Progress report for GNE21-270
Objective 1: To Identify the effect of cover cropping legacy on mycotoxin accumulation and disease severity in maize during Fusarium and Gibberella stalk and ear rot.
Specific objectives are to:
a. Assess the effect of cover crop legacy on disease severity (lesion size) of maize due to Gibberella (F. graminearum) and Fusarium (F. verticillioides) stalk rot.
b. Assess the effect of cover crop legacy on the DON contamination of ears of maize due to Gibberella ear rot disease (F. graminearum).
c. Assess the effect of cover crop legacy on the Fumonisin B1 and Fumonisin B2 contamination of ears of maize due to Fusarium ear rot disease (F. verticillioides)
Objective 2: To identify the interaction between cover cropping legacy, mycorrhizal colonization, and ear rot disease in maize.
The purpose of this project is to quantify the effect of cover cropping legacy on mycotoxin accumulation and disease severity of maize infected with F. graminearum and F. verticillioides. These are important maize pathogen species in the Northeast, contributing to both stalk and ear rot1. While fusarium infection can contribute to yield loss during both ear and stalk rot, it is the production of mycotoxins which is of greatest concern2,3. Mycotoxins are non-enzymatic metabolites that are toxic to animals or humans during consumption4. Deoxynivalenol (DON), is a major mycotoxin produced by F. graminearum and can cause vomiting, feed refusal and reduced immune functioning in livestock (deoxynivalenol). The major group of mycotoxins produced by F. verticillioides are fumonisins, which cause fatal livestock diseases equine leukoencephalomalacia, porcine pulmonary edema, and cancer in laboratory animals and are correlated with esophageal cancer and embryonic developmental defects, and neurological disease in humans4–7. Because maize kernels are used in food products for humans and animals, and stalks may be used for livestock feed as silage, reduction of mycotoxin accumulation through Fusarium disease management is critical.
While management options for Fusarium ear and stalk rot disease exist, they are often limited in their efficacy and are typically focused on conventional production systems. Plant genetic resistance is the most important management tactic for mycotoxin reduction in maize. While targeted genetic resistance to Fusarium has proven to be illusive, Bt maize (a transgenic variety which produces insect toxins derived from the soil bacterium Bacillus thuringiensis8) has effectively reduced both ear and stalk rot through reduction in insect herbivory8–15. Nonetheless, Bt maize is currently not an option for use in organic production and is also becoming less effective for conventional growers due to increasing insect resistance to Bt16,17. Alternative approaches must be explored for the reduction of Fusarium disease.
Various cultural management practices have been shown to impact mycotoxin development in maize and other field crops4,18. While the implementation of cover cropping systems (the cultivating of non-cash crops over winter periods between cash crop season) in PA has been steadily increasing, limited research has been conducted on the effect of this practice on Fusarium disease19–21. Cover crops provide many agronomic benefits including increased fertilizer and irrigation retention, weed suppression, and in some cases have been shown to reduce insect pest and pathogen damage in the following crop22–24. These disease interactions are nuanced and the exact effect of the cover crop for disease suppression is dependent on the cover crop species, pathogen, and host plant. I hypothesize that different cover cropping species will have differing effects on mycotoxin and Fusarium disease severity due to the unique ecosystem services they provide. Species which reduce disease can be leveraged as a sustainable management option for mycotoxin reduction which would contribute to improved human and animal health, and increased farmer income, as these diseases result in an annual yield loss in maize of 340,170 bushels costing $1.4 million in PA25.
To quantify the effect of cover cropping on Fusarium disease severity and mycotoxin contamination in maize, a combination of field and greenhouse experiments were performed. Experiments investigate ear and stalk rot disease in maize grown in legacies of different cover crop monocultures. The field experiment and soil collection for greenhouse experiments will be conducted at the Cover Crop Cocktails site at The Russell E. Larson Agricultural Research Center at Rock Springs, PA30. This research site consists of a randomized complete block design with four replications of the following crop rotation: cover crop -> maize -> rye cover crop -> soybean -> wheat -> cover crop. Cover crops used include two cereal cover crops (triticale, oats), two leguminous cover crops (winter pea, clover) and two brassicaceous cover crops (canola, forage radish).
With the financial support of this grant, I hired undergraduate students to assist in establishing and maintaining greenhouse experiments, Fusarium inoculations, disease severity, and mycorrhizal colonization analyses. Through these experiences, undergraduate students were involved with multiple stages of the scientific process. They gained the laboratory and field research skills necessary to conduct independent scientific research in the future.
For Objective 1a (Assess the effect of cover crop legacy on disease severity of successive maize due to Gibberella and Fusarium stalk rot), soil was collected from the following cover crop plots; triticale, radish, pea, and fallow plots directly after cover cropping and before maize planting in May 2022. In September 2022, forty corn plants (MC-3890 variety) were grown directly in each soil type in the greenhouse. Stems from twenty plants in each soil treatment were inoculated with PDA agar plugs containing F. verticillioides tissue or sterile PDA agar (control). Inoculations occurred at the V9-10 growth stage once nodes were detectable by touch. A sterile needle was used to create a hole in the center of each stem, and a plug of treatment agar was pressed on top of the hole and secured with Parafilm19. After two weeks, the stem was split in half vertically along the line of the hole, and photographs were taken with a ruler for calibration. The area of the rot lesion was measured by using ImageJ software. The relationship between cover crop legacy and lesion area will be analyzed using a one-way ANOVA in Rstudio (v4.2.1) with the p-value set to 0.05. Mean comparisons of treatments will be made using a Tukey-HSD test.
Fig 1. Field soil from the cover crop cocktail field site was collected with the help of members of a collaborating lab and stored in a cold room until experimental use.
To address Objective 1b (Assess the effect of cover crop legacy on the DON contamination of ears of successive maize due to Gibberella ear rot disease), maize ears were inoculated with F. graminearum in each maize plot as part of the cover crop cocktail trial during the 2021 and 2022 growing seasons. Ten plants per plot will be inoculated with F. graminearum or H2O (control). This will be performed on all four replicate plots of individually cover cropped treatments (triticale, oats, canola, forage radish, winter pea, clover), as well as the four fallow treatment plots.
In 2021, inoculations were performed via silk channel injection during the first 6 days after silk emergence, when ears are the most susceptible32. A liquid spore suspension (5 x 105 spores/mL) was injected directly into the silks with a blunt tipped 18.5 gauge syringe. Maize ears were harvested from the field at maturity, and ear rot severity was evaluated using the rating scale described by Reid et al. (1992)33. Severity will be estimated as 1 = no infection, 2 = 1–3% of kernels infected, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 = 76%+.
Due to observations of extremely high disease pressure in 2021, the inoculation strategy and disease severity measurement was modified for 2022. A toothpick inoculation method was performed rather than a silk channel injection. Sterilized toothpicks were colonized with F. graminearum by incubating for three weeks in flasks with an 0.5mm layer of liquid Czapek-Dox media and an F. graminearum spore suspension or just Czapek-Dox media (control). Fourteen days after silk emergence, when kernels were developing and beginning to milk, one toothpick was stabbed into the side of each ear and left in the ear until harvest (Fig 2).
During both years, inoculated ears from each replicate plot were pooled, as well as a group of non-inoculated ears in each plot. These samples will be ground and analyzed for mycotoxin levels. Two analyses will occur per sample to ensure analysis accuracy. The major mycotoxin associated with F. graminearum colonization of maize is deoxynivalenol (DON). The level of DON contamination will be measured through high pressure liquid chromatography – UV detection (HPLC-UV) as described by Yoshizawa et al. (2001)34.
The relationship between cover crop legacy, ear rot severity, and DON contamination will be analyzed using a Kruskal-Wallis HSD test in Rstudio with the p-value = 0.05. Mean comparisons of treatments will be made using a Tukey-HSD test. Regression models will be used to characterize the relationship between individual cover crop treatments and DON contamination.
Fig. 2A At the silking stage, corn plants were inoculated with Fusarium pathogens through silk channel injection method.
Fig. 2B The assistance of fellow M.S. student Tyler McFeaters and friend Ashley Fogelsanger was crucial to completing inoculations in one day.
Fig. 2C A toothpick inoculation method was performed in 2022 rather than a silk channel injection
To address Objective 1c (Assess the effect of cover crop legacy on the fumonisin B1 and fumonisin B2 contamination of ears of successive maize due to Fusarium ear rot disease), field inoculations and disease severity ratings will occur as described in Objective 1b. In this study, though, F. verticillioides will be inoculated through silk channel injection rather than F. graminearum, and these samples will be analyzed for FB1 and FB2 contamination. Sample preparation will be the same as for Objective 1b.Fumonisin extraction and quantification will occur through high-performance liquid chromatography (HPLC) based on the methods of Sydenham et al. (1992)35.
The relationship between cover crop legacy, ear rot severity, and fumonisin contamination will be analyzed using a two-way ANOVA in Rstudio with the p-value = 0.05. Mean comparisons of treatments will be made using a Tukey-HSD test. Regression models will be used to characterize the relationship between individual cover crop treatments and fumonisin contamination.
To address Objective 2 (To identify the interaction between cover cropping legacy, mycorrhizal colonization, and ear rot disease in successive maize) a greenhouse study will be conducted. An experiment was conducted to determine the effect of mycorrhizal colonization on the severity of stalk rot disease in maize. In this experiment, one hundred and twenty short flowering maize plants (Early Sunglow Variety, Burpee) were planted in pots containing sterile soil, half of which were inoculated with 1.0g of a commercial AMF inoculum, MycoGrow (Fungi Perfecti) placed beneath the seed at planting. This commercial inoculum contained six species of AMF fungi. Plants were allowed to grow until the V9-10 growth stages, and stalks were inoculated with pathogen treatments through a stab inoculation method as described in Objective 1a. Within each AMF treatment (+AMF and NoAMF), twenty plants were inoculated with F. verticillioides, twenty with F. graminearum, and twenty with a sterile PDA plug. After two weeks, stalks will be harvested, and lesion size will be measured as stated in Objective 1a.
A field experiment was conducted to describe the relationship between cover crop type, AMF colonization, and disease severity. The primary goal of this experiment is to identify whether the interaction between AMF x disease severity is also seen in cover-cropped field plots19. Inbred maize varieties (developed by Dr. Ruairidh JH Sawers – see attached letter of commitment) that have lost their ability to form mycorrhizal association due to the loss of the CASTOR gene will be used as a control (referred to as AMF resistant) and maize of the same variety with functioning CASTOR gene and having the ability to form AMF associations (AMF susceptible) will be used for all experimental groups36. Two cover crop cocktail plots of Triticale, Radish, Pea, and Fallow were chosen for this experiment. Thirty AMF-resistant plants and thirty AMF-susceptible plants were planted in the same row of each experimental plot. Ten plant stalks per treatment were inoculated with either F. graminearum, F. verticillioides, or control using the toothpick inoculation method described in Objective 1B, however, toothpicks were inserted into the second internode from the ground rather than the ear. Inoculation occurred in early September once stalks were as mature as they would be for the season.
To confirm AMF colonization in AMF susceptible treatments compared to AMF resistant plants and correlate the disease level to actual rates of mycorrhizal association, AMF colonization will be estimated through qPCR-based methods. Root samples of each plant will be harvested, washed, and stored in the freezer until analysis. A qPCR method will be used to estimate the abundance of Glomera sp. DNA relative to a genetic marker in the plant tissue gives a measure of relative colonization of AMF in the Glomera genus, accounting for a vast majority of AMF that associates with corn.
The relationship between cover crop legacy, AMF association, and disease severity will be analyzed using a two-way ANOVA in Rstudio with the p value = 0.05. Mean comparisons of treatments will be made using a Tukey-HSD.
Greenhouse experiment results to address objective 1a
Fusarium stalk rot (F. verticillioides) disease severity was not significantly structured by cover crop soil type when grown in greenhouse pots containing cover crop soil (Fig. 3). These plants were grown in 100% field soil, and a high level of stunting and nutrient deficiency was observed in the plants. In other greenhouse experiments, we use a soil mix containing only 60% soil and a 40% mix of Turface, sand, and Osmocote to mitigate this. However, we wanted to reveal the effect of the cover crop soil without amendment in this experiment. Due to the stress that these plants experienced, it is likely that any possible effect of the cover crop soil on plant defense and disease severity was masked by the effects of plant stress.
(Fig. 3) Fusarium stalk rot (FSR) and control Lesion areas of split stalks grown in different cover crop soils. Diamonds represent the mean lesion area, and letters of significance are based on a p < 0.05.
2021 Field experiment results to address objectives 1b and 1c
(Fig. 4) Example image of ears infected with F. graminearum in 2021. These ears were harvested from a pea cover crop plot.
(Fig. 5) Example image of ears infected with F. verticillioides in 2021. These ears were harvested from a triticale cover crop plot.
(Fig. 6) Example image of ears in the control (water) treatment group in 2021. These ears were harvested from a fallow treatment plot.
(Fig. 7) Example image of ears that remained uninoculated in 2021. These ears were harvested from a clover cover crop plot.
Disease severity was not significantly structured by cover crop legacy (at a confidence level of p =0.05) in corn inoculated with either F. graminearum or F. verticillioides (Fig. 8, 11) However, trends in the distribution of disease severity between cover crop treatments are described in boxplot visualization. A high frequency of high F. graminearum disease severity was observed in corn grown in a pea legacy compared to all other cover crop types and fallow treatment (Fig. 9). There was also a higher frequency of low disease ratings in the Brassica and Grass cover crops in comparison to Fallow and Legume treatments (Fig. 8).
F. verticilliodes disease severity was lower on average than the disease severity caused by F. graminearum. This was expected as F. verticillioides infection is usually characterized by isolated clusters of fungal growth on ear. In contrast, F. graminearum infection is characterized by complete coverage of the ear as the fungal mycelium grows downward from the ear tip. While not significant, it was observed that higher frequencies of low disease severity were observed in legume, brassica and grass in comparison to fallow plots (Fig. 10). Specifically, higher frequencies of low observations were seen in pea, clover, canola, and oat plots in comparison to fallow (Fig. 11).
An overall lack of significant correlation between mean disease severity and cover crop type may be a function of extremely high disease severity in F. graminearum-infected ears. It is possible that the high inoculum load and, therefore, high disease pressure overwhelmed any influence the cover crop systems may have on disease severity and susceptibility. The inoculation strategy was therefore modified for the 2022 season to reduce overall disease pressure in the hope of better revealing any influence of cover crop type on disease severity.
Eight subsamples of grain inoculated with F. verticillioides from each cover crop legacy were analyzed through HPLC-FLD for fumonisin B1 (FB1) and fumonisin B2 (FB2) accumulation. Total fumonisin, fb1, and fb2 accumulation were not significantly structured by cover crop type (Fig. 12). Trends in total fumonisin are not grouped by cover crop type and seem to be driven by individual species instead. There was a significant effect of field location on fumonisin accumulation, so we are currently exploring the results based on this effect.
(Fig. 8) F. graminearum disease severity grouped by cover crop types in maize ears that received F. graminearum silk channel injection. Diamonds represent mean disease rating values, and boxplots indicate the distribution of disease severity observations. p = 0.9651
(Fig. 9) F. graminearum disease severity grouped by cover crop species in maize ears which received water control silk channel injections. Diamonds represent mean disease rating values, and boxplots indicate the distribution of disease severity observations. p =0.1849 based on Kruskal-Wallis test of mean association.
(Fig. 10) F. verticillioides disease severity grouped by cover crop type in maize ears that received F. verticillioides silk channel injections. Diamonds represent mean disease rating values, and boxplots indicate the distribution of disease severity observations. p = 0.7831 based on Kruskal-Wallis test of mean association.
(Fig. 11) F. verticillioides disease severity grouped by cover crop species in maize ears that received F. verticillioides silk channel injections. Diamonds represent mean disease rating values, and boxplots indicate the distribution of disease severity observations. p = 0.5029 based on Kruskal-Wallis test of mean association.
(Fig. 12) Mean total fumonisin accumulation in ears inoculated with F. verticillioides and grown in different cover crop legacies. Total fumonisin is calculated based on the sum of fumonisin B1 (FB1) and fumonisin B2 (FB2). Error bars are based on the standard error of the mean sum of fumonisin and Letters of statistical significance are based on p < 0.05.
Greenhouse experiment results to address objective 2
While not significant, a trend was observed that there was greater disease severity in stalks that were inoculated with AMF in comparison to plants that were not inoculated with AMF (p = 0.38 for F. verticillioides infected stalks and p = 0.61 for F. graminearum infected stalks based on a Tukey HSD test) (Fig. 13). The mean lesion area was significantly higher in stalks inoculated with either pathogen than inoculated with a control treatment (p <0.05). This experiment is being replicated to see if the same trend is observed. The replication of this result would support the hypothesis that mycorrhizal colonization can increase stalk rot disease severity.
(Fig. 13) Lesion area of split stalks grouped by pathogen x mycorrhizal treatment interaction. Diamonds represent mean lesion areas and letters of significance are based on p < 0.05.
Education & Outreach Activities and Participation Summary
Results from my research will be shared with a broad audience of stakeholders, including other researchers, extension educators, industry representatives, and farmers. By sharing our results with the research community, we hope to add to the knowledge base on the ecosystem services provided by cover cropping systems and promote further study of the relationship between cover crops and plant pathogens. Through this project I hope to be able to provide foundational information on the interactions between cover crops and Fusarium disease. This information can directly contribute to recommendations made by extension educators and decisions made by farmers regarding cover crop selection. Therefore communication of these findings directly with extension educators and farmers will enhance the impact of this project.
I am committed to making the findings of this research easily accessible to other students and researchers. As such, my results will be incorporated into educational modules on cover crops, which were designed by Dr. Jason Kaye (collaborator on the cover crop cocktail field plot) and other cover crop researchers at Penn State30. These modules are designed for college courses to share as an open resource, but can be modified for other groups.
The results of my research project will be communicated to audiences at the APS (American Phytopathological Society) Northeastern division meeting in 2022, as well as the 2023 PASA (Pennsylvania Sustainable Agriculture) conference. The audience of the APS meeting will include researchers, extension specialists, and industry representatives who specialize in plant pathology. These professionals have a particular interest in plant disease management, therefore my research will be of interest due to it’s novel investigation of cover cropping for Fusarium disease mitigation. By presenting at the PASA conference, I will share my research with a distinctly different demographic which consists of a growers, extension educators, and industry representatives focused on sustainable agriculture and food systems. This audience will have vested interest in sustainable agriculture and will therefore have an understanding of cover crop systems as they relate to soil health and weed suppression. By sharing my findings with this audience I hope to enhance their understanding of how cover crops can also be leveraged for disease mitigation, which they may implement into their own sustainable agriculture operations.
I also hope to communicate by findings on the influence of cover crops on stalk and ear rot to PA farmers by writing an extension article for Field Crop News. Field crop news is a biweekly to weekly outlet for articles and issues about field and forage crops, published through the Penn State Field and Forage Crops Extension team, and is aimed at reaching PA field crop farmers. A peer – reviewed article will also be written at the conclusion of our research to communicate our findings on the influence of cover cropping systems on Fusarium to other researchers as well.