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 oC) 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

Crops, Novi Sad Sunflower

18 Mph So 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.

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

Pathogenicity tests

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

• 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.

Symptom Grade

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

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

Feathery

Restricted

Dense

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

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.