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Characterization and Molecular Phylogeny of Magnaporthe Oryzae Causing Rice Blast Disease in Eastern India

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Characterization and Molecular Phylogeny of Magnaporthe Oryzae Causing Rice Blast Disease in Eastern India essay

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Abstract

Rice blast, caused by the hemibiotrophic fungus Magnaporthe oryzae, is one of the most potent threats to rice production and causing tremendous yield loss. In the present study, seventy one M. oryzae isolates were collected from three states of eastern India. Based on colony color, the blast isolates were grouped in five; grayish (11), grayish black (9), grayish white (19), blackish (20), and white (12). Most of the isolates were smooth (60) and few were rough (11) in colony appearance. The blast isolates produced characteristic spindle shaped symptoms on susceptible plant. Five isolates were recorded as highly virulent, eight as moderately virulent isolates and only two isolates were observed as mild isolates. The sequence similarities among fifteen M. oryzae isolates varied from 78 to 98.8%. The phylogenetic analysis showed uniform distribution of 71 blast isolates into two clusters and indicated the existence of high genetic variation among blast isolates originated from the same location and existence of genetic similarity among blast isolates originated from different geographical origins. The outcome of the present work would help to formulate strategies for improved disease management against rice blast through resistance breeding, genetic studies and host-pathogen interaction.

Introduction

Rice (Oryza sativa L.) is one of the most important food crops that help in the survival of more than 50% of the world population1. Rice is cultivated in all the continents except Antarctica. It played a significant role in food security of Asia, where more than 90 per cent of the rice is produced and consumed. The rice productivity is influenced by numerous biotic and abiotic factors2. Among biotic stresses, fungal diseases are of major concern causing significant yield losses in the rice crop all over the world3,4. Amongst diseases, rice blast disease caused by Magnaporthe oryzae is one of the most destructive diseases in rice5.The blast pathogen is a heterothallic fungus and the new races are constantly evolving with diverse phenotypic virulence6. M. oryzae can infect a large number of plant species including, rice, wheat, and barley and finger millet7. Under blast-conducive environment, the pathogen can infect the paddy crop at all the stages of growth and development8. Wide geographic distribution, continuous evolution of new races, and high yield losses make this fungal disease a severe threat to rice production. It is estimated that every year rice blast is responsible to causes 10-30 percent of yield losses in rice7, that resulted in severe epidemics in major rice growing regions of the world.

Although, the fungicide application provided a short-term solution but, it is practically un-economical for resource poor farmers. Utilization of broad spectrum blast resistant varieties is the most successful, economical and environmentally responsive approach to manage blast disease9. However, M. oryzae is known for its high genetic variation that leads to breakdown of blast resistant varieties within 2–3 years of their release in the farmer’s field10. The M. oryzae genome possessed abundant transposable elements that create chromosomal rearrangement, gene duplication and gene evolution11. This leads to the continuous evolution of new pathogenic races that are able to infect previously reported blast resistant varieties. The disease management approach required the better understanding of the phenotypic and genetic diversity of the M. oryzae populations, that would help in the devising the strategies to manage this disease through gene stacking and gene deployment.

Understanding the genetic variability of the pathogen population not only help in understanding the co-evolution in the plant pathosystem but, also enhanced the durability of resistance cultivars. However, limited information is available about the phenotypic and genetic variation of blast pathogen populations from eastern India. Therefore, present study was designed to study the variation in morphological characters, virulence analysis and genetic diversity of M. oryzae isolates from eastern India. The information obtained from this study will guide to develop strategies to manage the havoc caused by blast disease in the endemic areas of the eastern India.

Material and methods

Collection and long term storage of rice blast pathogen: Blast infected rice samples were obtained from the different rice growing regions of eastern India, particularly, Chhattisgarh (31), Odisha (30) and Jharkhand (10). The blast infected samples were cut into small pieces and surface sterilized by dipping into ethanol (70%) for 45 sec followed by dipping in a mercuric chloride solution (0.1%) for 2–3 min, washed with autoclaved distilled water and finally leaf bits were dried with sterile filter paper and incubated for 5-7 days at 25°C. The blast fungus cultures were purified using single spore isolation and hyphal tip method. Single-conidial isolates were stored in sterile eppendorf tubes at -20°C.

Morphological characterization and pathogenicity of M. oryzae: The cultural characteristics viz; texture and colony color of all the M. oryzae isolates were studied by growing them on oat meal agar (OMA) medium for 7-10 days at 25±2°C. After attaining full growth, conidia were harvested and concentration was adjusted to 5×104 spores/ml. The highly susceptible variety (HR12) was grown in plastic trays and inoculated at three to four leaf stages. The inoculated plants were maintained in darkness for 24 hours at 25±2°C and more than 90% relative humidity. The disease reaction was recorded 7 days after inoculation using 0-5 scale scoring system12.

DNA isolation and quantification: Fungal mycelia (100-150 mg) was weighed and ground in liquid nitrogen using sterilized mortar pestle. The powdered mycelium was transferred to a 2 ml eppendorf tube containing 880 µl of extraction buffer (2% CTAB buffer, 4M NaCl, 0.5M EDTA, 1M Tris-Cl, 0.02% β-Mercaptoethanol). After incubation at 65°C for 1 hour, equal volume of phenol: chloroform: isoamylalcohol (25:24:1), was added and centrifuged @ 12000 rpm for 10 minutes. After proper mixing, transfer the clear supernatant to an eppendorf tube, an equal volume of chloroform: isoamylalcohol (24:1) was added followed by mixing and centrifuged @ 12000 rpm for 10 minutes. Then, add chilled absolute alcohol to the supernatant, mixed well and kept at -20°C for 2 hrs. After centrifugation, DNA pellet was washed with 70% ethanol, air dried and resuspended in 100μl TE buffer. The gel electrophoresis and Nanodrop Spectrophotometer were used to determine the quality and quantity of fungal DNA. After quantification, the DNA samples were diluted to a concentration of 30-50 ng/μl for use in PCR reaction.

PCR amplification and gel elution: The ITS primer pairs were used for molecular identification of M. oryzae isolate (Table 1). The PCR reaction was performed in a 25 μl reaction volume with, 0.5 μM of primers, 10 mM dNTP, 1.5 mM MgCl2, 50 ng of template DNA, 1X Taq buffer and 1U of DNA Taq polymerase. The PCR was performed as following parameters: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec, followed by a final extension at 72°C for 10 min. The amplified PCR products were analyzed in gel electrophoresis and documented under UV using gel documentation system (AlphaImager, USA). The desired band was cut from the gel clean, sterile scalped blade and DNA was eluted as per standard protocol.

Sequencing and phylogenetic relationships: The PCR product obtained using the primer pairs, (ITS 1 and ITS4) were purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA) and T&A cloning vector kit was used to clone. The positive clones were sequenced in AB 13130 Genetic analyzer (Xcelris, India). The ClustalX software was used for version 1.81 multiple sequence alignment and pair wise alignment was made using13. The neighbor-joining tree was constructed using Mega5 software14.

Results and Discussion

Rice blast is the most destructive fungal disease of rice that affects its global production. Due to the existence of numerous races, resistance to rice blast break down within 2-3 years of their release thus imposed a constant challenge in breeding for resistance. The co-evolution in the plant pathosystem can be best understood by analyzing the genetic variation in plant pathogen populations15. Infected samples showing characteristic symptoms of spindle-shaped lesions were collected from different locations of eastern India (Table 2). The blast pathogen was isolated from the infected leaf blast samples on OMA medium. The colony color varied from grayish to grayish white and blackish. Further, the blast isolates were purified using single spore and hyphal tip method and stored in OMA slants on sterilized filter paper discs at -20°C for medium term storage. Similarly, infected leaves, necks and nodes were collected to isolate M. oryzae and purified through dilution techniques and single spore isolation16 and through single spore isolation on water agar17. Likewise, blast infected rice leaf samples from different rice growing tracts of Tamil Nadu and Chhattisgarh were collected and purified using hyphal tip and single spore isolation method18,19.

Colony characteristics: Seventy one isolates of M. oryzae were grown on OMA mediu

Abstract

Rice blast, caused by the hemibiotrophic fungus Magnaporthe oryzae, is one of the most potent threats to rice production and causing tremendous yield loss. In the present study, seventy one M. oryzae isolates were collected from three states of eastern India. Based on colony color, the blast isolates were grouped in five; grayish (11), grayish black (9), grayish white (19), blackish (20), and white (12). Most of the isolates were smooth (60) and few were rough (11) in colony appearance. The blast isolates produced characteristic spindle shaped symptoms on susceptible plant. Five isolates were recorded as highly virulent, eight as moderately virulent isolates and only two isolates were observed as mild isolates. The sequence similarities among fifteen M. oryzae isolates varied from 78 to 98.8%. The phylogenetic analysis showed uniform distribution of 71 blast isolates into two clusters and indicated the existence of high genetic variation among blast isolates originated from the same location and existence of genetic similarity among blast isolates originated from different geographical origins. The outcome of the present work would help to formulate strategies for improved disease management against rice blast through resistance breeding, genetic studies and host-pathogen interaction.

Introduction

Rice (Oryza sativa L.) is one of the most important food crops that help in the survival of more than 50% of the world population1. Rice is cultivated in all the continents except Antarctica. It played a significant role in food security of Asia, where more than 90 per cent of the rice is produced and consumed. The rice productivity is influenced by numerous biotic and abiotic factors2. Among biotic stresses, fungal diseases are of major concern causing significant yield losses in the rice crop all over the world3,4. Amongst diseases, rice blast disease caused by Magnaporthe oryzae is one of the most destructive diseases in rice5.The blast pathogen is a heterothallic fungus and the new races are constantly evolving with diverse phenotypic virulence6. M. oryzae can infect a large number of plant species including, rice, wheat, and barley and finger millet7. Under blast-conducive environment, the pathogen can infect the paddy crop at all the stages of growth and development8. Wide geographic distribution, continuous evolution of new races, and high yield losses make this fungal disease a severe threat to rice production. It is estimated that every year rice blast is responsible to causes 10-30 percent of yield losses in rice7, that resulted in severe epidemics in major rice growing regions of the world.

Although, the fungicide application provided a short-term solution but, it is practically un-economical for resource poor farmers. Utilization of broad spectrum blast resistant varieties is the most successful, economical and environmentally responsive approach to manage blast disease9. However, M. oryzae is known for its high genetic variation that leads to breakdown of blast resistant varieties within 2–3 years of their release in the farmer’s field10. The M. oryzae genome possessed abundant transposable elements that create chromosomal rearrangement, gene duplication and gene evolution11. This leads to the continuous evolution of new pathogenic races that are able to infect previously reported blast resistant varieties. The disease management approach required the better understanding of the phenotypic and genetic diversity of the M. oryzae populations, that would help in the devising the strategies to manage this disease through gene stacking and gene deployment.

Understanding the genetic variability of the pathogen population not only help in understanding the co-evolution in the plant pathosystem but, also enhanced the durability of resistance cultivars. However, limited information is available about the phenotypic and genetic variation of blast pathogen populations from eastern India. Therefore, present study was designed to study the variation in morphological characters, virulence analysis and genetic diversity of M. oryzae isolates from eastern India. The information obtained from this study will guide to develop strategies to manage the havoc caused by blast disease in the endemic areas of the eastern India.

Material and methods

Collection and long term storage of rice blast pathogen: Blast infected rice samples were obtained from the different rice growing regions of eastern India, particularly, Chhattisgarh (31), Odisha (30) and Jharkhand (10). The blast infected samples were cut into small pieces and surface sterilized by dipping into ethanol (70%) for 45 sec followed by dipping in a mercuric chloride solution (0.1%) for 2–3 min, washed with autoclaved distilled water and finally leaf bits were dried with sterile filter paper and incubated for 5-7 days at 25°C. The blast fungus cultures were purified using single spore isolation and hyphal tip method. Single-conidial isolates were stored in sterile eppendorf tubes at -20°C.

Morphological characterization and pathogenicity of M. oryzae: The cultural characteristics viz; texture and colony color of all the M. oryzae isolates were studied by growing them on oat meal agar (OMA) medium for 7-10 days at 25±2°C. After attaining full growth, conidia were harvested and concentration was adjusted to 5×104 spores/ml. The highly susceptible variety (HR12) was grown in plastic trays and inoculated at three to four leaf stages. The inoculated plants were maintained in darkness for 24 hours at 25±2°C and more than 90% relative humidity. The disease reaction was recorded 7 days after inoculation using 0-5 scale scoring system12.

DNA isolation and quantification: Fungal mycelia (100-150 mg) was weighed and ground in liquid nitrogen using sterilized mortar pestle. The powdered mycelium was transferred to a 2 ml eppendorf tube containing 880 µl of extraction buffer (2% CTAB buffer, 4M NaCl, 0.5M EDTA, 1M Tris-Cl, 0.02% β-Mercaptoethanol). After incubation at 65°C for 1 hour, equal volume of phenol: chloroform: isoamylalcohol (25:24:1), was added and centrifuged @ 12000 rpm for 10 minutes. After proper mixing, transfer the clear supernatant to an eppendorf tube, an equal volume of chloroform: isoamylalcohol (24:1) was added followed by mixing and centrifuged @ 12000 rpm for 10 minutes. Then, add chilled absolute alcohol to the supernatant, mixed well and kept at -20°C for 2 hrs. After centrifugation, DNA pellet was washed with 70% ethanol, air dried and resuspended in 100μl TE buffer. The gel electrophoresis and Nanodrop Spectrophotometer were used to determine the quality and quantity of fungal DNA. After quantification, the DNA samples were diluted to a concentration of 30-50 ng/μl for use in PCR reaction.

PCR amplification and gel elution: The ITS primer pairs were used for molecular identification of M. oryzae isolate (Table 1). The PCR reaction was performed in a 25 μl reaction volume with, 0.5 μM of primers, 10 mM dNTP, 1.5 mM MgCl2, 50 ng of template DNA, 1X Taq buffer and 1U of DNA Taq polymerase. The PCR was performed as following parameters: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec, followed by a final extension at 72°C for 10 min. The amplified PCR products were analyzed in gel electrophoresis and documented under UV using gel documentation system (AlphaImager, USA). The desired band was cut from the gel clean, sterile scalped blade and DNA was eluted as per standard protocol.

Sequencing and phylogenetic relationships: The PCR product obtained using the primer pairs, (ITS 1 and ITS4) were purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA) and T&A cloning vector kit was used to clone. The positive clones were sequenced in AB 13130 Genetic analyzer (Xcelris, India). The ClustalX software was used for version 1.81 multiple sequence alignment and pair wise alignment was made using13. The neighbor-joining tree was constructed using Mega5 software14.

Results and Discussion

Rice blast is the most destructive fungal disease of rice that affects its global production. Due to the existence of numerous races, resistance to rice blast break down within 2-3 years of their release thus imposed a constant challenge in breeding for resistance. The co-evolution in the plant pathosystem can be best understood by analyzing the genetic variation in plant pathogen populations15. Infected samples showing characteristic symptoms of spindle-shaped lesions were collected from different locations of eastern India (Table 2). The blast pathogen was isolated from the infected leaf blast samples on OMA medium. The colony color varied from grayish to grayish white and blackish. Further, the blast isolates were purified using single spore and hyphal tip method and stored in OMA slants on sterilized filter paper discs at -20°C for medium term storage. Similarly, infected leaves, necks and nodes were collected to isolate M. oryzae and purified through dilution techniques and single spore isolation16 and through single spore isolation on water agar17. Likewise, blast infected rice leaf samples from different rice growing tracts of Tamil Nadu and Chhattisgarh were collected and purified using hyphal tip and single spore isolation method18,19.

Colony characteristics: Seventy one isolates of M. oryzae were grown on OMA medium and morphological characteristics were recorded viz; colony color and surface appearance. Based on colony color, the isolates were grouped in five; grayish (11), grayish black (9), grayish white (19), blackish (20), and whitish (12) (Table 2). The M. oryzae isolates showed significant variation in colony characteristic. Interestingly, most of the observed isolates were smooth (60) and a few were rough (11) in colony appearance. The present study indicates the existence of substantial variation among blast isolates belonged to eastern India for mycelial color and texture. The present study corroborated the previous findings where the colony color of the M. oryzae isolates varied from grayish, grayish white and black color with most of them have smooth colonies19,20. Similarly, blast isolates collected from different regions of Karnataka showed the buffy, grayish black colony color with medium and raised growth of mycelium21.

Pathogenicity of rice blast isolates: The existence of strains of the Pyricularia oryzae differing in pathogenicity was first noticed by Sasaki22. The virulence analysis of the blast isolates were evaluated on the susceptible cultivar HR12. After inoculation, first small specks symptoms develop that subsequently enlarge into spindle shaped lesion with the ashy grey centre. Finally, several spots coalesce to form big irregular patches. Based on the lesion length and affected leaf area, selected 15 blast isolates from three states (5 from each state) were classified into three distinct groups i.e. MG-I, MG-II and MG-III. The first group, MG-I consisted of highly virulent strains included five isolates (MG-4, MG-13, MG-71, MG-73 and MG-112). MG-II included eight moderately virulent isolates (MG-10, MG-12, MG-14, MG-70, MG-72, MG-110, MG-111 and MG-114), whereas MG-III consisted of only two mild blast isolates (MG-74 and MG-113). Our results are in agreements with the previous studies where the blast isolates were categorized based on their pathogenicity and observed considerable variation in their virulence spectrum19,20.

Sequence identity and phylogeny: The internal transcribed spacer (ITS) region is the most commonly used molecular method for identification of the fungal species. The primer pair’s, ITS-1 and ITS-4 generated an amplicon of approx 520 bp in selected fifteen isolates. Sequence similarities among fifteen M. oryzae isolates varied from 78 to 98.8%. The minimum sequence similarity was observed between Mg-10 and Mg-16 whereas, maximum sequence identity was observed between Mg-74 and Mg-113 (Table 3). Similar results were observed with Chhattisgarh isolates and showed sequence similarities ranged from 78 to 95.6%, with other blast isolates19.

The neighbour joining tree was constructed using Mega5 software based on ITS sequences of fifteen (present study) and nine (obtained from NCBI) isolates (Table 4). The phylogenetic analysis categorized the blast isolates into two clusters (Figure 1). Major cluster I consisted of fourteen blast isolates. It included three isolates from Chhattisgarh, two from Odisha and two from Jharkhand. Major cluster II included ten isolates, of which three from Odisha, two from Chhattisgarh and three from Jharkhand. Interestingly, Major cluster I included most of the world isolates except blast isolates from China and Japan. Similarly, the blast isolates of same region did not belong to the same cluster, whereas genetically similar isolates of each cluster were consisted of isolates of different location. Our study showed equal distribution of blast isolates into two clusters which indicated the existence of high genetic variation among isolates originated from different location of eastern India. Our study corroborated the findings of previous workers who observed high genetic variation among the blast isolates originated from different endemic areas of Andhra Pradesh and Chhattisgarh19,23. Similarly, a high genetic similarity was observed among blast isolates originated from Himachal Pradesh and Uttaranchal as compared to isolates collected from Karnataka and Madhya Pradesh24.

Conclusion

The blast pathogen showed considerable morphological and genetic variation among blast isolates.The blast isolates of the same location did not belong to the same cluster, whereas each cluster consisted of isolates of different location which reflects the existence of genetic variation independent of geographical locations. The genetic similarity observed among blast isolates collected from different location might be due to movement of pathogen through seed as M. oryzae is seed-borne in nature. This shows the importance of seed borne nature of the blast pathogen which helps in its survival under unfavorable condition and its movement to different geographical regions.

The authors are extremely grateful to the Director, ICAR-National Rice Research Institute, Cuttack, India for his support and facilitation for carrying out the research work successfully. We do not have any conflict of interest to declare.

Declaration to be given by authors: The facts and views in the manuscript are ours and we are totally responsible for authenticity, validity and originality etc. I / We undertake and agree that the manuscripts submitted to your journal have not been published elsewhere and have not been simultaneously submitted to other journals. I / We also declare that manuscripts are our original work and we have not copied from anywhere else. There is no plagiarism in our manuscripts. Our manuscripts whether accepted or rejected will be property of the publisher of the journal and all the copyrights will be with the publisher of the journal’.

m and morphological characteristics were recorded viz; colony color and surface appearance. Based on colony color, the isolates were grouped in five; grayish (11), grayish black (9), grayish white (19), blackish (20), and whitish (12) (Table 2). The M. oryzae isolates showed significant variation in colony characteristic. Interestingly, most of the observed isolates were smooth (60) and a few were rough (11) in colony appearance. The present study indicates the existence of substantial variation among blast isolates belonged to eastern India for mycelial color and texture. The present study corroborated the previous findings where the colony color of the M. oryzae isolates varied from grayish, grayish white and black color with most of them have smooth colonies19,20. Similarly, blast isolates collected from different regions of Karnataka showed the buffy, grayish black colony color with medium and raised growth of mycelium21.

Pathogenicity of rice blast isolates: The existence of strains of the Pyricularia oryzae differing in pathogenicity was first noticed by Sasaki22. The virulence analysis of the blast isolates were evaluated on the susceptible cultivar HR12. After inoculation, first small specks symptoms develop that subsequently enlarge into spindle shaped lesion with the ashy grey centre. Finally, several spots coalesce to form big irregular patches. Based on the lesion length and affected leaf area, selected 15 blast isolates from three states (5 from each state) were classified into three distinct groups i.e. MG-I, MG-II and MG-III. The first group, MG-I consisted of highly virulent strains included five isolates (MG-4, MG-13, MG-71, MG-73 and MG-112). MG-II included eight moderately virulent isolates (MG-10, MG-12, MG-14, MG-70, MG-72, MG-110, MG-111 and MG-114), whereas MG-III consisted of only two mild blast isolates (MG-74 and MG-113). Our results are in agreements with the previous studies where the blast isolates were categorized based on their pathogenicity and observed considerable variation in their virulence spectrum19,20.

Sequence identity and phylogeny: The internal transcribed spacer (ITS) region is the most commonly used molecular method for identification of the fungal species. The primer pair’s, ITS-1 and ITS-4 generated an amplicon of approx 520 bp in selected fifteen isolates. Sequence similarities among fifteen M. oryzae isolates varied from 78 to 98.8%. The minimum sequence similarity was observed between Mg-10 and Mg-16 whereas, maximum sequence identity was observed between Mg-74 and Mg-113 (Table 3). Similar results were observed with Chhattisgarh isolates and showed sequence similarities ranged from 78 to 95.6%, with other blast isolates19.

The neighbour joining tree was constructed using Mega5 software based on ITS sequences of fifteen (present study) and nine (obtained from NCBI) isolates (Table 4). The phylogenetic analysis categorized the blast isolates into two clusters (Figure 1). Major cluster I consisted of fourteen blast isolates. It included three isolates from Chhattisgarh, two from Odisha and two from Jharkhand. Major cluster II included ten isolates, of which three from Odisha, two from Chhattisgarh and three from Jharkhand. Interestingly, Major cluster I included most of the world isolates except blast isolates from China and Japan. Similarly, the blast isolates of same region did not belong to the same cluster, whereas genetically similar isolates of each cluster were consisted of isolates of different location. Our study showed equal distribution of blast isolates into two clusters which indicated the existence of high genetic variation among isolates originated from different location of eastern India. Our study corroborated the findings of previous workers who observed high genetic variation among the blast isolates originated from different endemic areas of Andhra Pradesh and Chhattisgarh19,23. Similarly, a high genetic similarity was observed among blast isolates originated from Himachal Pradesh and Uttaranchal as compared to isolates collected from Karnataka and Madhya Pradesh24.

The blast pathogen showed considerable morphological and genetic variation among blast isolates.The blast isolates of the same location did not belong to the same cluster, whereas each cluster consisted of isolates of different location which reflects the existence of genetic variation independent of geographical locations. The genetic similarity observed among blast isolates collected from different location might be due to movement of pathogen through seed as M. oryzae is seed-borne in nature. This shows the importance of seed borne nature of the blast pathogen which helps in its survival under unfavorable condition and its movement to different geographical regions.

The authors are extremely grateful to the Director, ICAR-National Rice Research Institute, Cuttack, India for his support and facilitation for carrying out the research work successfully. We do not have any conflict of interest to declare.

Declaration to be given by authors: The facts and views in the manuscript are ours and we are totally responsible for authenticity, validity and originality etc. I / We undertake and agree that the manuscripts submitted to your journal have not been published elsewhere and have not been simultaneously submitted to other journals. I / We also declare that manuscripts are our original work and we have not copied from anywhere else. There is no plagiarism in our manuscripts. Our manuscripts whether accepted or rejected will be property of the publisher of the journal and all the copyrights will be with the publisher of the journal’.

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