A Basic Guide to the Growth and Manipulation of the Blast Fungus, Magnaporthe oryzae

Abstract

The blast fungus, Magnaporthe oryzae, is a devastating plant pathogen that threatens global food security. The social and economic importance of blast disease has contributed to this filamentous fungus becoming a model organism for the study of host-pathogen interactions. Availability of the complete genome sequences of many strains of the pathogen, as well as rice and wheat cultivars, coupled with the tractability of M. oryzae to classical and molecular genetic manipulation have contributed to its widespread study. Although M. oryzae has been extensively investigated for the past two decades, procedures for storing, maintaining, and manipulating the blast fungus in the laboratory had not been compiled and updated. As a consequence, there is considerable disparity in how the fungus is stored and manipulated between studies. In this article, we present a collection of protocols providing clear explanations of how to preserve filter stocks of M. oryzae; how to grow the fungus in both liquid and solid media; how to extract genomic DNA from fungal mycelium; how to induce appressorium formation on coverslips for visualization and tissue collection; and how to perform two distinct types of plant infection assay for virulence assessment. By sharing our most used laboratory procedures, we aim to address some of the knowledge gaps in current M. oryzae protocols and contribute to uniformity and robustness in studies by the Magnaporthe research community. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Storage of M. oryzae strains Basic Protocol 2: Revival and regular maintenance of M. oryzae cultures in solid medium Alternate Protocol 1: Regular maintenance of M. oryzae cultures in liquid medium Basic Protocol 3: Genomic DNA extraction from M. oryzae mycelium Alternate Protocol 2: Quick DNA extraction from M. oryzae mycelium Basic Protocol 4: M. oryzae induction of appressorium development on glass coverslips for microscopy Alternate Protocol 3: M. oryzae induction of appressorium development on glass coverslips for tissue collection Basic Protocol 5: M. oryzae rice infection assay through spray inoculation Alternate Protocol 4: M. oryzae leaf-drop plant infection assay.

Keywords: M. oryzae; Pyricularia; appressorium; conidia; culture; fungal development; mycelium; virulence.

Figure 1

Left, a 7‐day‐old plate culture of M. oryzae (Guy11) after incubation at 24°C with a 12‐hr photoperiod. The red square indicates the growth margin where the mycelium should be cut to be transferred onto fresh CM agar for subculture. Middle, a CM medium agar plate with a piece of M. oryzae mycelium inoculated in the center and Whatman filter paper squares for the generation of fungal filter stocks. Right, a 12‐day‐old M. oryzae culture grown on CM agar with filter papers through which fungal mycelium has grown (at top right, a filter through which mycelium has grown has been collected from the plate).

Figure 2

M. oryzae liquid culture of strain Guy11 in liquid CM medium before (left) and after (right) incubation for 30 hr at 24°C with a 12‐hr photoperiod in a rotary shaker set at 100 rpm. A typical liquid culture is shown, made using a 250‐ml Erlenmeyer flask covered with a foam stopper and aluminum foil to prevent contamination.

Figure 3

Schematic representation of the induction of M. oryzae appressorium development on hydrophobic coverslips. 30‐μl droplets of conidial suspension (5 × 10⁴ conidia/ml) are inoculated on hydrophobic coverslips and incubated from 2 hr up to 24 hr on square petri dishes containing water‐soaked tissue paper to create a humid environment; they are later visualized by microscopy. On the right, a typical microscopy image (×100) of germinated appressoria (of strain Guy11) on coverslips after 8 hr of inoculation. Scale bar, 10 μm. Figure created with BioRender (biorender.com).

Figure 4

Schematic representation of M. oryzae spray‐inoculation protocol. Conidial suspensions are evenly sprayed onto 3‐week‐old rice (CO‐39) seedlings using an artist's airbrush. Plants are then incubated for 6 days (2 days covered with polyethylene bag and 4 days uncovered) in a controlled chamber set at 24°C with a 12‐hr photoperiod and 85% relative humidity. At right, an example of CO‐39‐infected leaf tip infected with M. oryzae Guy11 spores 6 days after spray inoculation. Figure created with BioRender.com.

Figure 5

M. oryzae leaf‐drop plant infection assay using 1‐week‐old barley (cultivar Golden Promise). Left, Schematic representation. Right, barley leaf fragments (∼4 cm) inoculated with 30‐μl droplets of Guy11 conidial suspension (5 × 10⁴ conidia/ml) and incubated at 24°C with a 12‐hr photoperiod, before and 5 days after inoculation. Figure created with BioRender (biorender.com).