Acquisition of the Grasshopper Retro Transposon by Rice <em>Magnaporthe</em> Isolates Indicates a Dynamic Gene Flow between Rice and Non-Rice <em>Magnaporthe</em> Population

Research Aricle

J Pathol & Microbiol. 2016; 1(2): 1011.

Acquisition of the Grasshopper Retro Transposon by Rice Magnaporthe Isolates Indicates a Dynamic Gene Flow between Rice and Non-Rice Magnaporthe Population

Mahesh HB1,2, Meghana S¹, Shailaja H²*, Prasannakumar MK³, Mahadevu P4, Channabyregowda MV5 and Malali G¹*

¹Center for Genomics Discovery, TransDisciplinary University, India

²Marker Assisted Selection Laboratory, Department of Genetics and Plant Breeding, University of Agricultural Sciences, India

³Department of Plant Pathology, University of Agricultural Sciences, India

4All India Coordinated Research Project on Fodder Crops, Zonal Agricultural Research Station (ZARS), India

5All India Coordinated Small Millets Improvement Project, Zonal Agricultural Research Station (ZARS), University of Agricultural Sciences, India

*Corresponding author: Shailaja Hittalmani, Marker Assisted Selection Laboratory, Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bengaluru, India

Malali Gowda, Center for Genomics Discovery, TransDisciplinary University, Foundation for Revitalization of Local Health Traditions (FRLHT), Bengaluru -560064, India

Received: November 30, 2016; Accepted: December 18, 2016;Published: December 22, 2016


Blast disease caused by Magnaporthe species is major problem faced by rice and finger millet cultivation across the world. Knowing the genetic diversity and population structure of Magnaporthe species is important for designing blast management strategies, to understand the evolution of virulent pathotypes and basis of host shifts. In this study, we used multi-marker system including Simple Sequence Repeats (SSRs), repetitive DNA based markers (Pot2 and Grasshopper), pathogenicity genes and mating locus to study genetic variability of Magnaporthe species in rice and finger millet ecosystems from southern India. Data from multiple markers revealed high genetic diversity and clustering based on geographical location and host species. This study also revealed two rice specific SSR markers (MGM246 and MGM286) and absence of AVR-Co39 and AVR-Pita1 in rice and finger millet, respectively. Interestingly, our study identified multiple copies of grasshopper repeat elements in rice isolates. This element intruded Magnaporthe population infecting finger millet, after evolution of host-specific forms of Magnaporthe. The molecular data obtained using multimarker system, indicated presence of dynamic Magnaporthe population in location of our study. While the clonal nature is known to predominate in field conditions, active gene flow and sexual recombination cannot be denied in cropping zones where different Magnaporthe host crops are co-cultivated. Recurrent characterization of Magnaporthe populations from such locations will help to keep check on emergence of more virulent pathotypes with broad host spectrum.

Keywords: Magnaporthe; Multi-marker; SSR; Pot2; Grh; Pathogenicity genes


Magnaporthe oryzae is an Ascomycetes fungal pathogen, which causes blast disease in rice. The genus Magnaporthe consists of several species, which parasitize a wide spectrum of hosts (>50 grass species) including rice, wheat, barley, finger millet and grasses (Panicum italicum; Cenchrus ciliaris; E. indica and E. tristachya) [1-3]. The blast on rice was first described by Cavara as fungal disease caused by Pyricularia oryzae (teleomorph of Magnaporthe oryzae (Hebert)) in 1891 [2]. In 2002, Couch and Kohn designated Digitaria infecting isolates as M. grisea, which is morphologically indistinguishable to M. oryzae. Sexual recombination in Magnaporthe is controlled by a single MAT locus. Fungus of two opposite mating types, MAT1-1 and MAT1-2 are required to produce fertile structure called as perithecia [4,5]. The sexual stage (teleomorph) of M. oryzae has not been found in nature, however sexual reproduction has been reported in vitro conditions between strains of opposite mating types [6,7]. Himalayan foothills are considered to be the center of origin of the M. oryzae [8-11]. Kumar, et al. (1999) have analyzed M. oryzae populations from Indian Himalayas and reported high genetic diversity, presence of both mating types MAT1-1, MAT1-2 and hermaphrodite strains. More recently various genetic events such as deletion, translocation, duplication of Avr gene, or chromosomal rearrangements of field isolates are reported [12]. These genetic variations lead to genome reshuffling which contribute to genome evolution and continuous emergence of virulent strains. High genetic variability coupled with broad host range makes it an ideal model system to study plantpathogen co-evolution [13].

To understand the Magnaporthe population dynamics, many studies have used repetitive DNA based molecular markers such as MGR 586 [14], Pot2 [15] and Grasshopper [16]. Recently, SSR became the most popular molecular marker for genetic mapping [17] and genetic diversity analysis in fungi [18]. However, there are no reports of comprehensive study of Magnaporthe population using combination of marker systems. In this study, we assessed the Magnaporthe species diversity using multiple marker systems including microsatellites, repetitive DNA elements (Pot2 and Grh), and pathogenicity genes and mating locus. In addition, we tested the efficacy of each marker to select best marker system, which can give maximum information about genetic diversity within and between Magnaporthe populations. This is the first study of rice and non-rice Magnaporthe characterization using multiple marker approach.

Materials and Methods

Design of experiment

We selected ten rice (HR12, Co-39, Tadukan, Tetep, MAS26, MAS946-1, Jaya, Intan, KHP9, Rasi, Karizaddu and 27 IRDLs), ten wheat (B. Yellow, DWR-1006, DWR-162, DWR-185, DWR-2006, DWR-39, Kern, UAS-304, UAS-316 and UAS-415) ten finger millet (GPU26, GPU28, GPU45, GPU48, GPU66, GPU67, Indaf5, Indaf9, KMR204, Uduru Mallige and PR202), eleven foxtail millet (Co-7, ISC1162, ISC1209, ISE-995, Narasimharaya, PS-4, RAU-2, RFM- 14, SIA-326, Srilaxmi and TNAU-59) two from each of kodo millet (GPUK-3 and RBK-155), proso millet (TNAU-145 and TNAU-151), little millet (JK-8 and OLM-203) and barnyard millet (VL-172 and VL-207) varieties and planted them in Mandya, Ponnampet and Bengaluru during monsoon season (July–October 2011-2013) for three consecutive years. The seeds of all host varieties were disinfected to avoid seed borne contamination. Disease symptoms were recorded after 21 days of sowing and symptoms were scored (0- 9 scale) as per the Standard Evaluation System (SES), International Rice Research Institute (IRRI), Philippines.

Diseased leaf sampling and single spore isolation

Magnaporthe infected leaves from rice, finger millet, foxtail millet and grasses were collected in 2011, 2012 and 2013 in rainy season (from August to November) from four locations (Bengaluru, Mandya, Ponnampet and Hyderabad). Single spore isolation from infected lesions was performed using method optimized in our laboratory. The infected lesions were surface sterilized by 0.1% Sodium hypochlorite solution followed by two successive washes with sterile water in aseptic laminar hood. A micro humid condition was created by petriplates, pasting sterilized Whatman filter paper on inner surface of upper lid and lower plate filled with sterile water. The surface sterilized leaf samples were pasted on upper lid of these petri plates and incubated under dark at 28oC in an incubator (Innova42, New Brunswick Scientific, USA) for 2-3 days to induce sporulation. Magnaporthe spores were suspended in sterile water and 20 μl of spore suspension was spread evenly using sterile spreader on 2% Agar (Agar-agar, CAS No. 9002-18-0, Fisher Scientific) plate amended with 2 mg of Kanamycin in 100 ml of medium (Kanamycin Sulfate, K1377-5G, Sigma, USA). The single spore was pinpointed under light microscope and scooped using fine tip of sterile Borosilicate glass Pasteur pipets (Cat No. 13-678-20A, Fisher Scientific). Scooped agar with single spore was transferred to oatmeal agar (OMA, cat # M397-500G, Himedia) and allowed to grow for 3-4 days incubated at 28oC under alternate light and dark conditions. The pure cultures of Magnaporthe thus obtained were stored on filter paper discs at -20°C for long term storage.

Genomic DNA extraction

Magnaporthe isolates were grown in a liquid culture (0.2% yeast extract and 1% sucrose) incubated in a shaker incubator at 28oC at 200 RPM for three days. The fungal mycelium was filtered through sterilized miracloth (cat # 475855, Calbiochem, CA, USA) and grounded in liquid nitrogen using Pestle and Mortar. DNA was isolated as per the protocol [19] and DNA quality was checked in Nanodrop ND2000 (Thermo Scientific, DE, USA).

PCR amplification of SSR and MAT locus

We set up 10 μl volume PCR reactions containing 20 ng of genomic DNA, 1 μl of 10 x buffers, 0.4 μl of 20 mM of dNTPs mix, 0.5 μl of 10 mM of each forward and reverse primers, 0.15 μl of Dream Taq (Fermentas, # EP0712, PA, USA). PCR amplification was performed in 2720 Thermal cycle (Applied Biosystems, Foster city, CA, USA) with initial denaturation temperature at 94°C for 5 minutes and followed by 30 cycles with 30 seconds at 94°C, 30 seconds of annealing temperature (variable as per primer provided in the (Table 1)), 1 minute of 72o°C, final extension for 5 minutes at 72°C. PCR products were resolved on 3.5% low Electroendosmosis (EEO) Agarose gel (Himedia, CAS # 9012-36-6, India) stained with GelRed (cat # 41003-1-10 ml, Biotium) and visualized using gel documentation unit (FlourChem, Alpha Innotech, California, USA).