This project is geared toward investigating the impact of cover crops and soil characteristics on the occurrence and diversity of Metarhizium. Accordingly, the project consists of the four objectives listed below. We collected soil samples from 12 cover crop treatments and conducted a sentinel insect assay to detect insect-pathogenic fungus in the soil. Relative abundance of the insect-pathogenic fungus, Metarhizium, was estimated as the percentage of sentinel Galleria mellonella infected with the fungus 10 days after exposure to the soil. We used binomial regression to determine the relationship between Metarhizium and soil properties. We established 91 Metarhizium isolates from the soil samples and characterized them using molecular methods. According to molecular characterization data, M. robertsii predominated in the soil at the site.
Biological control agents play an integral part in keeping pest populations in check. On organic farms, natural enemies including predators, parasitoids, and pathogens are even more important because farmers cannot spray their crops with synthetic insecticides. The beneficial soil fungus, Metarhizium, is a pathogen that attacks arthropods and can help keep pest populations at bay. The prevalence of Metarhizium is effected by soil characteristics such as organic matter, active carbon, and pH level. Cover crops used in sustainable agriculture influence these soil characteristic levels. Recently Metarhizium was found to also grow in plants endophytically and promote plant growth. The goal of my project is to study how feeding behavior and growth rate of an agricultural pest insect is affected by Metarhizium colonization in plants. This project was conducted within the context of a larger study, the Cover Crop Cocktails Project, which is researching the effects cover crops have on weed and insect management, nutrient cycling, soil quality, crop productivity, and profitability in an organic feed-grain rotation.
Objective 1: Determine the effects of cover crop treatments and soil characteristics on the occurrence and species diversity of endemic Metarhizium isolates using sentinel insect assays and molecular techniques. Completed by Puneet Randhawa in 2016.
Objective 2: Determine the ability of endemic Metarhizium species to establish growth in corn and selected cover crop species. Ongoing.
Objective 3: Measure the effects of Metarhizium-infection of corn on the growth of corn and a corn pest. Ongoing.
Objective 4: Develop extension products for outreach and extension events relating to the effects of cover crops on Metarhizium. Ongoing.
Completed by Puneet Randhawa in 2016.
Over the course of two replicates of a choice feeding assay, 141 corn plants were grown from seed exposed to Metarhizium and 53 control plants were grown without seed exposure to Metarhizium. The corn seeds were inoculated with spores from M. robertisii originally collected from soil in which a monoculture of canola was growing. Seed was surface sterilized, then placed in Metarhizium spore suspension (1×108 CFU/mL in 0.05% Triton X-100) for 2 hours. The control seeds were soaked in the 0.05% Triton X-100 solution without the Metarhizium spores for 2 hours. To confirm endophytic colonization by Metarhizium in plants grown from exposed seed, I collected their second true leaves and two root fragments from each plant, surface sterilized them, and plated tissue sections onto selective media containing chloramphenicol, thiabendazole, and cycloheximide (CTC media). Endophytic infection was considered positive if Metarhizium grew from these plant tissues. When Metarhizium was not recovered, the plant was considered exposed but not endophytically colonized.
Ho: Corn plants grown from seed exposed to Metarhizium will not support endophytic root or leaf colonization.
HA: Corn plants grown from seed exposed to Metarhizium will support endophytic root or leaf colonization.
I measured the above-ground height and chlorophyll content of sixty corn plants (40 inoculated with Metarhizium and 20 control plants) every 2-6 days for 24 days beginning 6 days after planting. The chlorophyll content was measured with a SPAD-502 Plus chlorophyll meter in units of SPAD on three locations of the second true leaf: closest to the stem, the middle of the leaf, and the distal tip of the leaf. I average the three chlorophyll measurements and heights at each measurement. After the 24 days, I collected each of the plants’ second true leaves and two root fragments from each plant, surface-sterilized them, and plated them onto CTC media to determine if Metarhizium colonization was expressed in the plant.
Ho: Corn plants grown from seed exposed to Metarhizium will not differ in height and chlorophyll content from corn plants grown from seed that was not exposed to Metarhizium.
HA: Corn plants grown from seed exposed to Metarhizium will differ in height and chlorophyll content from corn plants grown from seed that was not exposed to Metarhizium.
OBJECTIVE 3B: Effects of Metarhizium on relative growth rate of fall armyworm
To obtain initial weights of neonate fall armyworm larvae, I weighed them in groups of 30 larvae and then divided by 30 to obtain the average initial mass of each larva. I divided the fourth true leaf from V4 corn plants into three 9 cm long leaf fragments for use in feeding assays. Thirty neonate fall armyworm larvae were fed leaf fragments from 10 corn plants grown from unexposed seed (control) and thirty larvae were fed leaf fragments from 10 plants that were grown from seed exposed to Metarhizium. After 4 days (96 hours) the fall armyworm larvae were individually weighed and their Relative Growth Rate (RGR) was calculated using the formula:
RGR = ln (final weight) – ln (initial weight)
number of days
After the leaf portions were fed to the larvae, portions of the leaf and two portions of the roots from each plants were plated onto CTC media to determine endophytic colonization by Metarhizium. We tested a total of 120 neonate fall armyworm larvae in two replicates (30 control, 30 experimental per replicate.) and 40 corn plants (10 control, 10 experimental per replicate).
Ho: Relative Growth Rate (RGR) of fall armyworm larvae fed on corn plants grown from seed exposed to Metarhizium will not differ from the RGR of fall armyworm fed on corn plants grown from seed that was not exposed to Metarhizium.
HA: Relative Growth Rate (RGR) of fall armyworm larvae fed on corn plants grown from seed exposed to Metarhizium will differ from the RGR of fall armyworm fed on corn plants grown from seed that was not exposed to Metarhizium.
OBJECTIVE 3C: Effects of Metarhizium on feeding preference of fall armyworm
The feeding choice assay included three treatments: 1) no choice with corn leaf tissue from plants grown from Metarhizium-exposed seed; 2) no choice with corn leaf tissue from plants grown from unexposed seed; 3) choice tests with corn leaf tissue of each type. Fifty second instar fall armyworm larvae were placed individually in petri dishes containing 6 corn leaf squares measuring 1.1 x 1.1 cm. Ten of these petri dishes contained 6 leaf squares from control plants, ten had 6 leaf squares from Metarhizium-exposed plants, and thirty contained 3 leaf squares from control plants and 3 leaf squares from exposed plants placed in an alternating pattern near the edge of the petri dish. The orientation of the leaf squares for the choice arenas was recorded to account for effect due to differences in light or other environmental factors that might influence the movement of the fall armyworm. The location of the larvae and feeding events were recorded every 30 minutes for 2 hours. To determine endophytic colonization of plants used in the assay, I collected, surface sterilized and plated on CTC medium portions of the same leaf used in assays, and two root sections. This experiment has been repeated twice, with a total of 100 larvae (10 control, 10 experimental, and 30 choice per replicate), and 28 corn plants (Rep. 1: 3 control, 5 experimental; Rep. 2: 10 control, 10 experimental). Data will consist of the frequency of leaves fed upon from each treatment, and the percentage of tissue eaten using ImageJ.
Ho: Fall armyworm feeding will not differ between corn leaf tissue grown from Metarhizium-exposed seed and leaf tissue from plants grown from unexposed seed.
HA: Fall armyworm feeding will differ between corn leaf tissue grown from Metarhizium-exposed seed and leaf tissue from plants grown from unexposed seed.
OBJECTIVE 2 – RESULTS:
Out of the 141 plants grown from Metarhizium-exposed seed, Metarhizium grew from at least one root fragment in 80 plants (56.7%); and from at least one leaf section in 89 plants (63.1%). In total, Metarhizium grew from the root or leaf sections of 106 plants (75.2%). Metarhizium was not recovered from any of the control plants.
OBJECTIVE 3A – RESULTS:
There were no significant differences in the average height or chlorophyll content between the Metarhizium-exposed plants and control plants over the course of the experiment. Of the 40 experimental plants, I recovered Metarhizium from at least one leaf or root fragment from 30 plants (75%). None of the control plants had Metarhizium recovered from their tissues.
OBJECTIVE 3B – RESULTS: Fall armyworm relative growth rate
There were no significant differences due to Metarhizium-exposure of corn on relative growth rate of fall armyworm. In the first replicate, fall armyworm RGR for the control plants was 0.734 and for Metarhizium-exposed plants was 0.725. In the second replicate, fall armyworm RGR for the control plants was 0.548 and for Metarhizium-exposed plants was 0.498. In the first replicate, Metarhizium was observed from leaf and root fragments in 3 out of 13 plants (23.1%). In the second replicate, I recovered Metarhizium from only one leaf section of one out of 10 exposed plants (10%); however, assessment was recent, and with more incubation time, Metarhizium may grow from these leaf and root sections.
OBJECTIVE 3C – RESULTS: Feeding choice assay
In the first replicate of the choice assay experiment, there were 85 instances of leaf feeding, of which 52 feeding events (61.2%) were on leaf squares from plants grown from Metarhizium-exposed seed. In the second replicate, there were 36 instances of leaf feeding, of which 18 feeding events (50%) were on leaf squares from plants grown from Metarhizium-exposed seed. In the first replicate, Metarhizium was expressed in leaf and root tissue in four of 7 experimental plants (57.1%). In the second experiment, Metarhizium was expressed in the leaf and root tissue in one of 10 experimental plants (10%); however, assessment was recent, and with more incubation time, Metarhizium may grow from these tissue sections. The orientation of the choice arenas did not influence which leaf squares the larvae chose.
I found that endophytic Metarhizium could be established in corn plants and detected in their leaf and root tissue (75.2%). Endophytic Metarhizium did not have a significant effect on the corn plant height or chlorophyll content. Metarhizium colonization in corn did not have a significant effect on fall armyworm relative growth rate (RGR), or their feeding preference. These experiments were performed in a laboratory setting using leaf sections. Additional experiments will be conducted in the greenhouse using whole corn plants to determine if the results from the detached leaf assays is similar when using entire plants. The effect of the Metarhizium-exposed corn plants on fall armyworm development and mortality will also be assessed in future experiments.
Education & Outreach Activities and Participation Summary
In 2017, we participated in two field days associated with the larger experiment: in October at Charvin Farms, and in May at Stone House farms. Forty seven participants attended the field day in October and 45 participated in May. At both field days, we featured cover crop research and fostered information sharing and co-learning among farmers about cover crop mixture design and ecosystem services. Additionally, four study circles convened to discuss organic production topics, including cover crop mixtures and associated ecosystem services. We contributed to spring and fall 2017 project newsletters and presented material in an extension event for agricultural professionals.
After more experiments are conducted with Metarhizium-infected corn to study the effect these plants have on corn pests, corn could be infected with this fungus by farmers to reduce damage to corn and reduce the use of chemical pesticides. With Metarhizium being used and conserved in organic farming, this fungus could become a biological control agent in sustainable agricultural practices, reducing the cost of crop production by becoming an alternative to chemical pesticide application and potentially increasing corn yield.
During my research, I have learned that Metarhizium can successfully colonize corn plant tissue and may eventually be able to be used as a biological control agent against corn pests such as fall armyworm. By continuing our research with Metarhizium-infection in corn, we will discover its influence on corn pests’ development and mortality. The Metarhizium-infected corn could allow farmers to use less chemical pesticides and aid organic farmers in keeping their pest populations in check. This project helped my advisor and I gain further insight into sustainable agriculture, microbiological processes, and agricultural ecosystems. In the future, I hope to obtain a career in sustainable agriculture using biological control agents as alternatives against crop pests.