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Maize significantly contributes to food and fuel production. Yields can be reduced due to foliar diseases, which reduce photosynthetic leaf area. The bacterial foliar disease Goss's wilt (caused byClavibacter nebraskensis) can cause significant yield losses in susceptible maize varieties.C. nebraskensiscan infect leaves through wounds and colonize the vascular tissue of the leaf. We present a protocol that replicates this process with the use of a “clapper” with pins on one end to create wounds and a sponge soaked in inoculum on the other end, which allows for efficient field inoculations of maize leaves. Disease severity is then rated on a percentage scale multiple times over the season to generate an area under disease progress curve (AUDPC). Genetic host resistance is one of the most effective forms of foliar disease control in maize, as there are few effective forms of chemical control for bacterial diseases that affect maize. Screening for resistance in diverse germplasm, or for fine mapping a specific resistance gene, requires inoculating large populations in the field for obtaining phenotypic data. Our high-throughput protocol allows for large-scale disease evaluations and is useful for finding forms of genetic resistance or to understand plant–pathogen interactions of bacterial foliar pathogens.
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Inoculation of Maize Ears with Fusarium graminearum to Study Gibberella Ear Rot
Maize is an important food and fuel crop globally. Ear rots, caused by fungal pathogens, are some of the most detrimental maize diseases, due to reduced grain yield and the production of harmful mycotoxins. Mycotoxins are naturally occurring toxins produced by certain fungal species that can cause acute and chronic health issues in humans and animals that consume mycotoxin-contaminated grain. Pathogens can infect the developing ear through silks, or through wounds in the ears produced by pests. Plants naturally develop genetic resistance to pathogens. The maize genes involved in resistance to the pathogen may be different, depending on whether the ear was infected via silks or wounds. To differentiate between these two forms of resistance, natural infections can be reproduced by injecting inoculum through the silk channel, or by producing wounds using a needle, and introducing inoculum directly onto developing ears. Our protocol describes a technique used to inoculate developing maize ears withFusarium graminearum, one of the fungal species that causes ear rot. We describe both silk channel and side needle inoculation techniques. Our protocol uses a backpack inoculator for both methods of infection, allowing for high-throughput inoculations, which are necessary for large field experiments. After harvest, the ears are visually rated on a percentage of disease scale. The protocol results in quantitative data that can be used for research on elucidating genetic resistance to fungal pathogens to assist breeding selections, and to understand plant–pathogen interactions of ear rots in maize.
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- Award ID(s):
- 2154872
- PAR ID:
- 10608741
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- Cold Spring Harbor Protocols
- ISSN:
- 1940-3402
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Gibberella ear rot (GER) of maize, caused byFusarium graminearum, poses a serious threat to human and animal safety as mycotoxins are deposited in the grain during fungal colonization and are hazardous to human and animal health. Currently, no completely resistant germplasm has been identified, and the underlying mechanisms of resistance remain unclear. In this study, we evaluated three near‐isogenic line (NIL) populations for resistance to GER—NC344 × H100 (DRIL), B73 × Oh43 (nNIL), and B73 × teosinte (tNIL)—across multiple environments. The recurrent parents, H100 and B73, were moderately resistant to GER. NC344 is susceptible to GER. Oh43 is moderately resistant to GER. The teosinte donor parent, PI 384071, had unknown resistance to GER. We employed two inoculation methods—kernel injection and silk channel injection—to assess their impact on genotype response to F. graminearum. The inoculation method did not significantly affect genotype response, although kernel inoculations produced more consistent disease levels. We identified lines with significantly increased susceptibility to GER compared to their recurrent parent in each population. We employed quantitative trait locus (QTL) mapping to identify markers associated with GER in the NC344 × H100 population. We identified QTL on chromosomes 4, 5, and 9. We highlight a large region on chromosome 5 that may harbor important alleles for GER resistance and for resistance to other ear rots. This study underscores the utility of NILs in dissecting the genetic basis of GER resistance and provides valuable resources for future fine mapping, gene discovery, and resistance breeding.more » « less
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Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused byExserohilum turcicum, is an important fungal foliar disease of maize that is highly prevalent and causes yield losses globally. Microscopy can be used to visualize plant–fungal interactions on a cellular level, which enables pathology and genetics studies. Host resistance and isolate aggressiveness can be characterized at different stages of disease development, which enables a more detailed understanding of the pathogenesis process and host–pathogen interactions. Our protocol outlines an efficient, cost-effective method for stainingE. turcicumtissue on inoculated maize leaves and visualizing samples using a compound fluorescence microscope. This protocol uses KOH treatment followed by aniline blue staining, which stains glucans present in plant and fungal cell walls, and samples are visualized using fluorescence microscopy. Quantitative data about fungal structures including the conidia, hyphal structures, and appressoria, the structures formed to push through the plant leaf surface after conidia have germinated, can be obtained from the images generated using this technique. Visualization of these structures can help pathologists understand plant–pathogen interactions for maize andE. turcicum. This method has advantages over other methods because the stain is less toxic than other available stains, samples can be processed in a more high-throughput manner than other protocols, and the required supplies are relatively inexpensive.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1101/pdb.prot108641
