Fungal endophytes: plant protective agents against herbivores Diana Castillo and Gregory Sword Entomology Texas A&M University ESA November 2013

Outline Materials and methods Results 1. Introduction Background and definitions Research objectives 2. Case study I: sucking piercing herbivore Materials and methods Results 3. Case study II: chewing herbivore 4. Conclusions and future directions

Introduction Endophyte-plant-herbivore interactions background First report in 1941: Neotyphodium coenophialum and tall fescue grass Cattle fescue toxicosis verified by Bacon et al., 1970 Plants infected with N. coenophialum were also more resistant to herbivory attack This interaction led to the “protective mutualistic” hypothesis 1980’s: research started investigating the effects of endophyte infection on host fitness

Introduction Broad endophyte definition Microorganisms living in asymptomatic plant tissue for all or part of their life cycle without causing detectable damage to its host More specifically… Obligate endophytes in the Clavicipitaceae family complete their life cycle in the above ground portions of the grass forming nonpathogenic, systemic intercellular associations and transmitted vertically Facultative endophytes are less well characterized

Introduction Potential ecological roles of endophytes We cannot expect that all organisms that occupy a particular niche (endophytes inside plants) would have the same role... Different species of fungal endophytes living in different host plants may have many and varied roles (obligate vs. facultative) (benign vs. pleitropic) What do we know so far?

Introduction Increase plant biomass (Marks 1991; Clay 1993) Increase tolerance to heat and water stress (Bacon & White 2000) Chemical defense against pathogens (Arnold 2003) Increase leaf senescence in trees (Wilson 1993) Adverse effects against various insect pests (Omacini 2001; Devi 2003, Shia and Feng 2004; Jung 2006; Quesada 2006; Hartley & Gange 2009; Jaber & Vidal 2010; Gurunlingappa 2011; Wakil 2011; Gange 2012)

Research questions Q: How is our research contributing to the area of plant-endophyte interaction? A: Study of unique type of endophyte, primarily known as entomopathogens Very little information available thus far…

Objectives To inoculate cotton plants with two entomopathogenic endophytic strains Investigate the negative effects, if any, across two feeding modes: (2.1) a sucking and a (2.2) chewing herbivore in greenhouse and field conditions Investigate positive or negative effects, if any, on cotton inoculated with the same endophytes: (3.1) developmental stage and (3.2) dry mass

Case study I Effects on a sucking-piercing herbivore: Wiki-images.com Case study I Effects on a sucking-piercing herbivore: Cotton aphid greenhouse and field studies

Experiment methodology Plant inoculation with endophytes: Plant seeds are surfaced sterilized Spore counts on haemocytometer Stock spore solution diluted into treatment solutions Sterilized seeds are soaked in spore solutions for 24hrs previous to planting

Experiment methodology Endophyte detection method: Cotton plant harvested Plant tissue surfaced sterilized (Posada et al., 2007) Plate plant tissue on PDA media Identify the endophyte morphologically or through PCR assay

Experiment methodology Aphid reproduction test 10 plants per treatment: LA122 (All-Tex Seed, Inc) Three seed treatments: 0, 1x106 and 1x107 spores/ml 10, 2nd instar: A. gossypii / plant Aphids counted at: 7 and 14 days (greenhouse) 14 days (field)

Endophyte A: reduced aphid reproduction Repeated measures Time P=0.001 Treatment P=0.003

Endophyte B: reduced aphid reproduction Repeated measures Time P=0.001 Treatment P=0.001

Endophyte B: reduced aphid reproduction in field conditions

Endophyte treated plants reduced aphid reproduction in field conditions Kruskal-Wallis, P=0.04

Plant Performance: No differences

Case study II Effects on a chewing herbivore: Entmdept.ufl.edu Case study II Effects on a chewing herbivore: Helicoverpa zea laboratory study

Experiment meth0dology Experiment design: Factorial (2x5) Insect variables: (1) daily mortality (2) larvae weight at 7 and 14 days (3) days to pupation (4) pupae weight (5) days to adult (6) adult emergence and fecundity Plant variables: (1) developmental stage and (2) dry mass

Insect Performance: Caterpillar survival Kaplan-Meier Sig. 0.07

Insect Performance: Endophyte B reduced larvae weight AB B B

Insect Performance: No pupae weight difference Power: 0.3 A A A A

Insect Performance Kruskal-Wallis P=0.166 Power: 0.4 A A A A

Plant Performance: No difference in plant stage Methods: Plants were grown in the same conditions as H. zea exposed plants AB A A A A

Plant Performance: Endophyte B had higher dry mass Methods: Plants oven dried for 7 days at 60°C B AB B A A

Conclusions & Future Directions Case study I: We successfully inoculated cotton plants with two different entomopathogenic endophytes and showed negative effects on aphid reproduction on both greenhouse and field conditions Case study II: There seemed to be a tendency of lower survivorship and weight in the caterpillars and lower emergence in the adults but no statistical difference was observed due to a low power in our tests (low number of samples) A second trial with higher sample number will be required to test treatment effects if any Future directions: Interdisciplinary research efforts are needed to address and better understand the ecological roles and mechanisms of fungal endophytes and host plant interactions (e.g. molecular biology biochemistry and genetics)

Thank you! Acknowledgements: Funding: Special thanks: Good Neighbor Scholarship Cotton incorporated Texas A&M University Committee members: Dr. Greg Sword Dr. Keyan Zhu-Salzman Dr. Spence Behmer Dr. Craig Coates Dr. Mike Kolomiets Special thanks: Cesar Valencia Dr. Julissa Ek-Ramos (Lab post-doc) All my lab mates!