Discovery and testing of corn silk associated probiotic microbes to combat Fusarium disease and mycotoxins: An exciting new frontier in an old battle

Objectives:

• Culture silk-microbes from diverse corn varieties from across the Americas, test them in vitro for suppression of Fusarium (dual-culture), then test candidates in greenhouse trials (silk spraying), measuring disease and mycotoxins. If time and funding permit, perform greenhouse and/or seed storage trials with wheat for suppression of Fusarium head blight (FHB).
• In parallel, since many microbes are non-culturable, determine the complete silk-microbe composition using culture-independent microbial DNA sequencing. Identify anti-Fusarium microbial DNA-profiles that could be selected directly by breeders.

• Apply for field-release permits for field trials at Ridgetown, ON and/or initiate licensing with a commercial partner for wider-scale trials.

Impacts:

• The development of new commercial probiotic microbes that reduce silk-mediated Fusarium entry into corn kernels and hence reduce DON and other mycotoxins will benefit Ontario’s corn producers, processors, livestock, and consumers.
• The identification of a silk-associated microbial community profile that is associated with suppression of Fusarium graminearum and mycotoxins will benefit Ontario’s corn producers, processors, livestock, and consumers.
• Microbial profiles may be used directly for selection purposes during corn breeding.

Scientific Summary:

Similar to humans, plants are inhabited and coated by probiotic microbes. The research team previously demonstrated that plant cells, which are immobile, have maintained probiotics (endophytes) that are mobile to act similar to human immunity cells to seek-and-destroy pathogens at pathogen-entry points. However, scientists have overlooked probiotics within pollen tubes (corn silks) – hollow channels that sperm use to fertilize eggs, resulting in grain. Fusarium pathogens also enter into grain using silk channels, creating mycotoxins that affect humans and livestock. In a preliminary experiment, the research team found diverse microbes within unexposed silks: Are some of these sentinels to combat pathogens?

The research team proposed to culture fungicide-compatible silk bacteria from diverse corn varieties from across the Americas, and then test them in vitro, then in greenhouse trials (silk spraying) to combat Fusarium and mycotoxins. In parallel, as many microbes are non-culturable, the complete probiotic composition of silks was determined using culture-independent microbial DNA sequencing. Finally, anti-Fusarium microbial DNA-profiles were identified that could be selected directly by breeders.

Results

With respect to the effect of the rachis/silk bacteria on preventing Fusarium disease in corn when sprayed onto silks under field conditions, in one of two field trials, two candidate biocontrol agents (BCAs) showed significant improvement in bulk kernel weight per cob over the average negative control, while two other BCAs looked promising.

Quantitative mycotoxin content analysis for deoxynivalenol (DON) was then adopted and undertaken in the Raizada Lab using Neogen’s Veratox Kit for DON 5/5 ELISA test. One of the above promising BCAs also significantly reduced DON mycotoxin in grain compared to the Fusarium-only treated samples in one of two field trials, consistent with the improved grain yields observed. Notably, there was tremendous block-to-block variation in DON mycotoxin concentrations, with the best blocks showing excellent anti-DON results, suggestive of high biocontrol potential once further optimized. The best BCA blocks showed reductions in DON of 60% compared to the best Fusarium-only blocks (70% reductions compared to the mean) – without any optimization. As one microbe increased grain yield but did not reduce DON mycotoxin, this strain may be having a general growth promoting effect which itself is independently valuable.

In terms of wheat, when looking at the effect of the wheat rachis/corn silk microbes on their ability to suppress Fusarium head blight (FHB) when sprayed on wheat grain heads under field conditions, prior to spraying with Fusarium graminearum spores, we found that none of the biocontrol agents significantly improved yield compared to the Fusarium-only negative control plants. However, a single strain from the wheat rachis numerically increased bulk grain weight when combined with fungicide, compared to fungicide-only controls. Furthermore, the best blocks of this promising microbial spray showed >50% reductions in DON compared to Fusarium-only treated plots. In the majority of blocks, fungicide + this promising BCA (combined) had DON levels below 1 ppm. 

Moving forward, it is important to know whether the promising anti-Fusarium rachis/silk microbes are already present in a particular wheat/corn variety, although a greater quantity of that microbe may still offer benefits. To determine this, the genomic DNA of these tissues were isolated (plant DNA containing any microbial genomes) and then analyzed for their entire microbial communities. Silk microbiomes were previously reported by our group (Khalaf et al. 2021 Scientific Reports 11, 13215). The project undertook the most detailed characterization of the wheat rachis microbiome, based on 53 wheat samples spanning 150 years of bread wheat breeding. The rachis microbiome showed tremendous diversity across samples and wheat genotypes, with the population possessing at least 276 bacterial taxa and 80 fungal taxa. The most conserved and abundant bacteria include Burkholderia, Enterobacter, Delftia, Buchnera, Escherichia-Shigella, Rhizobium, Pantoea and Clostridium species. The most abundant rachis-associated fungi include Tilletia, Blumeria, Alternaria and at least 80 other fungal taxa. The results will help us to understand the prevalence/rarity of the tested biocontrol agents in Canadian bread wheat grain heads.

Overall, the results are somewhat encouraging, given that the initial hypothesis was purely speculative, that corn silks (through which Fg enters) and the wheat rachis (through which Fusarium spreads from floret to floret) may possess naturally occurring anti-Fusarium microbes. The “best blocks” may represent plants with the best colonization of the BCAs, thus representing “microbial potential”, akin to the term “yield potential” used by breeders. Future optimizations (improving the formulation, inoculant viability, concentration and stacking microbes together) may help achieve this potential. It was interesting that the best BCA strains for corn were different than for wheat. Finally, for conventional farmers, mixing a rachis/silk bacteria with fungicide may hold promise, offering multi-function modes of action to prevent a buildup of pathogen resistance. 

In conclusion, a total of three microbes are showing promise in terms of corn or wheat grain challenged with the Fusarium pathogen under field conditions, of which two are showing promise in terms of reducing DON (one each in corn and wheat). However, tremendous plot to plot and year to year variation was observed with these and all other tested strains. To be helpful to growers, the most promising microbes and their spray formulations will need further improvement. The research team has selected one promising microbe to improve in the coming years with support from GFO and has had contact with two companies who have shown interest in future commercialization. Finally, the project has identified DNA barcodes for a consortium of anti-Fusarium bacteria in corn silks that breeders can potentially use to select & promote endogenous beneficial bacteria packaged in seed.

External Funding Partners:

Organic Federation of Canada (OSC)

Agriculture and Agri-Food Canada (AAFC)

Prairie Heritage Seeds

Alberta Wheat Commission

Saskatchewan Wheat Development Commission

In-kind contributors:

Martin Meinert

Dwayne Smith

Denis Brisebois

Project Related Publications:

Khalaf, E.M., Shresha, A.R.J., Lynch, M.D.J., Shearer, C.R., Limay-Rios, V, Reid, L.M. and Raizada, M.N. 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports. 11:13215.

Shrestha, A., Limay-Rios, V., Brettingham, D.J.L. and Raizada, M.N. 2023. Bacteria existing in pre-pollinated styles (silks) can defend the exposed male fertilization channel of maize against an environmental Fusarium pathogen. Frontiers in Plant Science. 14:1292109.

Thompson, M.E.H., Shrestha, A., Rinne, J., Limay-Rios, V., Reid, L. and Raizada M.N. 2023. The cultured microbiome of pollinated maize silks shifts after infection with Fusarium graminearum and varies by distance from the site of pathogen inoculation. Pathogens. 12:1322.

Thompson, M.E.H., Shrestha, A., Khalaf, E.M., Rinne, J., Limay-Rios, V., Reid, L.M. and Raizada, M.N. 2024. Analysis of the cultured microbiome of fertilization-stage silks (styles) reveals taxonomic relationships across North American maize genotypes and heterotic groups. Bacteria. 3:476-498.

Thompson, M.E.H. and Raizada, M.N. 2024. Biologically relevant methods to test how microbes colonize maize styles (silks): Case study of a Pantoea strain. Bacteria. 3:287-298. 

Thompson, M.E.H. and Raizada, M.N. 2024. Protocols to enable fluorescence microscopy of microbial interactions on living maize silks (style tissue). Journal of Microbiological Methods. 10:1016.

Thompson, M.E.H. and Raizada, M.N. 2024. The microbiome of fertilization-stage maize silks (style) encodes genes and expresses traits that potentially promote survival in pollen/style niches and host reproduction. Microorganisms. 12:1473. 

Theses completed and publicly available:

Thompson, M.E.H. 2023. Discovery and testing of pollinated maize silk-associated microbes including microbiome assisted selection of biocontrol agents against Fusarium graminearum. PhD thesis, University of Guelph, Canada.

Shrestha, A. 2023. Discovery and testing of bacteria from pollen and unpollinated silks of Pan-American maize to combat Fusarium graminearum. PhD thesis, University of Guelph, Canada.

Gregory, J.T. 2023. The efficacy of endophytes and epiphytes against Fusarium head blight in wheat and Gibberella ear rot in corn under field conditions. MSc thesis, University of Guelph, Canada.

Muileboom, J.R.P. 2022. Discovery and testing of wheat head rachis beneficial microbes from ancestral and historic wheats for biocontrol of Fusarium graminearum. MSc thesis, University of Guelph, Canada.