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

2. 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

3. 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

4. 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

5. 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?

6. 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)

7. 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…

8. 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

9. 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

10. 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

11. 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

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

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

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

15. Endophyte B: reduced aphid reproduction in field conditions

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

17. Plant Performance: No differences

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

19. 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

20. Insect Performance: Caterpillar survival
Kaplan-Meier
Sig. 0.07

21. Insect Performance: Endophyte B reduced larvae weightAB B B

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

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

24. 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

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

26. 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)

27. 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!