Indian Phytopath. 66 (4) : 341-350 (2013)
RESEARCH ARTICLE
Variability in Rhizoctonia solani (AG-1 IA) isolates causing sheath blight of rice in India K. SUSHEELA* and C.S. REDDY National Institute of Plant Health Management, Hyderabad 500 033, Andhra Pradesh, India ABSTRACT: Variability in 35 isolates of Rhizoctonia solani Kühn [Teleomorph: Thanatephorus cucumeris (Frank) Donk] causing sheath blight of rice and weeds, was examined by polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) analysis in conjunction with morphology and pathogenicity studies. Studies on morphological characterization of R. solani isolates showed that isolates were highly variable both in mycelial and sclerotial parameters, with no consistent characters related to virulence, origin, production system or geographic origin. Pathogenicity of R. solani was also evaluated and based on their virulence, isolates were classified into different virulent groups. The similarity values of RAPD profiles ranged from 0.37 to 0.85 with an average of 0.61 among all the isolates. The percentage polymorphism detected per primer varied from 76.9 to 100% which suggested their utility in DNA finger printing of the sheath blight isolates. Relation between cultural/morphological characteristics, aggressiveness and genetic variation based on RAPD markers, and possible reasons for high intra-group variability are discussed. Keywords: Genetic variation, morphology, RAPD, Rhizoctonia solani, rice, virulence
Sheath blight of rice caused by Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) has become a major constraint to rice production during the last two decades. The pathogen is cosmopolitan with a very wide host range and attacks large number of crop plants and weeds (Ou, 1972). It is claimed that there is hardly any plant species, which cannot be infected by R. solani (Singh et al., 1999). Further, the fungus has worldwide distribution (Ogoshi, 1987) and isolates of R. solani are highly variable in aggressiveness. Although earlier studies suggested that AG-1 IA represented a homogenous group of R. solani (Kuninga and Yokosawa, 1982), recent investigations support the hypothesis that the sheath blight pathogen is far more diverse than previously assumed (Neeraja et al., 2002; Singh et al., 2002; Yu et al., 2003, Susheela et al., 2004). Diversity within rice sheath blight isolates has been studied by morphological characterization (Vijayan and Nair, 1985), pathogenecity testing (Jones and Belmar, 1989) and various molecular techniques (Banniza et al., 1999; Pascual et al., 2000; Yi et al., 2002a,b; Neeraja et al., 2002; Singh et al., 2002; Yu et al., 2003). Isolates from rice plants infected with sheath blight have been assigned to AG-1 (Vijayan and Nair, 1985; Borthakur and Addy, 1988). Rhizoctonia solani, belongs to anastomosis group 1 intraspecific group IA (AG-1 IA). Further, the understanding of disease epidemiology and host-pathogen interaction is greatly dependent on knowledge of the diversity of the pathogen at the field level (Banniza et al., 1999). The methods used for characterization of fungal plant pathogens generally fall in two groups, (i) ‘genetic’, based on traditional genetics, DNA sequences and polymorphisms, and (ii) ‘functional’, *Corresponding author:
[email protected]
based on particular phenotypic activities, such as secondar y metabolism, enzyme activities or pathogenicity. Bridge et al. (1995) concluded that integration of genetic techniques with functional characters could provide a powerful tool for characterization of fungal plant pathogens, particularly in respect of host and cultivar-specific populations such as weeds. Therefore, though the pathogen is cosmopolitan with a very wide host range, isolates attacking different crops have been grouped as anastomosis groups. Recent studies based on such an approach include Rhizoctonia solani isolates from rice (Singh et al., 2002). With the advent of various molecular marker technologies, studies on genetic diversity in plant pathogens have become feasible (Neeraja et al., 2002). Therefore, studies were undertaken to characterize R. solani isolates at three levels i.e., morphology, pathogenecity and genetic.
MATERIALS AND METHODS Sheath blight infected rice and weed plants showing typical sheath blight symptoms were collected from the farmers’ fields at 35 different agro-ecological regions of India, where rice is an important staple crop. The pathogen was isolated from these samples (Johnson and Sekar, 2012) and named with their respective acronyms. The list of R. solani isolates with their respective sources of collection is given in table 1. Morphological characterization Isolates of R. solani were subcultured on PDA poured Petri dishes in triplicate and incubated at 25oC for 3-5 days. Mycelial and sclerotial parameters were recorded for each isolate.
Indian Phytopathology 66 (4) : 341-350 (2013)
342 Table 1. Rhizoctonia solani isolates and source of collection Isolate acronym
Location
State / UT
Rice Variety / Weed
ACT
Achanta
Andhra Pradesh
Swarna
MDP
Mandapaka
Andhra Pradesh
Swarna
MTU
Maruteru
Andhra Pradesh
Swarna
MTU-W
Maruteru
Andhra Pradesh
Echinochloa crusgalli
MED
Medchal
Andhra Pradesh
BPT 5204
MYG
Miriyalaguda
Andhra Pradesh
BPT 5204
NZB
Nizamabad
Andhra Pradesh
BPT 5204
RNR
Rajendranagar
Andhra Pradesh
BPT 5204
RCP
Ramchandrapuram
Andhra Pradesh
Swarna
TGM
Tadepalligudem
Andhra Pradesh
Swarna
TNK
Tanuku
Andhra Pradesh
Swarna
GRA
Gerua
Assam
Swarna
JDP
Jagdalpur
Chattisgarh
Swarna
JDP-w
Jagdalpur
Chattisgarh
Panicum repens
RPR
Raipur
Chattisgarh
Chapti
KUL
Kaul
Haryana
PR 106
MDY
Mandya
Karnataka
Mandya Vijaya
MCP
Moncompu
Kerala
Jyothi
MCP-w
Moncompu
Kerala
Echinochloa crusgalli
PTB
Pattambi
Kerala
Jyothi
BPN
Barapani
Meghalaya
Swarna
BBN-w
Bhubaneshwar
Orissa
Cynodon dactylon
CHP
Chiplima
Orissa
Swarna
GDP
Gurudaspur
Punjab
PR 113
KPT
Kapurthala
Punjab
PR 113
LUD
Ludhiana
Punjab
PR 113
CBT
Coimbatore
Tamil Nadu
IR 50
PDY
Pondicherry
Union Territory
White Ponni
TRP
Tripura
Union Territory
IR 50
FZB
Faizabad
Uttar Pradesh
Pusa Basmati 1 Pusa Basmati 1
MSD
Masoda
Uttar Pradesh
ALM
Almora
Uttaranchal
VLK 39
PNT
Pantnagar
Uttaranchal
Pant Dhan 4
BNK
Bankura
West Bengal
Swarna
CHN
Chinsurah
West Bengal
Swarna
Pathogenicity test and virulence analysis Plants of the susceptible cultivar (IR 50) were inoculated at maximum tillering stage with R. solani colonized typha pieces and were maintained in the glass house at 80 to 90% relative humidity. They were regularly examined for the appearance of the symptoms. The disease severity was assessed seven days after inoculation. Disease development was recorded by measuring both horizontal (No. of tillers affected) and vertical spread (0-9 scale) of the disease and the disease index was calculated by integrating both the phases of disease development. Other disease parameters related to mycelium, lesion and sclerotia were also observed. Experiments were carried out in four replications per isolate. Pathogenecity index of 70-90% is considered as highly virulent; 50-69%
as virulent; 30-49%, as moderately virulent and 10-29% as least virulent. Genomic DNA extraction and purification Fungal DNA was extracted from the lyophilized fungal mycelium employing CTAB method. RAPD analysis For RAPD analysis, a set of 25 primers obtained from Nucleic Acid-Protein Service (NAPS) Unit, University of British Columbia, Canada were selected randomly. Each amplification was set at 25 µl. Following cocktail mixture was prepared in a sterile PCR tube: 6.0 µl DNA template (5 ng/µl); 2.0 µl dNTPs (2.5 mM each), 0.15 µl Taq DNA
Indian Phytopathology 66 (4) : 341-350 (2013)
polymerase (5 u/µl); 2.0 µl 10x Reaction buffer containing 10 mM Tris-Cl (pH 8.3), 50 mM KCL, 15 mM MgCl2, 0.01% gelatin and 1.2 µl primer (5 ng/µl). Final volume was made up to 25 µl using sterilized distilled water. Amplification conditions in thermocycler (MJ Reasearch, PTC-200) were set as follows: 10 sec. at 94oC followed by 35 cycles of 1 min. at 94oC, 1 min. at 37oC and 2min. 40 sec. at 72oC, with a final extension of 7 min. at 72oC. RAPD reproducibility was confirmed by repeating the reactions at least twice. A 10 µl volume of each reaction mixture was electrophoresed on 1.5% agarose gel run at constant voltage (6 v/cm) in 0.5 x TBE and stained with ethidium bromide (10 µg/ml). The DNA marker used was 1Kb ladder. The gels were photographed under UV light with Polaroid film 667. Data analysis All gels were scored twice manually and independently. Band presence was indicated by 1 and its absence by 0. Presence or absence of unique and
343
shared polymorphic bands were used to generate similarity coefficients (Dice coefficient). The similarity coefficients were then used to construct a phenogram by SAHN (sequential agglomerative hierarchal nested)/ UPGMA (Unweighted pair-group method with arithmetical averages) using a computer Programme NTSYS-PC (Numerical taxonomy system computer) version 2.11a (Rohlf, 2000).
RESULTS AND DISCUSSION Morphological characterization An attempt was made to group all the isolates based on mycelial and sclerotial dimensions as per Singh and Singh (1994). The isolates selected for morphological studies showed a continuum of variability in type of mycelial dispersion, position of sclerotia, colour of sclerotia, clump formation of sclerotia, mycelial growth rate, size of sclerotia and number of sclerotia (Table 2). A weed isolate from a sheath blight endemic location (MTU-w) did not produce any sclerotium.
Table 2. Mycelial and sclerotial characteristics of R. solani isolates on PDA medium Mycelial Characters a. Type of dispersion Aggregated Moderate Spatial b. Growth rate* Fast Moderate Slow Sclerotial Characters a. Position Periphery Center Periphery & Center Scattered b. Colour Deep dark brown Dark brown Light brown c. Clump formation More Moderate Less Absent d. Size* Macro Micro e. Number* More Moderate Less
ALM, BBN-w, CHP, CBT, FZB, GDP, JDP, JDP-w, MDP, MTU-w, MED, MCP, MCP-w, NZB, PNT, PDY, RNR, RCP, TNK & TRP BNK, BPN, KPT, KUL, MDY, MTU, MYG, PTB, RPR & TGM ACT, CHN, GRA, LUD & MSD ACT, CHN, CHP, CBT, FZB, KPT, LUD, MTU, MTU-w, MSD, MED, MCP, RPR, TGM, TNK & TRP ALM, BNK, BPN, BBN-w, GRA, GDP, JDP, MDP, MDY, MYG, NZB, PTB & RNR JDP-W, KUL, MCP-w, PNT, PDY & RCP
ALM, BNK, BPN, CBT, FZB, GRA, JDP-w, MDP, MYG, MCP, PNT & PTB BBN-w, MTU & MCP-w ACT, CHN, JDP, NZB, PDY, RNR, RCP, TNK & TRP CHP, GDP, KPT, KUL, LUD, MDY, MSD, MED, RPR & TGM ALM, BBN-w, CBT, FZB, GRA, JDP-w, KUL, MED, MCP-w, PNT & TRP ACT, CHN, GDP, KPT, LUD, MDP, MYG, NZB, RCP, TGM & TNK BNK, BPN, CHP, JDP, MDY, MTU, MSD, MCP, PTB, PDY, RPR & RNR BBN-w, CBT, FZB, KUL, MCP-w & TRP ACT, BNK, BPN, CHN, CHP, GDP, JDP-w, MDP, MSD, MYG, PDY, RCP & TNK ALM, GRA, JDP, KPT, MDY, MED, MCP, NZB, PNT, PTB, RNR & TGM LUD, MTU & RPR ACT, ALM, BNK, BPN, BBN-w, CHP, JDP, KPT, LUD, MTU, MSD, MED, MYG, PNT, PTB, PDY, TGM & TRP CHN, CBT, FZB, GRA, GDP, JDP-w, KUL, MDP, MDY, MCP, MCP-w, NZB, RPR, RNR, RCP & TNK JDP-w, KPT, MDY & MSD ACT, BNK, BBN-w, CHN, CBT, FZB, GRA, JDP, KUL, LUD, MDP, MED, MYG, MCP, MCP-w, NZB, PTB, PDY, RPR, RNR, RCP, TNK & TRP ALM, BPN, CHP, GDP, MTU, PNT & TGM
*In case of mycelial growth rate, size and number of sclerotia, isolates were grouped based on numerical data (circumference of mycelial growth, surface area of the sclerotia and no. of sclerotia)
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Studies on characterization of R. solani isolates showed that, morphologically, isolates were highly variable, with no consistent characters that related to origin, production system or geographic origin which is in accordance with the studies conducted by Banniza et al. (1999). Pathogenicity test and virulence analysis All the test isolates of R. solani belonging to AG-1 IA exhibited varying degree of virulence on IR-50, a sheath blight susceptible variety. Out of 35 isolates tested, 34 isolates induced typical sheath blight symptoms except MTU-w isolate, where browning of the tissue was observed followed by small typical sheath blight lesions, which was considered for recording disease score and tillers infected. The isolates were finally grouped into four groups based on disease index values. Among the 35 test isolates, 17 isolates were highly virulent (> 70% DI), 14 were virulent (60-70% DI), 2 isolates viz., MCP, FZB were moderately virulent (50-60% DI) and the remaining isolates (MTU-w, JDP-w) were least virulent (