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 severity and mycotoxin contamination in maize, a combination of field and greenhouse experiments will be performed. Experiments will investigate ear and stalk rot disease in maize grown in legacies of various 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 will hire an undergraduate student to assist in the set up and maintenance of greenhouse experiments, Fusarium inoculations, disease severity, and mycorrhizal colonization analyses. Through these experiences, the undergraduate student will be involved with multiple stages of the scientific process and will gain 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 crops; triticale, radish, pea, and fallow plots directly after cover cropping and before maize planting in May 2021. In the Spring of 2022, thirty 60-day hybrid sweet corn plants will be grown directly in each soil in the greenhouse. A short flowering variety of sweet corn was selected for greenhouse study due to their short stature, fast generation time, and susceptibility to Fusarium pathogens. Stems from ten plants from each soil treatment will be inoculated with F. verticillioides, F. graminearum, or PDA agar (control) at the V6 growth stage consistent using methods previously published19. Inoculation will occur through a stab inoculation method. A sterile needle will be used to create a hole onto the center of each stem and fungal hyphae, harvested using a 5mm diameter cork borer from growth medium and will be pressed on top of the hole on the stem and secured with parafilm. At plant maturity, the stem will be split in half vertically along the line of the hole and photographs will taken with a ruler for calibration. The area of the rot lesion was measured by measuring the length and width of the lesion and calculating the area of the oval created. This experiment will be carried out twice, giving a total of 20 plants per treatment
The relationship between cover crop legacy and lesion area will be analyzed using a one-way ANOVA in Rstudio with the p-value set to 0.05. Mean comparisons of treatments will be made using a Tukey-HSD test .
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 will be 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 each per plot will be inoculated with F. graminearum or H2O (control) through silk injections. 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. The silks are most highly susceptible to F. graminearum infection during the first 6 days of emergence; therefore, inoculations will occur 5-6 days after silk emergence32. Ears were inoculated using a silk channel inoculation with F. graminearum. A liquid spore suspension (5 x 105 spores/mL) will be injected directly into the silks with a blunt tipped syringe. Maize ears will be harvested from the field at maturity for disease analysis.
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%+. Inoculated kernels from each replicate plot will be pooled, as well as the control group of non-inoculated kernels in each plot, and 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 gas chromatography – mass spectrometry (GC-MS) as described by Hallen-Adams et al. (2011)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.
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. Sample preparation will be the same as for Objective 1b, however, fumonisin extraction and quantification will occur through high-performance liquid chromatography (HPLC) based on methods of Sydenham et al. (1992)35. Levels of FB1 and FB2 will be measured for each treatment.
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) two greenhouse experiments will be conducted. A preliminary experiment was conducted in January 2021 to identify 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 for 5 weeks, and stalks were inoculated with pathogen treatments through a stab inoculation method as described in Objective 1a. Within each AMF treatments (+AMF and NoAMF), twenty plants were inoculated with F. verticillioides, twenty with F. graminearum, and twenty with a sterile PDA plug. After a week stalks will be harvested, and lesion size will be measured as stated in Objective 1a.
If the results from the previous experiment indicate that AMF colonization influences the disease severity of Fusarium stalk rot leasions, a second greenhouse experiment using cover cropped soils and AMF maize mutants will be conducted to describe the relationship between cover crop type, AMF colonization, and disease severity. The primary goal of this experiment is to identify whether increased AMF colonization of maize in a triticale cover crop treatments is responsible for increased Fusarium disease in comparison to a radish cover crop as demonstrated in Ray et al (2021)19. 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. Treatments will therefore consist of Triticale soil + AMF susceptible maize, Triticale soil + AMF resistant maize, radish soil + AMF susceptible maize, and radish soil + AMF resistant maize. Ten plants per treatment will be inoculated with either F. graminearum, F. verticillioides, or H2O through a silk channel injection as described for objective 1a. At plant maturity, ears will be harvested, and disease severity ratings will be performed using the rating scale described in objective 1b.
To confirm AMF colonization in AMF susceptible treatments compared to AMF resistant plants, as well as correlate the level of disease to actual rates of mycorrhizal association, AMF colonization will be measured at the roots of harvested plants. Root samples of each plant will be harvested, washed, and cut into 1 cm segments. Segments will be made clear with 10% sodium hydroxide and boiled in a solution of vinegar and 5% black Sheaffer ink for staining of mycorrhizae. Segments will be randomly selected from each sample and the number of root intersections will be enumerated and calculated as percent colonization.
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.
Results from the 2021 field experiment to address Objectives 1b and 1c
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. However, trends in the distribution of disease severity between cover crop treatments are described in boxplot visualization.
The overall distribution of F. graminearum disease rating observations was higher in legume cover crops than in all other cover crop types (Figure 5). High frequencies of high disease severity occurred in pea treatments specifically (Figure 6). There was also a higher frequency of low disease ratings in the Brassica and Grass cover crops in comparison to Fallow and Legume treatments (Figure 5). Low disease severity was seen in all control and uninoculated plots regardless of cover crop species or type (Figure 7, Figure 8).
F. verticilliodes disease severity was lower on average than disease severity seen in disease caused by F. graminearum. This was to be expected as F. verticillioides infection is usually characterized by isolated clusters of fungal growth on ear, while F. graminearum infection is characterized by a more complete and full coverage of the ear as the fungal mycelium grows downward from the ear tip. Disease severity was lower in legume, brassica and grass in comparison to fallow plots (Figure 9). Specifically, higher frequencies of low observations were seen in pea, clover, canola, and oat plots in comparison to fallow (Figure 10). In both control and uninoculated plots, there was some disease pressure noted, as all had mean ratings between 1 and 2 (Figure 11, Figure 12).
Overall lack of significant correlation between mean disease severity and cover crop type may be a function of extremely high disease severity and in F. graminearum infected ears. It is possible that the high inoculum load and therefore high disease pressure overwhelmed any influence that cover crop systems may have on disease severity and susceptibility. In future ear rot experiments, including the repetition of this field experiment in the summer of 2022, inoculation methods will be modified to reduce overall disease pressure in the hope of better revealing the influence of cover crop type on disease severity. Additionally, mycotoxin levels from these ears are in the process of being quantified and this data may reveal trends not seen through disease severity measures.
Results from greenhouse experiment to address Objective 2
While not significantly significant (p=0.1730), mean lesion area was higher in F. graminearum that was inoculated with AMF in comparison to plants which were not inoculated with AMF (Figure 13). This trend was however not observed in F. verticillioides treated stalks. Mean lesion area was significantly higher in stalks inoculated with F. graminearum and treated with AMF than both control treatments (p=0.0173, p=0.0125). All other treatments did not have mean lesion areas significantly higher than the control. There was however a high frequency of lesion areas which measured 0 cm2 across all treatments due to inoculation error. The internode which was inoculated in many plants was not yet mature and therefore the inoculated piece of tissue had moved up the plant, following the growth of the basal meristem, and was absent within the area of sampled stalk. Therefore, this experiment will be replicated using a different variety of corn and a later inoculation time to remedy this. Additionally, the time between inoculation and sampling will be increased from one to two weeks to increase the size of lesions, hopefully exaggerating differences in lesion area.
Given that there was a high frequency of 0 cm2 measurements across this data, and a trend was still observed where F. graminearum lesions were larger when treated with AMF than without AMF, it is promising that we may see this trend prove significant in future replications where the number of 0 cm2 lesions is reduced. This result would support the hypothesis that mycorrhizal colonization can increase F. graminearum disease severity.
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.