Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Summary

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Abstract

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Introduction

Magnaporthe oryzae, the rice blast fungus, infects an assortment of grain crops, including barley, wheat, and rice¹. This pathogen causes devastating diseases and poses a worldwide threat to these valuable crops, causing complete crop loss if not controlled. Many labs around the world focus on rice blast disease because of its global threat and its attributes as an excellent model for plant-fungal interactions². It has been fully sequenced, and the genetics of its infective cycle, particularly the early events, have been established^3,4. The life cycle be....

Protocol

1. Preparation of experimental materials

1. Prepare oatmeal agar (OMA) by blending oatmeal until it is a fine powder. Add 25 g of oatmeal powder and 15 g of agar to 500 mL of ddH₂O, and autoclave on media cycle (alternatively bring to a boil for 20 min). Pour the media into sterile 60 mm Petri dishes.
   NOTE: Other media types that induce sporulation, such as V8 agar, are acceptable for this protocol.
2. Plate M. oryzae filter stocks directly onto the OMA plates using sterile forceps, and allow them to cover the entire plate (9-12 days). Place the plates in a growth incubator at 25 °C with 12:12 h day:nigh....

Results

A depiction of the initial workflow for this technique is displayed in Figure 1. The sheaths were harvested from 14-day-old susceptible "Lacey" barley plants (H. vulgare). The conidia were harvested from 10-day-old sporulating M. oryzae OMA plates, with a conidial suspension prepared using sterile ddH₂O for a final concentration of 5 x 10⁴ spores per mL. The inoculum suspension was directly applied to the leaf sheaths, which were secured to sterile Pet.......

Discussion

There are many commonly used assays available to test M. oryzae strains that provide a macroscopic-level visual of a compatible or incompatible infection response, such as spray or droplet inoculations, and the use of rating systems to quantifylesion sizes^13,14. Another common assay for M. oryzae is to test the ability of the pathogen to form its specialized penetration structure, the apppressorium¹⁵. Described here is an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma-Aldrich	A6283
Cell phone	Google	Pixel 4A	Any smartphone with a rear facing camera that can be mounted in an a holder will suffice.
Cell phone Microscope adapter	Vankey	B01788LT3S	https://www.amazon.com/Vankey-Cellphone-Telescope-Binocular-Microscope/dp/B01788LT3S/ref=sr_1_2_sspa?keywords=vankey+cellphone+telescope+adapter+mount&qid=1662568182&sprefix= vankey+%2Caps%2C63&sr=8-2 -spons&psc=1&spLa=ZW5jcnlwd GVkUXVhbGlmaWVyPUFKNklBR jlCREJaMEcmZW5jcnlwdGVkSWQ 9QTA2MDMxNjhBRFYxQTMzNk9E M0YmZW5jcnlwdGVkQWRJZD1BM DQxMzAzOTMxNzI1TzE3M1ZGTEI md2lkZ2V0TmFtZT1zcF9hdGYmY WN0aW9uPWNsaWNrUmVkaXJlY3 QmZG9Ob3RMb2dDbGljaz10cnVl
Glycerol	Sigma-Aldrich	G5516
Microscope	AmScope	FM690TC	40x–2500x Trinocular upright epi-fluorescence microscope
Oatmeal old fashioned rolled oats	Quaker	N/A	https://www.amazon.com/Quaker-Oats-Old-Fashioned-Pack/dp/B00IIVBNK4/ref=asc_df_B00IIVBNK4/?tag=hyprod-20&linkCode=df0 &hvadid=312253390021&hvpos= &hvnetw=g&hvrand=98212627704 6839544&hvpone=&hvptwo=&hvq mt=&hvdev=c&hvdvcmdl=&hvlocint =&hvlocphy=9007494&hvtargid =pla-568492637928&psc=1
ProMix BX	ProMix	1038500RG
Rectangular coverglass	Corning	CLS2975245
Slides, microscope	Sigma-Aldrich	S8902
Stage micrometer	OMAX	A36CALM7	0.1 mm and 0.01 mm Microscope calibration slide
Trypan blue	Sigma-Aldrich	T6146