Morphological and Genetical Variability among Rhizoctonia solani Isolates Causing Sheath Blight Disease of Rice

doi:10.1016/j.rsci.2016.01.005

Abstract

Abstract:

Eighteen isolates of Rhizoctonia solani collected from infected rice plants in four different locations of Bangladesh were studied by using morphological characters and molecular markers. Anastomosis study with a reference isolate confirmed that all the isolates belonged to R. solani. Significant variation was observed in sclerotial size, shape and distribution. Un-weighted pair group method with arithmetic mean dendrogram constructed based on the Gower’s general similarity coefficient showed that these isolates were grouped into four clusters at the 0.68 similarity coefficent according to morphological characters. Cluster I was a major cluster consisting of 13 isolates, while clusters II to IV consisted of 1 or 2 isolates. Analyses by variable number of tandem repeat and amplified fragment length polymorphism markers showed that the isolates were grouped into five and three clusters at a similarity coefficient of 0.64 and 0.69, respectively. Although most of the variability was found between isolates from different regions as expected, significant variation was observed within the isolates collected from similar agro-ecological regions. Our results suggest the presence of different races of R. solani within the same local geographic regions.

Key words: rice, Rhizoctonia solani, fungal variability, molecular marker, morphological character

Rahman Moni Zakiah, Ansar Ali Md, Shahidul Alam Md, Asif Rahman Md, Rejwan Bhuiyan Md, Salim Mian Md, Md Iftekharuddaula Khandakar, Abdul Latif Md, Ashik Iqbal Khan Mohammad. Morphological and Genetical Variability among Rhizoctonia solani Isolates Causing Sheath Blight Disease of Rice[J]. Rice Science, 2016, 23(1): 42-50.

Figures/Tables 12

Table 1 List of R. solani isolates with geographic locations.

Code	Location	Agro-ecological zone
GA1, GA2, GA3, GA4, GA5	Gazipur	Madhupur tract
GO1, GO2, GO3, GO4, GO5	Godagari	High ganger river flood plain
TA1, TA2, TA3, TA4, TA5	Tanor	High ganger river flood plain
CO1, CO2, CO3	Comilla	Middle Meghna river flood plan

Table 2. Morphological characteristics description of R. solani isolates and their attributes.

		Character	Character state
Mycelial color on potato dextrose agar, color of sclerotia	0, hyaline; 1, cream or faint brown; 2, light brown; 3, medium brown; 4, dark brown
Superficial sclerotia (SS) dispersed on whole colony, exudate droplets on sclerotium surface	0, present; 1, absent
Colony reverse: pigment	0, not present; 1, cream or faint brown; 2, light brown
Sclerotia on lid	0, absent; 1, present
Topography of sclerotia	0, immersed; 1, superficial
Shape of sclerotia	0, flattened bottom and round top; 1, superficial; 2, irregularly globose with pitted surface; 3, irregular
Arial mycelial quality	0, absent; 1, all hyphae close to surface of agar; 2, air space in dish half filled; 3, almost all airspace filled
Quantity of sclerotia, pseudo sclerotia, SS discrete, SS aggregated, SS scattered, SS near inoculum, SS near margin, dark brown runner hyphae in aerial mycelium and/or on colony surface, growth on lid	0, absent; 1, few; 2, moderate; 3, abundant

Table 3. List of primers and their sequences.

		Primer	Sequence
Variable number of tandem repeat (VNTR)
MR	GAGGGTGGCGGTTCT
RY	CAGCAGCAGCAGCAG
GF	TCCTCCTCCTCCTCC
Amplified fragment length polymorphisms (AFLP)
AFLP-C	GACTAGGATACATGCAGGC
AFLP-D	GACTACGTACATGKACKGKAC

Fig. 1. Morphological variation of R. solani showing sclerotial distribution.

Fig. 2. Un-weighted pair group method with arithmetic mean dendrogram of R. solani isolates constructed with Multi Variaty Statistical Package ver. 3.1 using the Gower’s general similarity coefficient based on morphological characters.

Fig. 3. Variable number of tandem repeat fingerprinting patterns of total genomic DNA of R. solani isolates with primer MR.M, Kilo base (kb) ladder; Lanes 1 to 18, Isolates GA1, GA2,GA3, GA4, GA5, GO1, GO2, GO3, GO4, GO5, TA1, TA2, TA3, TA4,TA5, CO1, CO2 and CO3, respectively.Arrow indicates polymorphic bands.

Fig. 4. Variable number of tandem repeat fingerprinting patterns of total genomic DNA of R. solani isolates with primer RY. M, Kilo base (kb) ladder; Lanes 1 to 18, Isolates GA1, GA2, GA3, GA4, GA5, GO1, GO2, GO3, GO4, GO5, TA1, TA2, TA3, TA4, TA5, CO1, CO2 and CO3, respectively.Arrow indicates polymorphic bands.

Fig. 5. Variable number of tandem repeat fingerprinting patterns of total genomic DNA of R. solani isolates with primer GF.M, Kilo base (kb) ladder; Lanes 1 to 18, Isolates GA1, GA2, GA3,GA4, GA5, GO1, GO2, GO3, GO4, GO5, TA1, TA2, TA3, TA4, TA5,CO1, CO2 and CO3, respectively.Arrow indicates polymorphic bands.

Fig. 6. Un-weighted pair group method with arithmetic mean dendrogram of R. solani isolates constructed with Multi Variety Statistical Package ver 3.1 using the Gower’s general similarity coefficient based on DNA fingerprinting (variable number of tandem repeat analysis of primers MR, RY, and GF).

Fig. 7. Amplified fragment length polymorphisms fingerprinting patterns of total genomic DNA of R. solani isolates with primer AFLP-C.M, Kilo base (kb) ladder; Lanes 1 to 18, Isolates GA1, GA2, GA3, GA4, GA5, GO1, GO2, GO3, GO4, GO5, TA1, TA2, TA3, TA4, TA5, CO1, CO2 and CO3, respectively.Arrow indicates polymorphic bands.

Fig. 8. Amplified fragment length polymorphisms fingerprinting patterns of total genomic DNA of R. solani isolates with primer AFLP-D.M, Kilo base (kb) ladder; Lanes 1 to 18, Isolates GA1, GA2, GA3,GA4, GA5, GO1, GO2, GO3, GO4, GO5, TA1, TA2, TA3, TA4, TA5,CO1, CO2 and CO3, respectively.

Fig. 9. Un-weighted pair group method with arithmetic mean dendrogram of relatedness of R. solani AG1 IA isolates constructed with Multi Variaty Statistical Package ver 3.1 using the Gower’s general similarity coefficient based on amplified fragment length polymorphism analysis of primers AFLP-C and AFLP-D.

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