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Summary

Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Published: March 9th, 2021

DOI:

10.3791/62287

1Institute for Bioscience and Biotechnology Research, University of Maryland, 2Department of Nutrition and Food Science, University of Maryland College Park, 3Department of Plant Sciences and Landscape Architecture, University of Maryland College Park
* These authors contributed equally

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 µm mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 µm pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

Powdery mildew is one of the most common and important diseases of numerous food crops and ornamental plants1. Studies of powdery mildew diseases have been very popular, as evidenced by over 10,500 publications as the search result with "powdery mildew" as key word at the Web of Science (as of November 2020). Indeed, powdery mildew (represented by Blumeria graminis) is considered to be one of the top 10 fungal pathogens by the journal of Molecular Plant Pathology2. Quantification of disease susceptibility is a necessary step in characterization of plant genes contributing to disease resistance or suscep....

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1. Making a standard inoculation box with a removable top lid mounted with a mesh

  1. Purchase a roll of 50 µm nylon membrane mesh or 48 µm stainless steel mesh (recommended) from stores. Make sure to order enough for cutting into multiple pieces of 14 in x 26 in for replacement of worn mesh.
  2. Purchase one 1/4 in x 2 ft x 4 ft medium density fiberboard or plywood, and cut two 24-1/2 in x 10 in pieces and two 12 in x 10 in pieces for making an inoculation box.Use 8 corner clamps for supporting t.......

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Here, we present a new powdery mildew spore inoculation method that is easy to prepare, operate and adjust. Figure 1 shows the assembly of the standard inoculation box with emphasis on the make of the removable lid mounted with a 50 µm membrane mesh. Figure 2 shows the assembly of the inoculation chamber. Figure 3 illustrates the key steps of the inoculation process using this system. Figure 4 show.......

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Our meshed-box-based inoculation method has several advantages over other inoculation methods. First, it can achieve even distribution of spores if operated properly, as demonstrated in Figure 5. Second, the use of ~50 µm mesh, plus spore-dislodging by gentle shaking of infected leaves can reduce plant infection by thrips or other plant-infecting insects that are present in source plants. Third, the use of different-sized inoculation boxes for inoculating plants or detached leaves insid.......

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The work was supported by the National Science Foundation (IOS-1901566) to S. Xiao. The authors would like to thank F. Coker and C. Hooks for the maintenance of the plant growth facility, and Jorge Zamora for technical help associated with fabrication of the inoculation box and chamber.

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Name Company Catalog Number Comments
 48 µm stainless steel grid mesh screen; Size: 24" X 48"  Amazon NA For making the lid of an inoculation box
#6-32 x ¾" machine screws, flat washers and nuts  Home Depot NA For making an inoculation chamber
#6-32 zinc plated nylon lock nut (4-Pack) Home Depot NA For making an inoculation chamber
#6-32x3/8” Phillips flat head machine screws, flat washers and nuts  Home Depot NA For securing  magnet door catch plates
#8-32x1/2" machine screws, flat washers and nuts Home Depot NA For securing corner braces and door hinge
0.250 thick clear extruded acrylic film-masked sheet;  Size: 17 ½" X 20" Professional Plastics SACR.250CEF For making an inoculation chamber
0.250 thick clear extruded acrylic film-masked sheet; Size: 18" X 20"   Professional Plastics  SACR.250CEF For making an inoculation chamber
0.250 thick clear extruded acrylic film-masked sheet; Size: 18" X 30"  Professional Plastics SACR.250CEF For making an inoculation chamber
0.250 thick clear extruded acrylic film-masked sheet; Size: 20" X 29 ½ " Professional Plastics SACR.250CEF For making an inoculation chamber
1-5/8" cabinet door magnetic catch white Home Depot Model #P110-W For making an inoculation chamber
2" steel zinc-plated corner brace (8-Pack)  Home Depot  Model #13611  For making an inoculation box & chamber
3" Corner Clamp Harbor Freight Tools SKU 63653, 1852, 60589 For making inoculation chamber
3/4"  steel zinc plated corner brace (4-Pack) Home Depot Model #13542 For making an inoculation box & chamber
4-7/8" zinc-plated light duty door pull handles Home Depot Model #15184 For making an inoculation box
Fine fan-blender brushes Michaels Store M10472846  For inoculation
Kelleher 3/4" x 3/4" x 36" wood square dowel  Home Depot NA For making the lid of an inoculation box
Medium density fiberboard (1/4" x 2' x 4');  Home Depot Model# 1508104 For making an inoculation box
Round glass coverslips with a 500 µm grid ibidi USA Inc. 10816 For determining  spore density

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