Investigation on Cultural Characterization and Parasexual Recombination in Pyricularia Grisea, Causal Agent of Blast of Rice

Blast of rice caused by Pyricularia grisea is one of the most devastating diseases of rice. Because of importance of the disease and the fact that pyricularia grisea is considered to be notorious and model species. The variability in cultural characteristics of fty isolates of P. grisea were taken from different regions of Jharkhand state. Out of fty isolates of P. grisea, colony color of six isolates were found to be as greyish white color, three isolates were blackish grey, six isolates as white color, three isolates as whitish grey color, ve isolates were whitish black and twenty seven isolates were recorded as blackish white in color. The growth pattern of 47 isolates of P. grisea showed circular growth pattern and three isolates have irregular growth pattern but elevation of the mycelium differs from at to raised. Out of fty isolates of P. grisea, sector formation was observed in seventeen isolates and no sector formation was observed in rest isolates. The radial growth of fty isolates of P. grisea were ranged from 76.0 mm to 90.0 mm. Out of fty isolates, group I included six isolates, group II and III included eleven and thirty three isolates, respectively. Parasexual recombination rarely causes genetic and phenotypic variation through hyphal anastomosis in India. Parasexual recombination is the principle cause in rice blast fungus and cause infection of resistant rice variety (IR-64) in India . the present studies were undertaken as Investigation on cultural characterization and parasexual recombination in pyricularia grisea.


Introduction
Rice (Oryza sativa L.) is one of the most important cereal crops grown all over the world. Rice crop is attacked by about fty diseases including twenty one fungal, six bacterial, twelve viral, four nematodal and seven other diseases including physiological disorders (Hollier et al., 1993;Webster and Gunnell, 1992;Jabeen et al., 2012). Among fungal diseases, blast disease is the one of the most signi cant and destructive disease of rice which is caused by the ascomycetous fungus, Pyricularia grisea (Teleomorph Magnaporthe oryzae Couch) formerly known as Pyricularia oryzae. . The disease is more speci c when the pathogen affects the leaf, neck, panicle and collar (Bonman et al., 1989). First appearance of lesions as minutes of brown specks on leaf tissue and gradually growing spindle shaped (Hossain et al., 2017). The center is grayish with brown margin. The lesions may extend and thus eventually coalesce the entire leaf into necrotic. Even so, M. grisea has been reported to have high pathogenic variability in rice as well as other hosts and varietial speci city. The principal cause of breakdown of resistance in rice against blast disease is pathogenic variability (Thon etal., 2007). Blast disease can be categorized into different pathotypes based on infection pattern found on differentials in rice genotypes (Couch and Kohn, 2002). However, resistant varieties may sometimes become ineffective owing to evolutionary changes, mutation, parasexuality, sexual mating etc in the pathogen population (Noguchi, 2011). Understanding pathogenic variation of M. grisea is critical in overcoming through the modern rice breeding techniques face.
According to Common wealth mycological institute (CMI) cultures of Pyricularia grisea are greyish in color, conidiophores single or in group, simple or rarely branched, show sympodial growth. Single conidia developed at the tip of the conidiophores, arising sympodially and in succession, The shape of conidia either pyriform or obclavate, narrow towards tip, round at the base, three celled, rarely one or two celled, hyaline to pale olive, 19-23 × 7-9 μm, with a distinct protruding basal hilum. The sexual fruiting body of fungus is called perithecia formed within 21 days. Flask-shaped perithecia undergoes meiosis and forms ascospores. Ascospores are in large populations of randomly selected ascospores (Talbot, 2003). P. grisea isolates forms ring like structure having irregular colonies with rough and smooth margins on OMA media (Srivastava et al., 2014).
Magnaporthe oryzae is notorious for changeability in pathogenecity but it lacks sexual life cycle (Dean et al., 2012). M. oryzae is one of the most important pathogenic fungi having an ability to rapidly change in its genetic make-up resulting in new pathogenic races (Hossain et al., 2017). The teleomorph stage of the M. oryzae was discerned in cross experiments (Eung and Williams 1985). Due to infertility of the eld isolates of M. oryzae which is considered to be limited to asexual reproduction in nature. Sexual recombination causes little genetic variations in the pathogen. A number of hypotheses were made that can cause genetic variation from different sources in P. oryzae. Generation of genetic variability in fungi involves sexual genetic recombination. However, sexual reproduction of P. oryzae, which facilitates meiotic recombination (Hayashi et al., 1997). Therefore, sexual recombination, if any, contributes genetic variations in the M. oryzae. In imperfect fungi, the other source of genetic variation is parasexual recombination followed by vegetative hyphal fusion (Mehrabi et al., 2011), which allows minor gene or chromosome transfer among different isolates. Parasexual recombination in P. oryzae was rst suggested by Yamasaki and Niizeki (1965) who reported nuclear behavior in anastomosis and observed variants by combining two different strains of blast pathogen. That isolates are known as recovered recombinants (Fatemi and Nelson, 1978). It was also presumed that pathogenicity changes in M. oryzae were caused by parasexual recombination (Namai et al., 1999). Parasexual recombination is the only cause of genetic recombination in Japan. No information available on the Parasexuality mechanism in India. Therefore, the present studies were undertaken as Investigation on cultural characterization and parasexual recombination in pyricularia grisea.

Material And Methods
The laboratory work was done in research laboratory at ICAR-Indian Institute of Agricultural Biotechnology (IIAB) Ranchi, Jharkhand and Deptt. of Plant Pathology, Birsa Agricultural University (BAU), Ranchi. Geographically Ranchi is situated at 23 0 17' N latitude and 85 0 19' E longitude with an altitude of 625 meters above mean sea level (MSL) of Jharkhand (India).

Isolation of the pathogen
Diseased leaves of rice blast were brought to the laboratory and thoroughly washed with tap water to remove dust or soil particles, if any. After washing infected specimen was cut into small pieces measuring 2-3 mm in size with sterilized blade. These pieces (bits) were surface sterilized by dipping them in 0.1% Mercuric chloride (Hgcl 2) solution (Riker and Riker, 1936) for 30 seconds and then washed thrice in sterilized distilled water for 45 seconds each to remove the traces of Hgcl 2 . These bits were then placed on a sterilized lter paper to remove excess moisture. Finally these bits (pieces) were transferred to sterilized Potato dextrose agar (PDA) medium slants under aseptic conditions and incubated at 25±2 ºC.
Puri cation of the pathogen Fifteen mililitre clear sterilized Oat meal agar (OMA) was poured into the sterile Petriplates and allowed to solidify under aseptic conditions. Dilute spore suspension was prepared in sterilized distilled water from fteen days old culture. One ml of such suspension was sprayed uniformly on OMA Petriplates these plates were incubated at 25±2 ºC for four days then such plates were kept under compound microscope so as to locate germination of conidia. Single isolated germinated conidium was marked with in the lower surface of the plate the growing hyphal tip portion was transferred to fresh OMA slants with the help of needle under aseptic conditions and incubated at 25±2 ºC (Hansen, 1926). Such obtained pure culture tubes used as stock culture. Stock cultures were sub cultured at an interval of 25-30 days each for further studies.
We have conducted a survey on severity of rice blast disease and collected the diseased leaf samples with the permission of land owners. A total of fty leaf blast isolates were collected from two local/indigenous varieties (Supplimenatry Table 1) and 48 cultivated varieties of farmers rice elds in different villages of seven districts (Ranchi, Khunti, East Singhbhum, West Singhbhum, Saraikela Kharsawan, Garhwa and Palamou) of Jharkhand State during Kharif crop season, 2019-20. Sampling details were deposited in plant pathology department library, Ranchi Agricultural college, Jharkhand. The study complies with local and national regulations. The collected samples were wrapped in cellophane paper and brought to the laboratory for cultural studies. In order to isolate the pathogen, leaf portion exhibiting typical symptoms of leaf blast was isolated and puri cation of the pathogens were made each as above mentioned technique.
In order to study cultural characteristics of above fty isolates of Pyricularia grisea, was grown on OMA medium in Petriplates at 25±2 0 C under aseptic condition. 15 ml sterilized OMA medium was poured aseptically to each sterilized Petriplates under aseptic condition and allowed to solidify. Mycelia disc (3 mm dia) of ten days old culture of P. grisea were placed inverted in the centre of Petriplates having OMA media under aseptical comdition and incubated at 25±2 0 C. Three replications were made for each isolates. After fourteen days of incubation, the observations for radial growth of the mycelium were recorded. The radial growth was measured by drawing two lines at right angles to each other on the back side of each plate and the average of the two was expressed as diameter of colony. (Lilly and Barnett, 1951). In the case of irregular growth, wavy, the colony mean of the largest and shortest diameter was taken as the colony diameter (Brown, 1923). Growth pattern, colony color, colony sector, colony zonation and colony wrinkle were also recorded each after fourteen days of incubation of above isolates. Per day radial growth rate (mm) of each isolates of P. grisea was also calculated. On the basis of colony color, growth pattern, sector formation, zonation, wrinkles and radial growth of all the 50 isolates using R (Version 3.4.0) statistical tool (Fig.1). Details of the characters and their attributes were presented in the Table 3. The above isolates were divided into three major groups (I, II and III) and six subgroups (Ia, Ib, IIa, IIb, IIIa, IIIb).
Parasexual studies of Pyricularia grisea isolates obtained from typical rice blast symptoms collected from different rice growing regions of Jharkhand, made pure cultures of the pathogen for further studies. Out of above fty isolates parasexuality con ned to a local variety isolate of Potka village of West Singhbhum district of Jharkhand State. Geographically it is situated at an latitude 22°40'20.60'' N and longitude of 85°38'54.86'' E with an altitude of 209 m from above MSL of Jharkhand state. Parasexual nature was found in cultural studies, microscopic and pathogencity studies were further carried for variability.
Compound microscopy and Scanning electron microscopy (SEM) (Model-JOEL JSM-6390LV) observations was studied for analytical studies of fungi in relation with parasexuality.

Pathogenicity test of parasexuality in rice blast fungus
Pathogenicity of P. grisea causing rice blast with parasexual nature was con rmed by proving Koch's postulates. The culture of the Potka isolate with hyphal anastomosis vegetative hyphae was grown on OMA. After 15 days of incubation at 25 ± 2ºC, the culture was transferred to another plates having 10 ml sterilized distilled water to make a conidial suspension and make the concentration of 1.0 ×10 7 conidia ml -1 of water by serial dilution. Forty millimeter of the conidial suspension containing Tween-20 (0.2%) was sprayed onto twenty ve days old rice seedlings of IR-64 in a pot by using a hand atomizer. The inoculated plants were covered with polythene bags moistened inside for 24 h with a view to provide high humidity during initial stages of infection. After incubation, the plants were kept in glass house and observations were made regularly for the appearance and development of symptoms. When the symptom on leaf was observed after ve days of inoculation, the infected leaves were collected and the pathogen was re-isolated under aseptic condition. The fungus identity was determined by comparing with the original culture of P. grisea. Symptoms of the disease observed in rice variety (IR-64) and variability nature was found in cultural media.

Cultural Variability of Pyricularia grisea Isolates
The variability in cultural characteristics among fty isolates of P. grisea taken from different regions of Jharkhand, was carried out on OMA medium. The observation of cultural characteristics such as colony color, growth pattern, elevation, sectored or non-sectored growth, zonation and wrinkles formation, radial growth and grouping are presented (Table 1&3 and Fig. 1&2).

PARASEXUALITY:
The parasexuality in asexual lamentous blast fungus (hyphae) was producing genetic and phenotypic diversity. Parasexuality occurred in laboratory culture of the rice blast fungus from a local variety isolate. The lineage of isolate parasexual recombination occurs in nature directly from local variety infectious isolate of Potka village, Jharkhand state. Hyphal anastomosis i.e., parasexual recombination observed in isolate of above locality was identi ed in the rice blast fungus it may be due to genetic exchange of DNA through hyphal anastamosis i.e., parasexual recombination and segregation of pathogenicity. The parasexual recombination in rice blast isolate may be occured in eld populations of blast pathogen. The colony color of culture one half is blackish white and another half is white oppy in nature, the blackish white growth is observed as in zonation form and white oppy growth is observed as circular raised form. The potential hyphal fusion (Fig. 3A-C) was considered to be the reason for possible parasexual recombination.
Further SEM study in rice blast lesions with multiple infections of rice blast isolate, rice blast pathogen shows higher degree of variation in cultural characteristics and hyphal recombination in natural condition and under compound and electron microscopic study. In this study the variantion of Pyricularia sp. and parasexuality was found which is described as follow.
The explanation of genetical variation in P. grisea may be sought on the basis of the ultrastructural evidence of hyphal anastamosis which is presented below (Fig.4).
Hyphal fusion under 4000x of scanning electron microscopy from local variety isolate, anastomosis was seen in between tip cells from side branches of blast fungus (Plate-4). Based on the hyphal fusion of isolate differs in genotypic and phenotypic characters and causes variability in rice blast fungus. Two hyphal interaction results in complete anastomosis and compatibility allow the formation of a single cell joining of two hyphae. Although anastomosis is a very useful mechanism in which variability was seen. Using compound and SEM, anastomosis could be monitored and observation of successful anastomosis in rice blast fungus, resulting in a compatible reaction was recorded. Initially, as the type of fusion mechanism was unknown, the hyphal tips growing towards the side of another hyphae were observed and occurrence of hyphal fusion observed under 4000X magni cation and other magni cations under SEM.
From the pathogenicity test it was observed that parasexuality may cause breakdown of resistant rice variety (IR-64) with hypal anastomosis gene recombination in the rice blast fungus and spores were observed under SEM (Fig. 5) in Jharkhand state.

Discussion
Blast of rice caused by Pyricularia grisea is considered to be notorious and model species. The variability in cultural characteristics of fty isolates of P. grisea were taken from different regions of Jharkhand state. Out of fty isolates of P. grisea, colony color of six isolates were found to be as greyish white color, three isolates were blackish grey, six isolates as white color, three isolates as whitish grey color, ve isolates were whitish black and twenty seven isolates were recorded as blackish white in color. The growth pattern of 47 isolates of P. grisea showed circular growth pattern and three isolates have irregular growth pattern but elevation of the mycelium differs from at to raised. Out of fty isolates of P. grisea, sector formation was observed in seventeen isolates and no sector formation was observed in rest isolates. The radial growth of fty isolates of P. grisea were ranged from 76.0 mm to 90.0 mm. Out of fty isolates, group I included six isolates, group II and III included eleven and thirty three isolates, respectively. Cultural characteristics between leaf blast isolates were recorded and variability differentiated. Panda et al. (2017) characterized twenty isolates of P. oryzae and categorized based on the variation in morphological characteristics viz., colony color, surface appearance and type of growth. The isolates showed few surface, downy, at with few mycelium and submerged growth with smooth and rough margins on OMA media. The colony color varied from grey, greyish white, dark black, blackish white and greyish black. The colony diameters of different isolates varied from 27.0 mm to 48.0 mm. Among twenty isolates, maximum isolates have shown 16 at with little mycelium growth followed by little surface and downy growth. Srivastava et al., (2014) isolated ten isolates of P. oryzae from different rice eld. They differentiate into six morphological groups (PG-I to PG-VI) based on the differences in morphological characteristics (colony color, colony morphology and conidia shape). The four isolates were recorded in PG-III, two isolates in PG-I and one isolate in PG-II, IV, V and VI group. In parasexual studies, pathogenicity revealed that the parasexuality blast fungus was capable of infecting resistant rice plant i.e., IR-64. Pyricularia grisea, isolated resembled with the original isolated pathogen in morphological characteristics. Parasexual recombination rarely causes genetic and phenotypic variation through hyphal anastomosis. Parasexual recombination is the principle cause in rice blast fungus and cause infection of resistant rice variety (IR-64) in India. The above results which is supported by the following scientists i.e., Parasexual recombination causes variation in pathogenicity of rice blast fungus can break down resistant rice variety (Noguchi, 2011). Complex race can attack various resistance genes in cultivar (Vanderplank, 1963) Parasexual recombination may play important role in the evolution of rice blast fungus (Monsur and Kusaba, 2018

Conclusions
The variability in cultural characteristics among fty isolates of P. grisea taken from different regions of Jharkhand state which were clustered into three groups based on different cultural characterstics i.e., group I included six isolates, group II and III included eleven and thirty three isolates, respectively. Parasexual recombination rarely occurs in nature and cause genetic and phenotypic variation through hyphal anastomosis.   Figure 1 On the basis of colony color, growth pattern, sector formation, zonation, wrinkles and radial growth of all the 50 isolates using R (Version 3.4.0) statistical tool (Fig.1).

Figure 4
The explanation of genetical variation in P. grisea may be sought on the basis of the ultrastructural evidence of hyphal anastamosis which is presented below (Fig.4).