Anthony J. Wenndt Grinnell College, USA Global Plant Health Intern, UCR

1. Assessment in vitro of the preventative andalleviative capabilities of Trichoderma spp. as biological control against Ojo deGallo (Mycena citricolor) in coffee Anthony J. Wenndt Grinnell College, USA Global Plant Health Intern, UCR

2. The Disease • Name: Ojo de Gallo, American Leaf Spot • Causal Agent: Mycenacitricolor • Disease mechanics: Production of toxins, oxalic acid. • Symptoms: White/grey round lesions on foliage. • Effects: Defoliation, fruit loss, and reduction in yield size and quality (Leandro & Soto, 1980). • Treatment: Fungicide, shade manipulation.

3. Trichoderma spp. as Biocontrol • Genus Trichodermais widespread, and native to coffee production regions in Central America. • Several species in genus Trichoderma have been noted for their antagonistic, mycoparasitic behaviors (Macias, 2000). • Exposure of M. citricolorto Trichoderma has been shown to retard mycelial growth and mitigate toxin production (Vargas, 1984). • Relevance: • Economy and Community Development • Climate Change and Environmental Sustainability

4. Objectives • Assess in vitro the potential for utilization of Trichoderma spp. as biocontrol against Ojo de Gallo. • Gain insight regarding the conditions in which Trichoderma can most effectively be utilized in the field (factors: strain, application method, time of application, et cetera). • Perform molecular analyses to determine the in vitro competitive ability of pathogen Mycenacitricolor.

5. Experimental Design • Confrontation assays in vitro. “Proximal” configuration: explants cultured adjacent in the center of the Petri dish, incubated for 10 days. Molecular evaluation following incubation period. “Distal” configuration: explants cultured at opposite extremes for 8 days. • MismoTiempo format: explants of both species cultured at the same time. Measurements of mycelial growth every 2 days. • TiempoDiferente format: Explant of Trichoderma cultured three days after the establishment of M. citricolor. Measurements of mycelial growth every 2 days.

6. Distal Assay, MismoTiempo Mycenacitricolor Trichoderma spp. Analysis: Adaptation of “Rapilly Method” of formulaic evaluation of confrontation assays with Trichoderma(Benzohra, 2011).

7. Distal Assay, TiempoDiferente Mycenacitricolor Trichoderma spp. Analysis: Adaptation of “Rapilly Method” of formulaic evaluation of confrontation assays with Trichoderma.

8. Proximal Assay Analysis: Utilize ITS-PCR and gel electrophoresis to determine whether mycelia of both species were present after an incubation period of 10 days at a distance of 15 mm from the explants.

9. Assay Functions: • Distal-Same Time: Assess the preventative capacities of Trichoderma against the disease Ojo de Gallo. • Distal-Different Time: Evaluate the corrective and alleviative potential of Trichoderma application in a setting with already-established colonies of M. citricolor. • Proximal: Assess the competitive developmental capacity of the pathogen when grown directly with Trichoderma.

10. Experimental Design • Controls: • 5 cultures of each strain of both species, 25 total. • Experimentals: • 5 cultures of each of the 6 M/T strain combinations for each of the 3 assays (D-M, D-D, PROX), 90 total.

11. Data Collection: Distal Assays • Adaptation of “Rapilly Method.” • L, mycelial growth (mm) • V, Rate of Growth (mm/day) • Rate of Growth Inhibition (RGI) (%) *Measurements based on interior radius of discernablemycelial growth per colony from center of explant, in millimeters.

12. Results: Distal-Same Time • No statistically-significant difference between the three strains of Trichoderma. • In every case, exposure to Trichoderma negatively impacted mycelial growth of M. citricolor(P < 0.002).

13. Results: Distal-Same Time Point: Trichoderma exposure had a significantly greater inhibition effect on strain MW, M. citricolor from weed origins. Values P Day 2: 0.002 Day 4: 0.008 Day 6: 0.004 Day 8: 0.022

14. Results: Distal-Same Time Point: Rate of Growth Inhibition exhibited statistically-significant (P = 0.000) increases over time across every treatment.

15. Results: Distal-Different Time Point: MW strain was significantly more impacted by Trichodermaexposure than MC in every case except for the final day of measurement (P < 0.05; P = 0.6).

16. Results: Distal Compared Point: -In the “Different Time” format (left), effects in Mycena growth rate were not observed until the 5th day of Trichoderma exposure. -In the “Same Time” format (right), effects in Mycena growth rate were apparent after 2 days of Trichoderma exposure. .: Suggests Trichoderma may be more effective as a preventative measure than a curative one.

17. Results: Proximal Assay Point: Only one band was present for each sample, suggesting that Trichoderma suppressed totally the mycelial growth of M. citricolorwhen cultured in direct competition.

18. Summary of Results • Trichoderma exposure significantly inhibited Mycenacitricolor growth in all treatments. • M. citricolorcultures of material collected from weeds appeared to be more inhibited by Trichoderma than cultures from coffee hosts. • The “proximal” confrontation assay suggests that in direct competition with Trichoderma, Mycena growth may be* severely restricted. Analyses ongoing, further study needed.

19. Big Picture • Trichodermamay be most effective as biolcontrol when: • Applied to mulch/leaf litter instead of to the plant directly. • Applied as a preventative suppression measure than as a remedy for existing epidemic.

20. Thanks!