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