Rapid Detection of Wheat Blast Pathogen Magnaporthe Oryzae Triticum Pathotype using Genome-Specific Primers and Cas12a-mediated Technology

Abstract Wheat blast disease caused by a South American lineage of Magnaporthe oryzae Triticum (MoT) pathotype has emerged as a serious threat to wheat production in Bangladesh since its first emergence in 2016. Efficient and suitable methods for isolation, storage, inoculum production and molecular characterization of the pathogen can help in achieving the target of sustainable management of the disease in a relatively short period of time. In this study, we aimed to develop suitable methods for isolation, storage and morphological characterization and molecular identification of MoT isolates collected from the blast-infected wheat fields in Bangladesh. This process included modification of existing protocols that were available for a related fungal pathogen M. oryzae or de novo method development and validation. We developed suitable methods for isolation of MoT from field-infected plant samples using modified monoconidial isolation technique and produced abundant conidia from a single mycelial plate for in vivo pathogenicity assay in a reproducible manner. Cultural and morphological characterization of the isolates revealed that all Bangladeshi MoT isolates are of a single clonal lineage with similar cultural and morphological characters. Molecular detection of isolates with M. oryzae-specific primers Pot1 and Pot2 and MoT-specific primers MoT3F and MoT3R produced bands with the expected size from all wheat-infecting isolates. We also successfully established a PCR-based detection system based on a commercially available detection kit for field-infected leaf and seed samples by detecting Pot2- and MoT3-specific bands. Additionally, the simple method we developed in our study for producing abundant conidia in a very short period of time will be very helpful in studying biology of the wheat blast fungus. This method was also proven to be more user-friendly and cost-effective than previously available methods. Successful characterization of MoT isolates at morphological and molecular levels coupled with detection of the pathogen in infected field and seed lots should be useful for efficient surveillance and management of the fearsome wheat blast disease.

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Tracing seed to seeding transmission of the wheat blast pathogen Magnaporthe oryzae pathotype Triticum

Plant Pathology ◽

10.1111/ppa.13400 ◽

2021 ◽

Author(s):

S.I. Martinez ◽

A. Wegner ◽

S. Bohnert ◽

U. Schaffrath ◽

A. Perello

Keyword(s):

Magnaporthe Oryzae ◽

Wheat Blast

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First Report of Virulence and Effects of Magnaporthe oryzae Isolates Causing Wheat Blast in Argentina

Plant Disease ◽

10.1094/pdis-11-14-1182-pdn ◽

2015 ◽

Vol 99 (8) ◽

pp. 1177 ◽

Cited By ~ 20

Author(s):

A. Perelló ◽

I. Martinez ◽

M. Molina

Keyword(s):

Magnaporthe Oryzae ◽

First Report ◽

Wheat Blast

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The MoT3 assay does not distinguish between Magnaporthe oryzae wheat and rice blast isolates from Bangladesh

10.1101/345215 ◽

2018 ◽

Author(s):

Dipali Rani Gupta ◽

Claudia Sarai Reyes Avila ◽

Joe Win ◽

Darren M. Soares ◽

Lauren S. Ryder ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Rice Blast ◽

Dna Amplification ◽

Diagnostic Tools ◽

Diagnostic Assay ◽

Asian Countries ◽

Wheat Blast ◽

New Hosts ◽

Grass Hosts ◽

The Uk

ABSTRACTThe blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the UK. In addition, comparative genomics of the WB12 sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast.

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Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates

Phytopathology ◽

10.1094/phyto-06-18-0199-le ◽

2019 ◽

Vol 109 (4) ◽

pp. 504-508 ◽

Cited By ~ 8

Author(s):

Dipali Rani Gupta ◽

Claudia Sarai Reyes Avila ◽

Joe Win ◽

Darren M. Soanes ◽

Lauren S. Ryder ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Rice Blast ◽

Dna Amplification ◽

Diagnostic Tools ◽

Diagnostic Assay ◽

Wheat Blast ◽

The United Kingdom ◽

New Hosts ◽

Grass Hosts ◽

Further Development

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.

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Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia

PLoS ONE ◽

10.1371/journal.pone.0238724 ◽

2020 ◽

Vol 15 (9) ◽

pp. e0238724 ◽

Cited By ~ 2

Author(s):

Batiseba Tembo ◽

Rabson M. Mulenga ◽

Suwilanji Sichilima ◽

Kenneth K. M’siska ◽

Moses Mwale ◽

...

Keyword(s):

Triticum Aestivum ◽

Magnaporthe Oryzae ◽

Triticum Aestivum L ◽

Blast Disease ◽

Wheat Blast

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Rapid detection of Magnaporthe oryzae chrysovirus 1-A from fungal colonies on agar plates and lesions of rice blast

Journal of General Plant Pathology ◽

10.1007/s10327-014-0567-6 ◽

2014 ◽

Vol 81 (2) ◽

pp. 97-102 ◽

Cited By ~ 15

Author(s):

Syun-ichi Urayama ◽

Yu Katoh ◽

Toshiyuki Fukuhara ◽

Tsutomu Arie ◽

Hiromitsu Moriyama ◽

...

Keyword(s):

Magnaporthe Oryzae ◽

Rapid Detection ◽

Rice Blast

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Soil supplementation with Si, B and Zn and their synergetic effects in reducing severity of wheat blast (Magnaporthe oryzae Triticum)

International Journal of Agricultural Research Innovation and Technology ◽

10.3329/ijarit.v11i2.57259 ◽

2022 ◽

Vol 11 (2) ◽

pp. 76-84

Author(s):

MH Kabir ◽

HR Nayan ◽

MA Abedin ◽

MB Meah

Keyword(s):

Magnaporthe Oryzae ◽

Susceptible Variety ◽

Blast Disease ◽

Management Approach ◽

Limiting Factor ◽

Wheat Blast ◽

Soil Preparation ◽

Cropping Seasons ◽

House Condition ◽

Chemical Pesticides

Wheat blast (Magnaporthe oryzae Triticum) in Bangladesh and South America is recognized as one major limiting factor of wheat production. Its control using chemical pesticides raises concerns about food safety and pesticide resistance, which have dictated the need for alternative blast management approach, nutrient supplementation could be an ecofriendly alternative. Experiments were carried out under confined net house condition for two consecutive cropping seasons. Single doses of the nutrients (Si, B and Zn) were incorporated during soil preparation. Plants of the wheat blast susceptible variety BARI Gom-26 were inoculated with spores (1 x 107 spores ml-1) of Magnaporthe oryzae Triticum at blast vulnerable pre-heading stage of 52 days age. Typical wheat blast symptoms of spike bleaching from top to downward appeared on sight 14 days after inoculation i.e., 66 days age of the crop. Incidence and severity of blast bleaching of spike were scored for four times starting from 68 days age @ three day’s interval. None of the nutrients could stop the incidence of blast on wheat; however, some nutrients reduced the blast incidence significantly. Solo application of Si, B and Zn or combination of two caused significant reduction of spike bleaching. With the mixed application of Si, B and Zn, > 47% reduction of wheat blast severity was obtained. The results revealed that the soil application of silicon, zinc and boron had a synergistic effect on the intensity of blast disease of wheat. Int. J. Agril. Res. Innov. Tech. 11(2): 76-84, Dec 2021

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Screening and Biochemical Characterization of Wheat Cultivars Resistance to Magnaporthe oryzae pv Triticum (MoT)

Journal of Plant Stress Physiology ◽

10.25081/jpsp.2020.v6.6096 ◽

2020 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Chandra Shekhar Biswas ◽

Afsana Hannan ◽

Abul Monsur ◽

G H M Sagor

Keyword(s):

Magnaporthe Oryzae ◽

Biochemical Characterization ◽

Cell Damage ◽

Blast Resistance ◽

Wheat Variety ◽

Infection Process ◽

Fungal Diseases ◽

Cellular Damage ◽

Wheat Cultivars ◽

Wheat Blast

Global food security is seriously threatened due to increased frequency and occurrence of fungal diseases. One example is wheat blast caused by Magnaporthe oryzae is a fungal diseases of rice, wheat, and other grasses, that can destroy the whole food production to sustain millions of people. Wheat blast was first detected in february 2016 with a serious outbreak in Asia. Assessment of the available germplasms to stress tolerant/resistant is one of the best options for developing stress tolerant crop varieties. In this study, a total of sixteen wheat cultivars were collected and test their disease severity to blast pathogen Magnaporthe oryzae pv. Triticum (MoT). Among the varieties, BARI Gom 33 exhibited partially resistance against blast pathogen, whereas all other genotypes become susceptible to MoT. Different yield and yield contributing characters of both resistant and susceptible cultivars were also evaluated and found no significant differences among them. To understand the underlying mechanism of resistance in BARI Gom 33, antioxidant enzyme activity, concentration of reactive oxygen species and cellular damage after fungal infection were also evaluated and found that activities of ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were higher in BARI Gom 33 than BARI Gom 25 and BARI Gom 31. The hydrogen peroxide (H2O2) and malondealdehyde (MDA) content in BARI Gom 33 was low compare to BARI Gom 25 and BARI Gom 31, which may due to greater increase of the APX, CAT and POD in resistant genotypes. Thus, it may suggest that a more efficient antioxidative defense system in BARI Gom 33 during the infection process of M. oryzae restricts the cell damage caused by the fungus. The identified genotypes can either be used directly in the blast prone area or as a source of resistance to further development of blast resistance high yielding wheat variety.

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