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Morphological and Molecular Characterization of Rhizoctonia Oryzae Sativae in Bangladesh

S.B. Jahan
Govt. Teacher’s Training College, Mymensingh, Bangladesh

M.A. Ali
Bangladesh Rice Research Institute, Joydebpur, Gazipur-1701, Bangladesh

M.S. Alam
Department of Botany, Rajshahi University, Bangladesh

Z.R. Moni*
Bangladesh Agricultural Research Council, Dhaka, Bangladesh

M.A. Latif
Plant Pathology division, Bangladesh Rice Research Institute, Joydebpur, Gazipur, Bangladesh

Abstract/ Executive Summary:

Aggregate sheath spot disease of rice caused by Rhizoctonia oryzae sativae has emerged in higher incidence in North-Western region of Bangladesh. Thirty isolates of R. oryzae-sativae were studied by using morphological and molecular marker. Isolates were confirmed using specific primer where a single band of 1200 bp was amplified. Two distinct groups relatively slow and faster were found in mycelal growth. Molecular characterization was done using four primers and DNA band ranged from 0.25 to 2.21 kb. A combined dendrogram was constructed which separated the isolates into three groups at 69.6% similarity level. All isolates placed in two major clusters except isolate RA-1 placed in cluster group III but were not grouped according to their geographic origins. Fast growing isolates have been placed in Group II while slow growing isolates in cluster group I. The similarity coefficient values of the dendrogram profile ranged from 0.36 to 0.98 with an average of 0.67. Diversity of different isolate showed that significant variation was present among the isolate and were not genetically identical.

Keywords: Aggregate sheath spot Rhizoctonia oryzae-sativae; Variable number tandem repeat (VNTR); Repetitive-element polymerase chain reaction (Rep-PCR)

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

Multi-disciplinary approaches using diagnostics techniques were undertaken to characterize of morphological and genetic variability among the collected isolates of Rhizoctonia oryzae-sativae isolates of different locations of Bangladesh.

Methodology:

Collection, purification and identification of R. oryzae-sativae A total of 30 isolates of R. oryzae-sativae were isolated from rice sheaths with aggregate sheath spot symptoms in disease prone area of Bangladesh. Single hyphal tip method was followed for isolation and purification was confirmed by constricted hyphae and bi-nucleate cells. All isolates was also confirmed by molecular identification. DNA was extracted from 30 isolates of R. oryzae-sativae following the methods of (Raeder and Broda., 1985). For direct detection and identification of R. oryzae- sativae, PCR was performed using R. oryzae-sativae specific primers on 30 isolates. The PCR was conducted with the combination of forward Primer GMROS-6 (5'-GAA AGA GAG AGA GGT CGC CTC-3') and reverse primer R635 (5'-GGT CCG TGT TTC AAG ACG G-3'). PCR amplification was used to complete the reaction. Aliquots (10µl of the amplification products) were subjected to electrophoresis using 2% agarose gel with TBE buffer at 100 V for 1:20 min, stained with ethidium bromide and visualized using a UV trans-illuminator. Cultural and morphological characterization Mycelial disks of 6 mm diameter of 3 days old colonies were transferred aseptically to PDA plates for 2 weeks with three replications. Qualitative cultural characteristics viz., Sclerotial size placing the sclerotia were examined. Data were analyzed using MSTATC (version 2.10), in a two-way Analysis of Variance (ANOVA) of 30 treatments. Mean separation was performed using least significant difference (LSD) test at α=0.05. Somatic compatibility test Mycelial plugs having 6 mm diameter from 3 days old culture and placed on 9 cm diameter petri plates were approximately 2 cm apart from each other. Three isolates per plate were placed and preserved for 10 days. Isolates failed to show a barrage reaction at the colony junction were classified as same somatic compatibility groups (SCGs), while isolates exhibiting a barrage reaction were classified into different SCGs. Thirty isolates were tested in all possible combination with three replications. Molecular characterization For molecular characterization three primers, MR (GAG GGT GGC GGT TCT), RY (CAG CAG CAG CAG CAG) and GF (TCC TCC TCC TCC TCC) were used for VNTR analysis. BOXA1R primer (5’-CTACGGCAAGGCGACGCTGACG-3’) used for REP-PCR analysis. Genotypic data were obtained by considering different product sizes as different alleles at each locus, which were measured by AlphaEase FC 4.0 software and then scored for the presence (1) or absence (0) of the bands at the certain position for each isolate. A similarity matrix based on Dice’s similarity coefficient was used and cluster analysis of the matrix was done using unweighted pair group method with arithmetic mean (UPGMA) by numerical taxonomy and multivariate analysis system (NTSYS-PC) version 2.20e. Under SIMQUAL program similarity matrix was constructed using Dice coefficient method.

Source of Information: SAARC J. Agri., 16(2): 119-128 (2018)

Article Link (Online Journal): DOI: https://doi.org/10.3329/sja.v16i2.40264

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Achievement/Findings:

Comparison of two molecular marker systems showed the highest number of scored DNA bands and a higher percentage of polymorphism were produced by the GF primer indicated VNTR was more efficient compared to that of Rep-PCR. Because, fingerprints generated by BOXA1R seemed to overlap adjacent bands. Though the primers and isolates used were relatively few in number, it could be effectively establish the molecular variability among the isolates of Bangladesh. However, at 90% similarity level, the isolates were categorized into 26 groups, which indicated a high genetic diversity of rice aggregate sheath spot fungus as revealed by VNTR and Rep-PCR primers. Variation in the isolates of R. oryzaesativae indicated that various genotypes of R. oryzae-sativae present in Bangladesh and should be included in screens of resistance genes in rice and fungicides.

Knowledge Management: Journal