DISTRIBUTION OF MATING TYPE ALLELES AND FERTILITY OF MAGNAPORTHE ORYZAE ISOLATES IN SOUTH INDIA

* Khaled Fathy 1 and Prashanthi S. K. 2 . 1. Genetics Department, Molecular Genetics Lab., Faculty of Agriculture, Zagazig University, 44519, Egypt. 2. Department of Biotechnology, IABT, University of Agricultural sciences, Dharward, Karnataka, 580005, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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The fertile interactions only possible between individuals of opposite mating type designated MAT1-1 (Male fertile) and MAT1-2 (Female fertile). However, the capacity of M. grisea isolates to produce perithecia (female fertility) is apparently controlled by genes at several loci, and these segregate independently of mating type and pathogenicity on different hosts . In Magnaporthe oryzae, meiosis is immediately followed by a single mitotic division to generate four pairs of sister ascospores grouped into a single ascus. The ascospores are formed within the sexual structures (perithecia) produced by the female-fertile strain (or by both strains if both are female-fertile). Perithecia are flask shaped bodies that carry asci-bags containing ascospores, the products of meiosis-in abundance. Asci can be dissected to liberate the ascospores, which are arranged as un ordered octads. In either case the segregation patterns of genetic markers can be readily followed and the genetic basis of phenotypic traits determined (Valent et al., 1991).
In fertility survey results were generally consistent in that female sterility was the norm, fertile isolates were typically male-fertile only, and mating type ratios were skewed (Notteghem and Silué, 1992;Yaegashi and Nishihara, 1976). This rarity of female fertility was consistent with the reported abundance of transposable elements in Magnaporthe grisea rice pathogens (Zeigler, 1998;Zeigler et al., 1994). Translocations and non homologous crossover events can result in meiotic failure. Where the environment permits long-term persistence of well-adapted asexual lineages, mutations can accumulate at loci mediating fertility and result in a predominance of infertility in populations (Kistler and Miao,1992;Zeigler, 1998). In the late 1980s, hermaphroditic isolates were used as tester strains as well as in back-crossing programs to develop fertile rice pathogens of both mating types (Chao and Ellingboe, 1991).
Hermaphroditic isolates have been reported from southern China (Chengyun et al., 1992) and the Indian Himalayas (Kumar and Zeigler, 1995). However, a study from southern India reported no sexually fertile rice pathogens (Devulapalle and Suryanarayanan, 1995;Viji and Gnanamanickam, 1998). Mekwatanakarn et al. (1999) studied mating type distribution and sexual fertility of Magnaporthe grisea isolates collected from different locations in Thailand. Three hundred forty-one single conidium isolates of M. grisea collected from five sites in north, northeast, and central Thailand were evaluated in vitro for sexual fertility and mating type. Ascospores were detected in isolates which isolated from the north-eastern and northern regions. Sixty seven per cent of Magnaporthe grisea isolates were infertile when crossed with the hermaphrodite strains. In bioassay mating type, fifty to seventy five per cent of isolates showed male fertility MAT1-1 and fifty to eighty five per cent of isolates showed MAT1-2 mating type from all locations in Thailand, hermaphroditic isolates were also detected. Back crossing of Magnaporthe grisea with weeping love grass (Eragrostis curvularia) was developed to show high level of fertility (Valent et al., 1991).
Regulation of mating is controlled by a single mating type locus that contains genes that encode putative transcription factors which play role in transcription regulators of pheromone production and reception. Sexual mating compatibility in Magnaporthe oryzae is governed by the presence of two alleles of the mating type locus MAT1. The mating-type locus is found in the mating partner as an idiomorph, a non-homologues DNA sequence and gene set at the same chromosomal position. Ascogonia that are fertilized by male structures, which can be conidia, or spermatia (Metzenberg and Glass 1990;Coppin et al., 1997). MAT locus could be linked to an avirulence gene, with no compatibility with the prevailing Rice cultivar, and this could represents a drawback for fitness, being eliminated along time (Notteghem and Silué, 1992).
Mycelium tufts were observed between crossed Magnaporthe oryzae cultures, new haplotypes were found, indicating the presence of recombination (Zeigler et al., 1997). Crossing can occurs between fertile isolates, Magnaporthe oryzae isolates also found to be hermaphrodite (Zeigler, 1998).
When two Magnaporthe oryzae populations from Himalayas were analyzed, it was found that twenty two per cent of the isolates belonged to MAT1-1 and forty three per cent to MAT1-2, and hermaphrodite and male fertile isolates were found, suggesting a possible occurrence of sexual recombination in this Himalayan region. The structure of some Magnaporthe oryzae populations may be affected by sexual recombination which gives dynamic behave and diverse among populations (Kumar et al., 1999).
There was a consensus that the majority of the Magnaporthe oryzae populations lost its viability of sexual reproduction. It was proposed that only one mating type could be found when new rice cultivar was planted in a 1148 particular area (Zeigler, 1998). In Argentina, analysis of one hundred twenty five isolates collected between 2000 and 2003 showed that all of them belonged to mating type MAT1-1. Similar results were obtained in Korea, when the analysis of two hundred fifty four isolates demonstrated that all of them also belonged to MAT1-1 (Park et al., 2008). Twenty nine per cent isolates belongs to MAT1-1 and seventy one per cent belongs to MAT 1-2 in Africa, but none of the isolates was hermaphrodite, avoiding crossing among them (Takan et al., 2011).
DNA mobile genetic elements can cause chromosomic rearrangement and degeneration of sexual behavior without producing a drawback of fitness (Chuma et al., 2011). One role of the sexual reproduction is to form resistance structures to enable the fungi to adjust against unfavourable conditions (www.intechopen.com, Klaus, Brazil).
Populations of Magnaporthe oryzae worldwide were characterized genetically to identify sexual reproduction . The two mating types in one Chinese population and almost all strains were female-fertile. Viable progenies of Magnaporthe oryzae were produced in vitro, sexual reproduction in Magnaporthe oryzae population was confirmed with the help of genotypic richness and linkage disequilibrium data. Computer simulations confirmed that the observed genetic characteristics were unlikely to have arisen in the absence of recombination. The authors concluded that Magnaporthe oryzae, population reproduces sexually in nature in Southeast Asia and evidenced the loss of sexual reproduction by a fungal plant pathogen outside its centre of origin.
Mating type distribution and lineage diversity was studied; sexual recombination might be the one reason for lineage diversity in Magnaporthe oryzae in fields of rice growing regions in North-East and Eastern states of India (Imam et al., 2014). Soma et al. (2014) studied the mating type alleles as a marker to measure population diversity in forty six isolates of Magnaporthe oryzae from various ecosystems of coastal Odisha, India. Mating types MAT1-1 and MAT1-2 was found in all the ecosystems in uplands and in irrigated fields. In irrigated ecosystem fields MAT1-1 and MAT1-2 could be found. The disease spreads was very fast in rice fields resulting in blast lesions looking as green islands was found in isolates showed MAT1-2.

Materials and methods:
Analysis of mating type alleles in Magnaporthe oryzae isolates: In current study 97 Magnaporthe Oryzae isolates collected from different places in south India (Table 1) were studied for mating type analysis by Mat gene specific molecular markers. The pure culture of each isolate was grown in Rice straw Broth medium for approximately 8-15 days at room temperature (25-28 o C) to produce mycelia.
Mycelium mats of ninety seven blast isolates of Magnaporthe oryzae was used for genomic DNA isolation by adopting Cetyl Trimethyl Ammonium Bromide CTAB / NaCl method. Agarose gel electrophoresis was done to check the quality of DNA by running on 0.8% agarose (Lonza, USA) and DNA was quantified by using Nano drop spectrophotometer (ND-1000 V3.5.2, Nano Drop Technologies Inc., USA).

Amplification of MAT gene:
Mating type genes MAT1-1 and MAT1-2 were amplified according to Wang et al. (2004) using two pairs of specific primers.
MAT1-1 Forward Primer (TCAGCTCGCCCAAATCAACAAT) and Reverse primer (ACTCAAGACCCGGCACGAACAT) yield a product of 809 bp and MAT1-2 Forward Primer (GAGTTGCCTGCCCGCTTCTG) and Reverse primer (GGCTTGGTCGTTGGGGATTGT) yield a product of 940 bp. Amplifications were performed in a final volume of 20 μl of reaction mixture: 10x Taq assay buffer, 2.5 mM dNTPs, 10 picoMole for forward and reverse primers, Taq DNA polymerase XT-5 PCR system (GeNeI TM ,Merck Biosciences, Bengalure) 3 units/µl, Template DNA 50 ng/μl and sterile distilled Millipore water. The reaction mixture was given a momentary spin for mixing of the reaction components except DNA template. Master mixture was distributed to all 0.2 ml PCR tubes and finally 1 μl of respective DNA template was added and short spin was given to mix template with all reaction components and then tubes were loaded in a thermal cycler. Thermal cycler conditions; 95°C Initial denaturation for 3 min, followed by 35 cycles of 95°C for 45 sec, The annealing temperature 70 o C for MAT1-1 and 71 o C for MAT1-2 for 1.45 min, 72°C for 2 min and a final extension step at 72°C for 7 min. PCR reaction was carried out using Master gradient 5331-Eppendorf version 2.30.31-09, Germany. Two per cent (6 1149 g/ 300 ml) agarose was added to 1X TAE buffer (pH-8.0) for PCR separation, Ethidium bromide 12 μl was used as a staining agent. The gel was run at 8 V/cm for 1 to 1.5 hours and bands were visualized and documented in gel documentation system (Model Alpha Imager 1200, Alpha Innotech Corp, USA). Based on the results, 97 isolates were grouped into Male fertile, Female fertile, Hermaphrodite and Unknown based on presence or absence of MAT allele. Mating type frequencies were calculated.

Determination of mating type and fertility status of unknown isolates:
The

Results:
A total of ninety seven isolates of Magnaporthe oryzae collected from different locations of south India were evaluated to understand the mating type distribution and fertility of isolates by using MAT locus specific primers ( Table 2). MAT1-1 (Male fertile) mating type generated the amplicon of 809 bp (Fig.2) where as MAT1-2 (Female fertile) yielded amplicon size of 940bp (Fig.3). Among 97 isolates 46 isolates were MAT1-1 mating type accounting to 47.42 per cent frequency. 42 isolates were positive for MAT1-2 mating type with 43.29 per cent frequency. About seven isolates were hermaphrodite type with 7.21 per cent frequency and two isolates showed unknown mating type, which showed negative amplification for both MAT1-1 and MAT1-2 gene specific primers (Fig.4).
The highest frequencies of isolates that could be typed for MAT1-1 were detected in Dharward around 22 isolates with 75.86 per cent frequency (Fig.5 Both the mating types were observed in isolates collected from Raichur district which represents irrigated rice ecology and Uttara kannada district which is rainfed ecology encompassing MAT1-1 mating type with 60 per cent frequency and MAT1-2 mating type with 40%. Isolates collected from Koppal district contained MAT1-1, MAT1-2 and hermaphrodite mating types with 25, 58.33 and 16.66 per cent frequency respectively. Two isolates from Gangavathi and unknown location sample showed hermaphrodite mating type Similarly Chikkamangalore isolates showed MAT1-1, MAT1-2, hermaphrodite and unknown mating types with 50, 16.66, 16.66 and 16.66 per cent frequency respectively. In this location two of the isolates showed no amplification for either of the two mating types. Kerela isolates consisting of only two cultures which showed MAT1-1 and MAT1-2 type1:1 ratio. Tamil Nadu rice blast isolates results showed 40 per cent of samples presented as MAT1-2, 50 per cent frequency recorded for MAT1-1 and 10 per cent unknown mating types. No hermaphrodite type was observed in Tamil Nadu isolates. Cultures were checked for sporulation and all of them were highly sporulated cultures (Fig.6) which used in crosses experiment. Production of Perithecia were not seeing when the cross between isolates of the opposite was done.
1153 Until recently sexually fertile rice isolates were thought to be very rare and variations were attributed to parasexual recombination, mutations and clonal lineages. Sexual reproduction can reshuffle genetic material via recombination, thereby bringing together new alleles, which may influence pathogenicity to various rice cultivars and could influence the efficacy of fungicides. Capacity of M. grisea isolates to produce perithecia (female fertility) is apparently controlled by genes at several loci, and these segregate independently of mating type and of pathogenicity on different hosts (Mekwatanakarn et al., 1999). In fertility surveys of rice pathogens results were generally consistent in that female sterility was the norm, fertile isolates were typically male-fertile only, and mating type ratios were skewed (Notteghem and Silué, 1992;Yaegashi and Nishihara, 1976). This rarity of female fertility was consistent with the reported abundance of transposable elements in M. grisea rice pathogens (Zeigler, 1998;Zeigler et al., 1994). In the late 1980s, hermaphroditic ( male and female-fertile) rice isolates were found and subsequently used as tester strains against field isolates as well as in back-crossing programs to develop fertile rice isolates of both mating types (Mekwatanakarn et al., 1999). A study from Southern India reported no sexually fertile rice pathogens (Devulapalle and Suryanarayanan, 1995;Viji and Gnanamanickam, 1998).
However, in our present study among 97 isolates seven isolates showed the amplification for both MAT alleles indicating these isolates are male and female-fertile. Isolates Mo-si-076a, Mo-si-084, Mo-si-007, Mosi-215, Mo-si-15c, Mo-si-53, Mo-si-98a, are hermaphroditic and hence these can be used as tester strains to identify and clone new avirulence genes in several field isolates. Both male 75.86 per cent and female fertile 24.13 per cent isolates were recovered from a single field of A.R.S Mugad. Similarly in other surveyed locations both mating alleles were recorded. Samples collected from Kodagu, Mandya and Koppal location was dominated by female fertile isolates 58.33 per cent to 72.72 per cent.
Conventional approach to determine mating type in the pathogen population depends upon the appearance of mature perithecia in a cross between known tester and an unknown strain on culture media which is time consuming and requires high technical expertise. PCR amplification methods using MAT gene specific primers are a rapid method to explore the mating type population of M. oryzae (Priyadarisini et al., 1999;Notteghem and Silué, 1992 Analysis of mating type can provide an estimation of genetic diversity among M. oryzae populations from rice. Genetic diversity and mating-type studies suggested that M. oryzae exists as a recombinant population. However, detection of sexual form in natural field conditions and the pathogenicity of its progenies on rice still remain elusive despite the existence of firm populations (Saleh et al., 2012 b ). The presence of both mating-type as detected by PCR based molecular markers are basically useful to identify the mating type locus in the field isolates of M. oryzae but not effective to identify the fertile M. oryzae strains because fertility is also influenced by genes other than the mating-type genes (Dayakar et al., 2000).
Using this assay, we determined the mating type of 97 isolates from different locations in south India, 46 isolates showed mating type allele MAT1-1 (Male fertile), 42 showed mating type MAT1-2 (Female fertile), 7 isolates showed hermaphrodite and 2 unknown mating type. The observation that both mating-types were present approximately in equal proportion in the population sampled from South India suggesting the possibility of sexual recombination in nature which can affect diversity and dissemination. Two isolates Mosi-15a from Mudegeri (Gamsali variety, Leaf blast) and Mo-si-88 from Bhavanisagar (Deluxe variety L.B) yielded no amplification consistently under standard conditions by using both MAT primers.
Several previous studies discussed about mating types in Magnaporthe oryzae, from various ecosystems of coastal Odisha, India. MAT1-1 mating type was dominating in all the ecosystems and MAT1-2 was found to be present in uplands as well as in irrigated fields. Both mating types could be found in the same field in irrigated ecosystem (Soma et al., 2014).
In the present study MAT1-2 and MAT1-1 were found to be present in both in irrigated and upland fields in Raichur and Uttara Kannada respectively. Where ever MAT1-1 and MAT1-2 are there we could expect 1157 variation in the M. oryzae population due to recombination. In addition presence of hermaphrodite M. oryzae isolates in a location clues the occurrence of wide diversity in its population. Distribution of both the mating type in the same field among field isolates of M. oryzae was recorded by earlier workers and suggested the possibility of occurrence of sexual recombination in nature Kumar and Zeigler 1995). Kumar et al. (1999) suggested possible occurrence of sexual recombination in M. oryzae population in Himalayan region. Imam et al. (2014) reported the presence of both mating types in North-East and Eastern India, of 63 M. oryzae isolates analysed, 16 of the isolates were of the mating type MAT1-1 while 35 were of the mating type MAT1-2 and eleven isolates did not produce a PCR product with either of these two mating types.
If a single mating-type predominates in a particular rice growing region, M. oryzae may not be sexually reproducing in that region and this may be important in the population dynamics of this pathogen. In past studies, MAT1-1 has been identified as the dominant mating-type associated with rice (Dayakar et al., 2000). The presence of only MAT1-1 in the pathogens is not uncommon as it was also detected in the M. oryzae population in Japan and other regions (Notteghem and Silué, 1992). In this study, it is interesting to note that, in few locations Viz., Mandya, Chikkamangalore, Koppal districts both MAT1-1 and MAT1-2 mating types and hermaphrodite isolates were present. This information draws our attention to study in detail about hostpathogen population in these locations. Hence, in these locations variations due to sexual recombination may be expected in nature. Clonal reproduction and sexual recombination may be the possible reasons for the population dynamics of M. oryzae in South India. Populations showing evidence for both types mating types may be assayed for fertility using a range of tester isolates from different regions. As locally obtained hermaphrodite isolates of both mating types from this study may be used as testers for a systematic survey of local field isolates in future.