Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides

There is interest in studying microbes that colonize maize silks (style tissue, critical for reproduction) including the fungal pathogenFusarium graminearum(Fg) and its interactions with the microbiome and biocontrol agents.In plantaimaging of these interactions on living silks using confocal fluorescence microscopy would provide key insights. However, newly discovered microbes have unknown effects of on human health, and there are regulatory requirements to prevent the release of fluorescently-tagged microbes into the environment.Therefore, the microbe infection, colonization, and interaction stages on silks prior to microscopy must be contained. At the same time, silk viability must be maintained and experiments conducted that are biologically relevant (e.g. silks should remain attached to the cob), yet the silk tissue must be accessible to the researcher (i.e. not within husk leaves) and allow for multiple replicates. Here we present methods that meet these five contrasting criteria. We tested these methods usingFgand four silk-derived bacterial endophytes. The endophytes were previously known to have anti-Fgactivityin vitro, butin plantaobservations were lacking. In Method 1, a portion of the tip of a cob was dissected, and silks remained attached to the cob in a Petri dish. The cob was placed on a water agar disc to maintain hydration. DsRed-tagged bacteria and GFP-taggedFgwere inoculated onto the silks and incubated, allowing the two microbes to grow towards one another before staining with propidium iodide for confocal microscopy. A variation of the protocol was presented in Method 2, where detached silk segments were placed directly on water agar where they were inoculated with bacteria andFgto promote dense colonization, and to allow for many replicates and interventions such as silk wounding. The bacterial endophytes were successfully observed colonizingFghyphae, silk trichomes, and entering silks via cut ends and wounds. These protocols can be used to study other silk-associated microbes including several globally important fungal pathogens that enter maize grain through silks.

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Bacteria existing in pre-pollinated styles (silks) can defend the exposed male gamete fertilization channel of maize against an environmental Fusarium pathogen

Shrestha,

Limay-Rios,

Brettingham

et al. 2023

Front. Plant Sci.

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In flowering plants, fertilization requires exposing maternal style channels to the external environment to capture pollen and transmit its resident sperm nuclei to eggs. This results in progeny seed. However, environmental fungal pathogens invade developing seeds through the style. We hypothesized that prior to environmental exposure, style tissue already possesses bacteria that can protect styles and seed from such pathogens. We further hypothesized that farmers have been inadvertently selecting immature styles over many generations to have such bacteria. We tested these hypotheses in maize, a wind-pollinated crop, which has unusually long styles (silks) that are invaded by the economically-important fungal pathogen Fusarium graminearum (Fg). Here, unpollinated silk-associated bacteria were cultured from a wild teosinte ancestor of maize and diverse maize landraces selected by indigenous farmers across the Americas, grown in a common Canadian field for one season. The bacteria were taxonomically classified using 16S rRNA sequencing. In total, 201 bacteria were cultured, spanning 29 genera, 63 species, and 62 unique OTUs, dominated by Pseudomonas, Pantoea and Microbacterium. These bacteria were tested for their ability to suppress Fg in vitro which identified 10 strains belonging to 6 species: Rouxiella badensis, Pantoea ananatis, Pantoea dispersa, Pseudomonas koreensis, Rahnella aquatilis, and Ewingella americana. Two anti-Fg strains were sprayed onto silks before/after Fg inoculation, resulting in ≤90% reductions in disease (Gibberella ear rot) and 70-100% reductions in associated mycotoxins (deoxynivalenol and zearalenone) in progeny seeds. These strains also protected progeny seeds post-harvest. Confocal fluorescent imaging showed that one silk bacterium (Rouxiella AS112) colonized susceptible entry points of Fg on living silks including stigmatic trichomes, wounds, and epidermal surfaces where they formed thick biofilms. Post-infection, AS112 was associated with masses of dead Fg hyphae. These results suggest that the maize style (silk) is endowed with potent bacteria from the mother plant to protect itself and progeny from Fusarium. The evidence suggests this trait may have been selected by specific indigenous peoples, though this interpretation requires further study.

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Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides

Cited by 4 publications

References 46 publications

Seed biopriming with Ochrobactrum ciceri mediated defense responses in Zea mays (L.) against Fusarium rot

Seed biopriming with Ochrobactrum ciceri mediated defense responses in Zea mays (L.) against Fusarium rot

Protocols to enable fluorescence microscopy of microbial interactions on living maize silks (style tissue) that balance the requirements for containment, biological relevance, viability, and experimental accessibility

Bacteria existing in pre-pollinated styles (silks) can defend the exposed male gamete fertilization channel of maize against an environmental Fusarium pathogen

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