

MORPHOLOGICAL, CULTURAL AND
PATHOGENIC CHARACTERISTICS OF
MACROPHOMINA PHASEOLINA
ISOLATES FROM SUGAR BEET
Stojšin, V., Budakov, D., Bagi, F., Đuragin, N., Marinkov, R.
Department for Environmental and Plant Protection,
Faculty of Agriculture, University of Novi Sad, Trg Dositeja
Obradovida 8, 21000 Novi Sad, Serbia

INTRODUCTION
• Macrophomina phaseolina is a causing agent of charcoal root rot.
• In our country firstly described in 1967 on sugar beet in north and
central Banat.
• Significant occurrence was observed in 1971 in many localities in the
Vojvodina Province, and it was predominant in isolations from sugar
beet in 1992, 2003 and especially in 2009 (caused damages on over
50% sugar beet growing areas, estimated on app 2.5 million euro) -
(Stojšin et al., 1998, 1999, 2003, 2009, 2011).
• This fungus attacks sunflower, soybean, maize, rapeseed, tobacco,
alfalfa, red clover, potato, peas, beans, pepper, onion, cabbage,
watermelon, strawberry and many weed species.

Symptoms on sugar beet
• Start with wilting and usually end in complete
decay.
• Severely damaged roots in dry soils can become
mummified, while in moist soils rot can have a
wet appearance.

Symptoms on sugar beet
• Typical symptoms may appear on the crown, the central part and tip
of the root.
• Outer tissues become grayish brown to black with a silverish
reflection.
• Inner tissues turn into sponge like consistency, with colors ranging
from lemon yellow and finally brownish to black.

Epidemiology
• The pathogen survives in plant remains in the field as mycelia and
microsclerotia-overwintering structure.
• Microsclerotia may survive in the soil for many years and they are
always present in sufficient amount.
• Infection may occur on young roots, after which the pathogen spreads
through the vascular system, while characteristic symptoms appear
after plants are being stressed due to drought (Stojšin et al., 1999).
Ecology
• Lack of water and high soil temperatures (25-30 o C) are favorable
conditions for disease development.
• With more frequent growing seasons associated with high
temperatures and lack of precipitation this pathogen could become
predominant in the sugar beet root mycopopulation in Serbia, where
beets are primarily grown without irrigation (Stojšin et al., 2011).

• The aim of this research was to separate and
classify isolates of M. phaseolina on the basis
of their morphological and cultural
characteristics, as well as their pathogenicity
on young sugar beet plants.

MATERIAL AND METHOD
Collection of isolates
Isolates that were used in this research were chosen from the collection comprising
103 monohyphal isolates of M. phaseolina.
Sixteen isolates from sugar beet grown in leading production areas in Serbia were
tested, including one isolate from sunflower, soybean and maize each.
No Isolate Locality Host
1 ŠR 2/09 Žabalj Sugar Beet
2 ŠR 3/09 Sremska Mitrovica (Glac) Sugar Beet
3 ŠR 5/09 Vojka Sugar Beet
4 ŠR 7/09 Šimanovci Sugar Beet
5 ŠR 10/09 Boljevci Sugar Beet
6 ŠR 14/09 Orlovat Sugar Beet
7 ŠR 42/09 Jaša Tomid Sugar Beet
8 ŠR 45(4)/09 Pančevo Sugar Beet
9 ŠR 55(3)/09 Lepušnica Sugar Beet
10 ŠR 62/4 Ečka Sugar Beet
11 ŠR 9M/10 Ečka Sugar Beet
12 ŠR24M/10 Inđija Sugar Beet
13 ŠR 1/11 Beška Sugar Beet
14 ŠR 15/11 Beška Sugar Beet
15 ŠR 17/11 Ečka Sugar Beet
16 ŠR 23/11 Rimski Šančevi Sugar Beet
17 Mph Su Obtained from the Institute for Field and Vegetable Sunflower
18 Mph So Crops, Novi Sad
Soybean
19 MphKu Maize

Cultural and morphological
characteristics
• Cultural characteristics were evaluated on Potato Dextrose Agar (PDA)
and Minimal Media (MIN) using methods described by Pearson et al.
(1986).
• MIN was amended with chlorine by adding 0.015% of potassium
chlorate.
• Mycelial growth was evaluated after three days by measuring the
diameter of the culture.
• According to their growth patterns on MIN, isolates were divided into
three groups: dense, feathery and restricted growth pattern.
• The size of 100 microsclerotia was measured on all media after six
days.

Pathogenicity tests
• Inoculum was prepared by colonizing sorghum
seed with tested M. phaseolina isolates (Omar et
al., 2007).

Pathogenicity tests
• Sugar beet plants at the two leaf stage were
replanted in 500cm³ pots containing mixture of
sterile sand and inoculum in a 3:1 ratio (v/v).
• For the negative control, plants were grown in
non inoculated sterile sand.

Pathogenicity tests
• Plants were incubated in a growth chamber at 30°C with a
photoperiod of 16h/8h light/dark and watered daily.
• Symptom development on leaves was assessed daily,
while final evaluation of root and hypocotyl necrosis was
performed after eight days when over 50% of plants
showed symptoms of irreversible wilting.

Pathogenicity tests
Symptom
Healthy plant 0
Individual lesions on hypocotyl and root 1
Merging of lesions on hypocotyl and root 2
Section necrosis on hypocotyl and root 3
Complete necrosis of hypocotyls and root 4
Grade
• Based on average pathogenicity isolates were divided into
three groups:
i) low pathogenic 0-1,
ii) moderately pathogenic 1-2,
iii) highly pathogenic 2-4.

Data analysis
• Data were analyzed by analysis of variance (ANOVA)
using Statistica 10 (StatSoft, Tulsa, OK).
• Comparisons between means were made with
Duncan’s Multiple Range Test at significance level of
5%.

Table 3. Influence of media on mycelial growth and chlorate sensitivity of M. phaseolina
Mycelial growth (mm)
Isolates PDA Rank MIN+ Cl Rang Chlorate type
ŠR 2/09 90,00 C 40,56 C Sensitive
ŠR 3/09 90,00 C 42,33 C Sensitive
ŠR 5/09 90,00 C 37,33 C Sensitive
ŠR 7/09 78,00 A 22,17 B Sensitive
ŠR 10/09 90,00 C 63,00 D Sensitive
ŠR 14/09 90,00 C 16,50 B Sensitive
ŠR 42/09 90,00 C 24,83 B Sensitive
ŠR 55(3)/09 90,00 C 61,50 D Sensitive
ŠR 62/4 90,00 C 24,77 B Sensitive
ŠR 9M/10 90,00 C 60,33 DE Sensitive
ŠR24M/10 90,00 C 0,00 A Sensitive
ŠR 1/11 90,00 C 60,17 D Sensitive
ŠR 15/11 90,00 C 48,17 C Sensitive
ŠR 17/11 90,00 C 79,00 F Sensitive
ŠR 23/11 90,00 C 45,83 C Sensitive
Mph Su 80,67 B 0,00 A Sensitive
Mph So 90,00 C 0,00 A Sensitive
MphKu 90,00 C 18,50 B Sensitive
Dense
Feathery
Restricted

Diameter of microsclerotia

Pathogenicity

DISCUSSION:
• M. phasolina, causal agent of charcoal root rot is a plant pathogenic
fungus that causes great damages in arid regions. Additionally,
drought stress increases plant susceptibility to this fungus (Mayek-
Perez et al., 2002).
• Since M. phaseolina is highly polyphagous attacking over 500 species
(Dhingra and Sinclair, 1978), variations in chlorate sensitivity,
microclerotial size and formation as well as pathogenicity are not
surprising (Pearson et al., 1987).
• No pattern was detected in comparison of growth patterns on PDA
and MIN, microsclerotia formation on both media and pathogenicity
on sugar beet plants between isolates from sugar beet and other
hosts. Since various authors reported that M. phaseolina isolates
from different hosts can be differentiated using chlorate resistance
(Pearson et al., 1986; Rayatpanah et al., 2009), this implies the need
to increase the number of isolates from other hosts.

DISCUSSION:
• Identifying differences in pathogenicity among isolates is the most useful
tool for grouping isolates. Based on our results, isolates can be divided
into 3 groups: low pathogenic – 5 isolates (26.3%), moderately and highly
pathogenic – 7 isolates (36.8%) for each group.
• However, there are scarce data on occurrence and significance of
Macrophomina phaseolina in sugar beet, so the aim of this research was
to describe cultural, morphological and pathogenic characteristics of
isolates from sugar beet and compare them to isolates from other hosts
(sunflower, soybean and maize).

Discussion
• Preliminary studies showed that there are differences in
pathogenicity of isolates from sugar beet, and even more in
isolates that are from other hosts (sunflower, soybean,
maize). Regarding the fact that M. phaseolina is a
polyphagous and always present in the soil, and
environmental factors are hard to control in sugar beet
production without irrigation, one of solutions is breeding for
resistance (Stojšin et al., 2012).
• The described method for artificial inoculation can be used as
the optimized method for pathogenicity testing of large
number of isolates from Serbian sugar beet growing areas,
which enables further research of M. phaseolina population
as well as fast testing of sugar beet genotypes that are grown
in our region.