Suitable methods for isolation, culture, storage and identification of wheat blast fungus Magnaporthe oryzae Triticum pathotype

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.

Antagonistic Activity of Siderophore-Producing Bacteria from Black Rice Rhizosphere against Rice Blast Fungus Pyricularia oryzae

“…2C). These results are consistent with earlier studies which showed that the length of conidia ranged from 22.76−28.56 µm and width 7.85−9.21 µm [35]. The isolate was then subcultured to PDA medium and incubated for 7 days to undergone molecular identification (Fig.…”

Marine Natural Product Antimycin A Suppresses Wheat Blast Disease Caused by Magnaporthe oryzae Triticum

“…A fungal isolate BTJP 4–5 was obtained in pure culture from the field-infected wheat blast samples by picking up a single conidium and then preserving it at 4 °C on dry filter paper, following the method described earlier [ 47 ]. For this study, this virulent isolate BTJP 4–5 of MoT was retrieved from storage in potato dextrose agar (PDA) medium and incubated at 25 °C for conidia production [ 47 ].…”

“…The conidial suspension was prepared from 10-day-old culture plates and the spore concentration was adjusted to ca. 5 × 10 4 conidia mL −1 , as described by [ 47 ].…”

Taxonomic and pathogenic diversity of the blast pathogen populations infecting wheat and grasses in Minas Gerais

“…A sterilized sealed Pasteur pipette was scraped over the sporulation and loaded with conidia, which was streaked across the agar-water dishes supplemented with chloramphenicol and streptomycin at 100 µg/ml each. Plates were incubated at 25±5°C for 24 hours (12/12 h fluorescent light/darkness) (Farman et al 2017;Gupta et al 2020).…”

“…Blasted paper pieces were transferred to a new sterilized plastic dish filled with blue silica and left to dry at room temperature (25°C±5) for five days. Dried paper pieces were transferred to a 2 ml-microtube half-filled with new and sterile blue silica-gel (Figure 5G) and stored in a -10°C freezer (Farman 2002;Farman et al 2017;Gupta et al 2020). Isolates were stored in duplicates as a backup of the entire collection.…”