Source: PENNSYLVANIA STATE UNIVERSITY submitted to
CONSERVATION OF A MULTIFUNCTIONAL FUNGUS FOR PLANT PROTECTION IN ORGANIC CROPPING SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1020531
Grant No.
2019-51106-30198
Project No.
PENW-2019-03509
Proposal No.
2019-03509
Multistate No.
(N/A)
Program Code
112.E
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2023
Grant Year
2019
Project Director
Barbercheck, M. E.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Entomology
Non Technical Summary
Fungal endophytes are fungi that grow in plants, with neutral or beneficial effects. Endophytes inhabit the tissues of most plants, including crop plants, and can promote crop growth and suppress insect pests and plant disease. In field experiments, we will assess the effects of soil and crop management practices on the occurence of a naturally-occurring, beneficial insect-pathogenic and endophytic fungus, Metarhizium robertsii. Factors that we will examine include frequency, timing, and intensity of tillage, cover crop species, and soil characteristics. In greenhouse experimetns, we will determine how competitive this soilborne fungus is with other soil organisms, and how water stress affects the ability of the fungus to form a beneficial endophytic relationship with corn plants. We will also look at the interaction of stress from feeding by an insect pest, the black cutworm, and moisture stress interact, and how this interaction affects the ability of corn to host and benefit from endophytic Metarhizium. Overall, we will gain a better understanding of the benefits and trade-offs of M. robertsii to crop plants, andhow to conserve this fungus in the soil to improve crop production.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21515101130100%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
Our long-term goal is to understand how to exploit the multiple benefits of an endemic soil fungus, Metarhizium robertsii (Hypocreales: Clavicipitaceae) as an insect pathogen and as a beneficial fungal endophyte in organic production systems.Our immediate research objectives are to determine: 1) the prevalence of endophytic colonization of corn grown from non-inoculated and M. robertsii-inoculated seed in the context of an organic reduced tillage systems experiment; 2) the relative ability of M. robertsii to colonize corn by seed and soil inoculation under non-competitive and competitive conditions in greenhouse tests; 3) the effects of drought and soil saturation on recruitment and establishment of endophytic M. robertsii by corn, and on plant growth and plant defense gene expression, and 4) the effects of the interaction of abiotic stress and biotic stress from black cutworm on corn growth and defense gene expression.
Project Methods
We will use standard aseptic technique to create single- spore isolates of Metarhizium spp. from sporulating cadavers of G. mellonella used as sentinel insects of soil from a long-term field experiment designed to assess the effects of tillage and cover cropping treatments on soil health and agronomic crop performance. In field experiments to test the effects of tillage and cover crops on the prevalence of endophytic M. robertsii in corn,we will manually plant M. robertsii-treated and non-treated corn seeds in early June into experimental plots with newly germinating corn.The date and proportion of seed emergence will be recorded. We will harvest half of the plants at V2 (~12 d after germination) and half at V4 (~25 d after germination). Root and leaf tissue from ampled plants will be evaluated for endophytic colonization in the laboratory. Timed with corn sampling, we will collect mineral soil from each replicate of each treatment plot and will use one subsample each for: 1) standard sentinel insect assays with G. mellonella to estimate relative prevalence of entomopathogenic fungi,2) determination of soil electrical conductivity (EC, μS/cm) and pH; 3) determination of permanganate oxidizable carbon concentrations as an indicator of labile, biologically active soil carbon and soil moisture as gravimetric soil water content;and, 4) submission to the PSU Agricultural Analytical Services Laboratory (AASL) for analyses percent sand, silt, and clay, phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), Zinc (Zn), copper (Cu), sulfur (S), cation exchange capacity (CEC), salts, and soil organic matter by loss-on-ignition (LOI). Soil properties will be used as covariates in analyses to describe the prevalence of Metarhizium in soil and plants.DNA will be extracted from all Metarhizium isolates from plants and sentinel insect using a standard protocol. Isolates will be processed for molecular analysis by standard methods.To determine the effects of inoculation method and competition with soil biota on the degree of endophytic colonization of corn, we will conduct greenhouse experiments to compare 6 treatments each in a "competitive" and "non-competitive" environment, for a total of 12 treatments:1) Seed inoculation by soaking in M. robertsii spore/ Triton X-100 solution [Trt 1]; 2) Seed soaked in Triton X-100 solution [control for Trt 1]; 3) Soil inoculation by application of M. robertsii spore/Triton X-100 solution to soil with seed soaked in Triton X-100; 4) Soil inoculation by application of M. robertsii spore/Triton X-100 solution to soil with seed soaked in sterile distilled water (SDW) [control for Trt 3]; 5) Soil inoculation by application of M. robertsii spore/Triton X-100 solution to soil with dry seed [control for Trt 3]; and 6) Dry, untreated seed, watered with tap water [control for Trts 1 - 5]. To determine the effects of water stress on the prevalence of endophytic Metarhizium, we will perform a greenhouse assay to assess endophytic recruitment by corn plants under stress from deficit and excess water. Treatments will include: 1) Soil inoculation by application of M. robertsii spore/0.05% Triton X-100 solution, adequate moisture [Trt 1]; 2) Application of 0.05% Triton X-100 solution, adequate moisture [Trt 2, control for Trt1]; 3) Soil inoculation by application of M. robertsii spore/0.05% Triton X-100 solution, moisture deficit [Trt 3]; 4) Application of 0.05% Triton X-100 solution, moisture deficit [Trt 4, control for Trt 3]; 5) Soil inoculation by application of M. robertsii spore/Triton X-100 solution, moisture excess [Trt 5]; 6) Application of Triton X-100 solution, moisture excess [Trt 6, control Trt 5].We will collect and process ~100 mg sample of corn leaf and root from each plant for analysis of gene expression in response to abiotic and biotic stress treatments. We will measure expression patterns of genes belonging to the JA, SA, ET and ABA pathways that accumulate in response to stress conditions in corn using quantitative real-time PCR (qRT-PCR).We will determine the expression pattern of MYB transcription factor, dehydration responsive element binding (DREB) proteins, responsive to drought 22 (RD22) that are involved in ABA-dependent drought stress response.We will also determine the expression pattern of the plant defense-related genes LOX1 and allene oxide synthase 1 (AOS1), ethylene responsive ACC synthase 6 (ACS6), phospholipase 1 (PLD1), maize aquaporin 1 (PIP1), and PR5. We will test the interaction of moisture stress, M. robertsii colonization of corn, and black cutworm in whole plant feedingassays.We will also sample ~100 mg of leaf and root tissue from each plant to assess interactions between the JA and SA pathways in response to herbivory by measuring the expression of maize protease inhibitor (MPI), ribosome inactivating protein 2 (RIP2), lipoxygenases (LOX) and oxo-phytodienoate reductases (OPR), allene oxide synthase 1 (AOS1), phospholipase 1 (PLD1), maize aquaporin 1 (PIP1), and the pathogenesis-related protein 5 (PR5).

Progress 09/01/19 to 08/31/23

Outputs
Target Audience:Farmers, extension educators and other agricultural professionals, scientists, undergraduate students, graduate students, general public Changes/Problems:For objective four, we were not able to recover all of the black cutworm that were applied to plants. Therefore, we were not able to determine the effects of water and M. robertsii treatments on black cutworm growth. We were able to record the presence and level of feeding damage to corn and will use those values in statistical analyses. What opportunities for training and professional development has the project provided?During the reporting period, one graduate students and two undergraduate students received training associated with this project. The graduate student presented information from the project at one scientific meeting. Information to this project was presented in two undergraduate courses and one graduate course. Information from the project was presented at eight extension events. How have the results been disseminated to communities of interest?Extension presentations and written materials, scientific conference, academic courses. Barbercheck, M. 2022. Organic pest management in grains with a focus on noctuids. OGRAIN Virtual Conference, University of Wisconsin. 4 February 2022. Barbercheck, M. 2022. Better pest management through soil health. Virtual Crops Conference. 3 March 2022. 34 attendees Barbercheck, M. 2022. Soil health and IPM. Perry Co. Corn Day. 28 Feb. 2022. 64 attendees Barbercheck, M. 2022. Endophytes for Crop Protection and Growth Promotion. Perry Corn Day. 28 Feb. 2022. 64 attendees Barbercheck, M. 11 March 22. Soil health research update. Central Susquehanna Organic Growers Network meeting. New Columbia, PA. 25 attendees. Barbercheck, M., Brasier, K. 17 March 22. A growing concern: Supporting women farmers through meeting their extension needs. Penn State Extension Update. 300 attendees Barbercheck, M. 3 August 2022. Insect Pathogens for Crop Protection and Pest Management. Lunch and Learn, SEAREC, Manheim, PA. 10 interns. Barbercheck, M., R. Hoover, J. Myers. 13 Sept. 22. Soil Health Q & A. Happy Valley vineyard and Winery. Centre Co. Conservation District Event. Peterson, H., Barbercheck, M. 2022. Impact of Water Stress on the Establishment and Persistence of Endophytic and EntomopathogenicMetarhizium robertsii. Eastern Branch ESA Meeting, Feb. 19-21, Philadelphia, PA Academic courses: AGECO144 Principles and Practices of Organic Agriculture, Fall 2022 and Fall 2023 INTAD Global Agricultural Systems, Fall 2022 and Fall 2023 SOIL071 Sustainability, Fall 2022 and 2023 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Obj. 1:In 2020 and 2021, we inoculated maize seed with spores of M. robertsii and grew the plants in a certified organic corn field until V4 stage (~26 days after planting). We evaluated leaf and root samples from inoculated and uninoculated control plants for endophytic colonization by M. robertsii. In 2020, we detected M. robertsii from root tissue of 1 plant out of ~300 M. robertsii-treated plants. We did not detect endophytic M. robertsii from corn foliage. Obj. 2: Endophytic colonization of maize by soil and seed inoculation in sterilized and non-sterilized soil. We conducted a series of greenhouse experiments to compare frequency and intensity of endophytic colonization of corn inoculated with Metarhizium robertsii through seed inoculation and application of conidia to soil in sterile and non-sterile growth medium. For seed inoculation, we soaked seed in a M. robertsii spore solution (1 x 108 spores/ml) for 2h. For soil inoculation, we inoculated the base of corn plants growing individually in potsat the V1 growth stage with 10 ml of spore suspension (1 x 108 spores/ml). We sampled all the plants at V4 growth stage and plated 6 leaf and 6 root sections (surface-sterilized) on CTC growth medium for evaluation of endophytic colonization. The experiment was repeated three times. We detected endophytic colonization in 52 of the 141 corn plants treated with M. robertsii (36.9%). 27 of the endophytic 52 plants (51.9%) were colonized following seed soaking and 25 (48.1%) following soil application. Among the 52 endophytically colonized plants, we detected colonization in 7 plants (13.5%) in the non-sterile soil treatment, and 45 (86.5%) in the sterile soil treatment. Inoculation method, by soaking seed in a spore solution or applying spores to the soil, did not significantly affect the proportion of corn tissues (6 root + 6 leaf sections/plant) in which endophytic colonization was detected (seed soaking mean±st. err. = 12.8±1.24%; soil application 9.7±1.16%; F1,316 = 1.29, P= 0.2566). Colonization rate in sterile soil (10.2±0.8%) was greater (F1,315 = 62.9, P= 0<0.0001) than in non-sterile soil (1.5±0.8%). There was a positive relationship between proportion of corn tissues (root + leaf sections) in which endophytic colonization was detected and corn height (r2adj = 0.06, F=10.65, P=0.0014) and biomass (r2adj = 0.09, F=2.44, P=0.0079), but not chlorophyll content (P = 0.6069). Obj. 3.In greenhouse experiments, corn (Zea mays) was grown to V3 and then assigned to three soil moisture level treatments: deficit, adequate, and excess. In each water-level treatment, plants were divided into Metarhizium-treated and untreated groups and inoculated with M. robertsii. Water stress was maintained until plants reached V7, then root and leaf tissue were assayed for endophytic growth of M. robertsii. Presence of M. robertsii in the soil was confirmed by baiting with waxworm larvae. Mean root colonization in inoculated deficit, adequate, and excess water treatments was 31±1.1%, 44±1.2%, and 25±1%, respectively. Mean leaf colonization in uninoculated deficit, adequate, and excess water treatments was 0%, 0%, and 1.2±1.2%, respectively. Linear regression showed a significant positive relationship between height and intensity of maize root colonization in the deficit water treatment (r2adj =0.091, F1, 52= 6.301, P =0.01), but not in the adequate (P =0.451) or excess (P =0.1714) water treatments. The relative expression of ZmLOX1 in the jasmonic acid (JA) biosynthesis pathway was significantly greater in M. robertsii-inoculated than in non-inoculated plants, but water treatment had no effect. There was a significant interaction between M. robertsii and water treatments on foliar concentrations of JA and jasmonoyl isoleucine (JA-ILE), suggesting that water stress impacts M. robertsii as a modulator of plant defense. Water stress, but not inoculation with M. robertsii, had a significant effect on the expression of MYB (p=0.021) and foliar concentrations of abscisic acid (p<0.001), two signaling molecules associated with abiotic stress response. Obj. 4:We conducted greenhouse and lab-based experiments to determine the effects of endophytic M.robertsiion growth and defense in corn inoculated with second instar larvae of the black cutworm, Agrotis ipsilon. At V2, we inoculated corn plants with spores of M. robertsii by soil drenching. Three days after inoculation, we placed early instar black cutwormonto plants and allowed them to feed for five days during which time we imposed deficit, adequate and excess water treatments. At ~21 days post-inoculation, we measured plant performance and expression of selected defense genes and phytohormone content. We also evaluated the relative prevalence of M. robertsii spores in the soil using sentinel baiting with Galleria mellonella. Among the 92 corn plants treated with M. robertsii and BCW, endophytic colonization was detected in 51.5%, 53.1%, and 34.5% of the corn plants in the deficit, adequate, and excess water treatments, respectively. There was no difference in intensity of colonization among water treatments (P=0.2011). Mean intensity (proportion of root and leaf section endophytically colonized) was 13.1±3.6%, 12.8±3.6%, and 7.0±3.7% in the deficit, adequate, and excess water treatments, respectively. There was no effect of water treatments on the prevalence of M. robertsii in soil, as determined by infection rate of G. mellonella at the end of the experiment (P=0.1217). Mean intensity prevalence of M. robertsii in soil was 46.1±6.7%, 59.8±6.6%, and 58.7±6.8% in the deficit, adequate, and excess water treatments, respectively. The relative quantification (RQ) of plant defense-related genes, benzoxazinoid 7 and ribosome inactivating protein 2 (RIP2), differed significantly among water treatments (benzoxazinoid, F2,2=4.8917, P=0.0201; RIP2, F2,2 = 9.1288, P = 0.0018) but there were no significant differencedue to endophytic colonization by M. robertsii. The RQ of benzoxazinoid 7 was greater (P= 0.0153) in the excess (2.89±0.47), compared with the deficit ( 0.82±0.47) water treatment, but neither were different from the adequate (1.79±0.47) water treatment. The RQ of RIP2 was significantly (F2,2= 9.1288, P=0.0018) greater in the deficit (mean=2.57±0.44) compared to the adequate (mean=0.92±0.44, P=0.0040) and excess (0.96±0.44, P=0.0049) water treatments. The RQ of RIP2 in the adequate and excess water treatments did not differ. There was a non-significant trend (F2,2=3.2789, P=0.0611) for water treatment to affect the RQ of allene oxide synthase (AOS). The mean RQ of AOS in the deficit, adequate and excess water treatments were 16.12±3.81, 4.98±3.81, and 3.60±3.81, respectively.

Publications

  • Type: Other Status: Published Year Published: 2022 Citation: Barbercheck, M.E., Borrelli, K. A., Wallace, J. 2022. Organic Crop Production. Part 1, Section 11. Penn State Agronomy Guide. AGRS-026
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ahmad I, Jim�nez-Gasco MdM, Luthe DS, Barbercheck ME (2022) Endophytic Metarhizium robertsii suppresses the phytopathogen, Cochliobolus heterostrophus and modulates maize defenses. PLoS ONE 17(9): e0272944. https://doi.org/10.1371/journal.pone.0272944
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Peterson, H, Ahmad, I., Barbercheck, ME (2023) Maize response to endophytic Metarhizium robertsii is altered by water stress PLoS ONE: pone.0289143 (in press, to be published 27 Nov. 2023)
  • Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Peterson, Hannah. 2022. Water Stress Causes Context Dependent Effects In Endophytic Relationship Between Metarhizium robertsii And Maize. https://etda.libraries.psu.edu/?search_field=all_fields&q=Hannah+Peterson


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Farmers and other agricultural professionals, extension educators and specialists, scientists, graduate and undergraduate students, governmental and non-governmental agricultural agenceis and organizations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Information from this project was incorporated into classroom lectures and extension presentations. One graduate student attended a scientific meeting and presented results from this project. How have the results been disseminated to communities of interest?Extension presentations, What do you plan to do during the next reporting period to accomplish the goals?The experiments to meet the objectives of this research are complete. We will now focus on analysis, interpretation, and publication in scientific journals and extension outlets.

Impacts
What was accomplished under these goals? Obj. 1: Determine the prevalence of endophytic colonization of corn grown from non-inoculated and M. robertsii-inoculated seed in an organic cropping systems experiment. This experiment is complete. Data are being analyzed and a manuscript is in preparation. Obj. 2: Endophytic colonization of maize by soil and seed inoculation in sterilized and non-sterilized soil Research under this objective is complete and results were reported previously. Results will be incorporated into a manuscript in preparation. Obj. 3. Research for this objective is complete and is being incorporated into a Master's thesis. In greenhouse experiments comparing the effects of deficit, adequate, and excess soil moisture on the interaction between maize and endophytic M. robertsii, mean colonization of maize by M. robertsii in deficit, adequate, and excess water treatments was 31(±1.09)%, 44 (±1.19)%, and 25 (±1.01)%, respectively. Among all test plants, there was a significant relationship between maize root colonization and water treatment on plant height (p<0.001). There was a significant positive relationship between height and maize root colonization in the deficit water treatment (p=0.01), but not in the adequate (p=0.451) or excess (p=0.1714) water treatments. The defense gene, ZmLox1 was upregulated in plants that were inoculated with M. robertsii but were not affected by water stress. LOX genes are involved in the jasmonic acid (JA) pathway, which primarily respond to biotic stressors.Water treatment had a significant effect on relative expression of MYB genes. Plants in the deficit and adequate treatments had significantly higher relative expression of MYB compared to the excess water treatment. This is consistent with previous reports which have identified the MYB gene as a positive transcription factor for drought-related genes, making upregulation of MYB advantageous for plants during drought conditions. There was no significant difference in the phytohormones SA, cZ, GA19, GA53, and IAA content due to water treatment or inoculation with M. robertsii. In the deficit water treatment, JA content was significantly lower in maize inoculated with M. robertsii (p=0.009) compared to uninoculated plants. Plants that were in excess water treatment and inoculated with M. robertsii had higher levels of JA than uninoculated plants. The results of differing effects of soil moisture level suggest that the interaction between endophytic M. robertsii and maize is context-dependent. The difference in phytohormone response to M. robertsii colonization between water treatments demonstrates how the JA pathway functions differently under different environmental conditions, which may be in part due to the complex antagonistic and synergistic relationships between JA and other phytohormone pathways. Obj. 4. We conducted greenhouse and lab-based experiments to determine the effects of endophytic M.robertsiiJ.F. Bisch., Rehner & Humberon growth and defense in maize (Zea mays L.) infected with C. heterostrophus. We inoculated maize seeds with spores of M. robertsii and, at the 3 to 4-leaf stage,the youngest true leaf of M. robertsii-treated and untreated control plants with spores of C. heterostrophus. After 96 h, we measured maize height, above-ground biomass, endophytic colonization by M. robertsii, severity of SCLB, and expression of plant defense genes and phytohormone content. We recovered M. robertsii from 74% of plants grown from treated seed. The severity of SCLB in M. robertsii-treated maize plants was lower than in plants inoculated only with C. heterostrophus. M. robertsii-treated maize inoculated or not inoculated with C. heterostrophus showed greater height and above-ground biomass compared with untreated control plants. Height and above-ground biomass of maize co-inoculated with M. robertsii and C. heterostrophus were not different from M. robertsii-treated maize. M. robertsii modulated the expression of defense genes (lox1, lox3, endochitinase 3, pr5) and the phytohormone content (cis-zeatin, gibberellin 19, and salicylic acid) in maize inoculated with C. heterostrophus compared with plants not inoculated with C. heterostrophus and control plants. These results suggest that endophytic M. robertsii can promote maize growth and reduce development of SCLB, possibly by induced systemic resistance mediated by modulation of phytohormones and expression of defense and growth-related genes in maize. We conducted greenhouse and lab-based experiments to determine the effects of simultaneous stresses on endophytic colonization by M. robertsii and the effects of endophytic M. robertsii on maize defense and growth. At the 2-leaf stage, we inoculated maize (Zea mays L.) with spores of M. robertsii by soil drenching. Three days after inoculation with M. robertsii, we placed early instar black cutworm (Agrotis ipsilon) onto plants and allowed them to feed for five days during which time we simultaneously imposed deficit, adequate and excess water treatments. After ~21 days post-inoculated with M. robertsii, we measured maize height, above-ground biomass, relative water content of leaf tissue, endophytic colonization of leaf and root tissue, plant nutrient content, and expression of selected defense genes and phytohormone content. At the end of the experiment, we also evaluated the soil for nutrient content and estimated relative prevalence of M. robertsii spores using sentinel baiting with Galleria mellonella. Data are being analyzed and a manuscript is in preparation.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: " Ahmad , I. M.d. M.Jim�nez-Gasco, D. S. Luthe and M. E. Barbercheck. Endophytic Metarhizium robertsii suppresses the phytopathogen, Cochliobolus heterostrophus and modulates maize defenses. In press. PLoS One. Ms/ # PONE-D-22-11766.
  • Type: Other Status: Published Year Published: 2022 Citation: Barbercheck, M.E., Borrelli, K. A., Wallace, J. 2022. Organic Crop Production. Part 1, Section 11. Penn State Agronomy Guide. AGRS-026 (revision)
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Ahmad, I., Jim�nez-Gasco, M. D. M., Barbercheck, M. (2021). The role of endophytic insect-pathogenic fungi in biotic stress management. In: Giri, B. & Varma A. (Eds). Plant Stress Biology. Springer Nature, Singapore.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Flonc, B., Barbercheck, M., Ahmad, I. (2021). Observations on the relationships between endophytic Metarhizium robertsii, Spodoptera frugiperda (Lepidoptera: Noctuidae), and maize. Pathogens, 10, 713.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Graduate and undergraduate students, scientists, farmers, agricultural professionals Changes/Problems:There have been no major changes, but our research has been slowed somewhat due to restrictions related to COVID-19. What opportunities for training and professional development has the project provided?5 undergraduate students have participated in research activities associated with this project. How have the results been disseminated to communities of interest?Information from this project has been included in academic course lectures and in extension presentations. What do you plan to do during the next reporting period to accomplish the goals?We have initiated both field-based experiments and greenhouse experiments and these are on-going. A graduate student joined the project in Fall 2020 and she will conductresearch to meet project objective 3.

Impacts
What was accomplished under these goals? Obj. 1. In Summer 2020, we inoculated the seed of maize with spores of M. robertsii and grew the plants until V4 stage (~26 days after planting). We evaluated the plants for endophytic colonization by M. robertsii in leaf and root. We detected M. robertsii from root tissue of only 1 plant out of ~300 M. robertsii-treated plants. We repeated the experiment in Summer 2021 and are currently processing the samples. 80 soil samples were collected from the four tillage intensity treatments at the experimental site and are being analyzed formatric potential, gravimetric moisture, entomopathogenic organisms, pH, EC, active C, andfertility.Soil samples collected in June in corn and soybean treatments will be submitted to Cornell Soil Health for testing.A subsample of each soil sample was baited with waxmoth larvae to assess effect of treatments on prevalence of Metarhizium. Fungal spores from infected cadavers have been used to create isolates, which are stored at -20 until identifications can be made. Obj. 2. We collected the soil from the field and mixed with potting mix (1:1). We sterilized half of the soil medium. We used spores of M. robertsii to compare the degree of endophytic colonization in maize by seed vs soil inoculation. For seed inoculation, we inoculated the treated seed with spores of M. robertsii (1 x 108 spores/ml) and control seed with 0.1 % triton X-100 for 2h. For soil inoculation, we inoculated the base of each plant at V1 growth stage with 10 ml of spore suspension (1 x 108 spores/ml) and control plant with 10 ml of 0.1 % triton X-100. We sampled all the plants at V4 growth stage and plated at CTC growth media for evaluation of endophytic colonization. We recovered more frequently from soil- compared with seed-inoculated plants (p<0.002; F1,20=12.3; n=22). We recovered M. robertsii more frequently from the roots of soil-inoculated plants compared with seed-inoculated plants (p<0.03; F1,15=5.89; n= 17). Plant height (p<0.16; F1,21=2.1; n=22) and aboveground biomass (p<0.69; F1,22=0.16; n=22) of plant grown from M. robertsii treatment were not different between the inoculation methods. We recovered M. robertsii more frequently from plants grown in sterilized medium than non-sterilized medium. None of the larvae of wax worms were infected when baited in sterilized soil whereas we detected infection when larvae were baited in non-sterilized soil suggesting the presence of Metarhizium in the soil. Obj. 3. In greenhouse experiments, corn (Zea mays) was grown to the three-leaf stage and hen assigned to three soil moisture level treatments: deficit, adequate, and excess. In each water-level treatment, plants were divided into Metarhizium-treated and untreated groups and inoculated with M. robertsii. Water stress was maintained until plants reached the seven-leaf stage, then root and leaf tissue were assayed for endophytic growth M. robertsii. Presence of M. robertsii in the soil was confirmed by baiting with waxworm larvae. To confirm water stress treatments, we determined the relative water content of leaf tissue of each plant at the end of the experiment. To determine the effect of treatment on plant growth, plant height, chlorophyll content, and infrared temperature of the leaves were measured daily. Dry aboveground biomass and plant and soil nutrient analysis were conducted was measured at the end of the experiment. To determine the effect of treatments on plant defense response, leaf and root sections were collected and frozen during plant harvest and transferred to a -80 degree freezer for storage until analysis. A subsample will be sent to the University of Nebraska Integrated DNA Technologies Lab for phytohormone analysis of compounds associated with metabolic pathways linked to water and herbivory stress. To determine gene expression levels we will conduct PCR on leaf and root samples with Metarhizium-specific primers to amplify, gene products for analysis of the effect of treatment on gene expression in the plant phytohormone defense pathway. We have conducted one replicate of this experiment, another is currently underway, and a third is planned for December 2021. Obj. 4. The first replicate of this greenhouse experiment to meet this objective was initiated in early October 2021.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ahmad, I., Jim�nez-Gasco, M.D.M., Luthe, D.S., Shakeel S.N., & Barbercheck, M. (2019). Endophytic Metarhizium robertsii affects maize growth and gene expression and growth of black cutworm by eliciting plant defense. Society for Invertebrate Pathology 2019, Valencia Spain, Jul. 28-Aug.1, 2019, Invited talk.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Ahmad, I., Jim�nez-Gasco, M.D.M., Luthe, D.S., & Barbercheck, M. (2020). Mighty Microbes: The tri-trophic interactions of endophytic Metarhizium in maize. Penn State Microbiome Center, PA, USA, Feb. 14, 2020, invited talk.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Ahmad, I., Jim�nez-Gasco, M.D.M., Luthe, D.S., & Barbercheck, M. (2020). Mighty Microbes: The tri-trophic interactions of endophytic Metarhizium in maize. XXVIII Plant and Animal Genome, 2020, San Diego, CA, USA, Jan. 11-15, 2020, invited talk.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Flonc, B., Barbercheck, M., Ahmad, I. (2021). Observations on the relationships between endophytic Metarhizium robertsii, Spodoptera frugiperda (Lepidoptera: Noctuidae), and maize. Pathogens, 10, 713.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahmad, I., Jim�nez-Gasco, M. D. M., Luthe, D. S., Barbercheck, M. (2020). Systemic colonization by Metarhizium robertsii enhances cover crop growth. Journal of Fungi, 6, 64.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahmad, I., Jim�nez-Gasco, M. D. M., Luthe, D. S., Shakeel, S. N., Barbercheck, M. (2020). Endophytic Metarhizium robertsii enhances maize growth, suppresses insect growth and alters plant defense gene expression. Biological Control, 104167.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Champagne, R., Wallace, J., Curran, B., and Barbercheck, M. 2021. Rotational no-till and tillage-based organic corn produce management tradeoffs in the Northeast. Agron. J., 1-14 DOI: 10.1002/agj2.20823
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wallace, J.M., Barbercheck, M.E., Curran, W., Keene, C.L., Mirsky, S.B., Ryan, M. and VanGessel, M. 2021. Cover crop-based, rotational no-till (CCORNT) management tactics influence crop performance in organic transition within the Mid-Atlantic U.S. Agron. J. Published on-line 11 Oct 2021, https://doi.org/10.1002/agj2.20822
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Ahmad, I., Jim�nez-Gasco, M.D.M., Luthe, D.S., Shakeel S.N., & Barbercheck, M. (2019). Endophytic Metarhizium robertsii enhances maize growth and suppresses insect growth by eliciting plant defense. Plant Biology 2019, San Jose, CA, USA, Aug. 3-7, 2019, poster presentation.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Farmers, agricultural professionals, scientists, graduate and undergraduate students. Changes/Problems:Our progress has been slowed somewhat due to restrictions related to COVID-19. What opportunities for training and professional development has the project provided?5 undergraduate students are conducting research associated with this project. A Master's student joined the project in Fall 2020. How have the results been disseminated to communities of interest?Information on this project has been included in an invited seminar :Ahmad, I., Jiménez-Gasco, M.D.M., Luthe, D.S., & Barbercheck, M. (2020). MightyMicrobes: The tri-trophic interactions of endophyticMetarhiziumin maize. Penn State Microbiome Center, PA, USA, Feb. 14, 2020, invited talk. Presentations at conferences:Ahmad, I., Jiménez-Gasco, M.D.M., Luthe, D.S., & Barbercheck, M. (2020). MightyMicrobes: The tri-trophic interactions of endophyticMetarhiziumin maize. XXVIII Plant and Animal Genome, 2020, San Diego, CA, USA, Jan. 11-15, 2020, invited talk. What do you plan to do during the next reporting period to accomplish the goals?We have collected baseline data on prevalence of M. robertsii at our reserach site and are currently conducting greenhouse experimetnt to meet project objectives. We will continue these efforts during the next reporting period. A graduate student joined the project in Fall 2020 and will conduct greenhouse experiments described in Objective 3 of the project.

Impacts
What was accomplished under these goals? We have initioatedproject experiments. We collected the soil from the field and mixed with potting mix (1:1). We sterilized half of the soil medium. We used spores of M. robertsii to compare the degree of endophytic colonization in maize by seed vs soil inoculation. For seed inoculation, we inoculated the treated seed with spores of M. robertsii (1 x 108 spores/ml) and control seed with 0.1 % triton X-100 for 2h. For soil inoculation, we inoculated the base of each plant at V1 growth stage with 10 ml of spore suspension (1 x 108 spores/ml) and control plant with 10 ml of 0.1 % triton X-100. We sampled all the plants at V4 growth stage and plated at CTC growth media for evaluation of endophytic colonization. We recovered more frequently from soil- compared with seed-inoculated plants (p<0.002; F1,20=12.3; n=22). We recovered M. robertsii more frequently from the roots of soil-inoculated plants compared with seed-inoculated plants (p<0.03; F1,15=5.89; n= 17). Plant height (p<0.16; F1,21=2.1; n=22) and aboveground biomass (p<0.69; F1,22=0.16; n=22) of plant grown from M. robertsii treatment were not different between the inoculation methods. We recovered M. robertsii more frequently from plants grown in sterilized medium than non-sterilized medium. None of the larvae of wax worms were infected when baited in sterilized soil whereas we detected infection when larvae were baited in non-sterilized soil suggesting the presence of Metarhizium in the soil. We will repeat this experiment three times. We conducted a preliminary experiment to assess relative prevalence of M. robertsii in the soil and natural levels of endophytic colonization in corn following AWP, triticale, or canola cover crops; and to compare the prevalence of naturally-occurring endophytic Metarhizium in organic corn with prevalence in corn grown from M. robertsii-inoculated seeds planted at the same site. Neither detection in soil nor prevalence in corn was affected by the preceding cover crop or seed treatment with M. robertsii. Mean prevalence in soil was 24.5±3.8%, and mean prevalence of endophytic corn plants was 4.4±1.3%. In 2018, the natural prevalence of endophytic corn was infrequent but positively related to prevalence of M. robertsii in soil at V4 (r2adj = 0.36, F1,14=9.3248, p = 0.0085) but not at V2. We suggest this relationship is only significant at V4 because time is needed for M. robertsii to colonize corn roots, and to sufficiently colonize corn tissue to allow detection of endophytic colonization through re-isolation from plant tissue. Mean prevalence of M. robertsii in soil across the site was lower (F1,139=26.43, p<0.0001) in 2018 (6.2±0.5%) than in the 2016 (17.5±1.8%) and 2017 (14.5±0.4%), and soil moisture was negatively related to the detection of M. robertsii in soil (r2adj = 0.123, p<0.0001). Mean soil moisture across all corn plots was 17.5±0.4% in 2018, compared with 10.7±0.4% and 15.4±0.4% in 2016 and 2017, respectively, with all years differing from each other (F2,279= 80.9, p<0.0001). Although soil moisture is generally positively related to germination of Metarhizium conidia and infection of insects in the soil, conidial survival is adversely affected by wet soil. We suggest that the unusually wet weather and associated high soil moisture in 2018 contributed to low prevalence of M. robertsii in soil, which resulted in low prevalence of endophytic M. robertsii in corn plants. We plan to conduct additional assays to understand soil factors that affect M. robertsii in soil and in plants.

Publications