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Ecology, management and conservation in natural and modified habitats
RESEARCH ARTICLE

Novel grass–endophyte associations reduce the feeding behaviour of invasive European rabbits (Oryctolagus cuniculus)

Christopher G. L. Pennell A , M. Philip Rolston B , A. David M. Latham C , Wade J. Mace D , Ben Vlaming A F , Chikako van Koten D , M. Cecilia Latham C , Samantha Brown C and Stuart D. Card E G
+ Author Affiliations
- Author Affiliations

A Plant–Fungal Interactions Team, Forage Improvement, AgResearch Ltd, Lincoln Research Centre, Christchurch, New Zealand.

B Forage Breeding Innovation Team, Forage Improvement, AgResearch Ltd, Lincoln Research Centre, Christchurch, New Zealand.

C Landcare Research, PO Box 69040, Lincoln 7640, Canterbury, New Zealand.

D Bioinformatics and Statistics Group, AgResearch Ltd, Lincoln Research Centre, Christchurch, New Zealand.

E Plant–Fungal Interactions Team, Forage Improvement, AgResearch Ltd, Grasslands Research Centre, Palmerston North, New Zealand.

F Current address: 8 Neeve Road, Hawkes Bay, New Zealand.

G Corresponding author. Email: stuart.card@agresearch.co.nz

Wildlife Research 43(8) 681-690 https://doi.org/10.1071/WR16114
Submitted: 14 June 2016  Accepted: 26 November 2016   Published: 1 March 2017

Abstract

Context: Small mammalian herbivores, such as European rabbits (Oryctolagus cuniculus), can have significant unwanted impacts on agriculture and horticulture and can attract birds of prey and avian scavengers to airports, increasing the risk of bird strike. Sustainable wildlife management tools that have high efficacy and animal welfare are needed to mitigate these impacts.

Aim: We assessed perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) cultivars associated with selected Epichloë endophytes, originally developed for bird management at airports, to determine their feeding-deterrent properties towards invasive rabbits.

Methods: A pellet feed trial using caged domestic rabbits and a pen trial using wild rabbits were conducted to assess rabbit responses to food pellets with novel endophyte-infected or uninfected grass seed and to plots planted with endophyte-infected or uninfected grasses, respectively.

Key results: Caged rabbits ate significantly less food pellets containing endophyte-infected grass straw than pellets containing endophyte-free grass straw. Wild rabbits consumed significantly less herbage from plots planted with endophyte-infected grasses than those of the equivalent endophyte-free grass cultivars. Temporal patterns of rabbit feeding behaviour support the hypothesis that deterrence in rabbits resulted from post-ingestion feedback associated with grass–endophyte secondary metabolites.

Conclusions: Although more research is required on the exact mechanism of action in rabbits, our results suggest novel endophyte-infected grass associations may substantially reduce rabbit feeding behaviour and possibly rabbit numbers in areas where these grasses are sown.

Implications: Novel grass–endophyte associations have great potential for deterring problem wildlife at airports and other amenity areas.

Additional keywords: Epichloë, ergovaline, Festuca arundinacea, invasive mammalian herbivores, Lolium perenne, post-ingestion feedback, vertebrate pests.


References

Ahmed, A., Nelson, T. A., Daniels, L. B., Piper, E. L., and Beasley, J. N. (1982). Fescue toxicosis: progress toward identification of its cause. Arkansas Farm Reseach 32, 7.

Bacon, C. W., Porter, J. K., Robbins, J. D., and Luttrell, E. S. (1977). Epichloë typhina from toxic tall fescue grasses. Applied and Environmental Microbiology 34, 576–581.
| 1:STN:280:DyaE1c%2FlvFaksg%3D%3D&md5=e859e24bc2d50546701f9ad29da0cda4CAS |

Bouton, J. H., Latch, G. C. M., Hill, N. S., Hoveland, C. S., McCann, M. A., Watson, R. H., Parish, J. A., Hawkins, L. L., and Thompson, F. N. (2002). Re-infection of tall fescue cultivars with non-ergot alkaloid producing endophytes. Agronomy Journal 94, 567–574.
Re-infection of tall fescue cultivars with non-ergot alkaloid producing endophytes.Crossref | GoogleScholarGoogle Scholar |

Caister, L. (2009). Raptors, rodents and rare weather: managing increased migratory raptor populations at McConnell AFB, Kansas. In ‘2009 Bird Strike North America Conference’, 14–17 September 2009, Victoria, BC Canada. Paper no. 9, p. 13. (University of Nebraska: Lincoln, NE.)

Card, S. D., Faville, M. J., Simpson, W. R., Johnson, R. D., de Bonth, A., Voisey, C. R., and Hume, D. E. (2014a). Mutualistic fungal endophytes in the Triticeae – survey and description. FEMS Microbiology Ecology 88, 94–106.
Mutualistic fungal endophytes in the Triticeae – survey and description.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXltF2ru74%3D&md5=1bcdef3a1ac7f35e1aa49d7ebcef4937CAS |

Card, S., Johnson, L., de Bonth, A., Tapper, B., Mace, W., Faville, M., Pennell, C., Caradus, J., and Hume, D. (2014b). Epichloë endophytes from cool season grasses – reaping the rewards from a well-tuned bio-prospecting pipeline. In ‘The 10th International Mycological Congress, Bangkok’. (IMA: Thailand, Bangkok.)

Chilvers, B. L., Ryan, C. J., and Hickling, G. J. (1997). Factors affecting pilot-reported bird-strike rates at Christchurch International Airport, New Zealand. New Zealand Journal of Zoology 24, 1–7.
Factors affecting pilot-reported bird-strike rates at Christchurch International Airport, New Zealand.Crossref | GoogleScholarGoogle Scholar |

Cleary, E. C., and Dolbeer, R. A. (2005). Wildlife hazard management at airports: a manual for airport personnel. Federal Aviation Administration, Office of Airport Safety and Standards: Washington, DC, and USDA, APHIS, Wildlife Services, National Wildlife Research Center, Denver, CO.

Coley, A. B., Fribourg, H. A., Pelton, M. R., and Gwinn, K. D. (1995). Effects of tall fescue endophyte infestation on relative abundance of small mammals. Journal of Environmental Quality 24, 472–475.
Effects of tall fescue endophyte infestation on relative abundance of small mammals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXlsFyks70%3D&md5=049aad572a3453da493502cda91f10cfCAS |

Conover, M. R. (1991). Herbivory by Canada geese: diet selection and effect on lawns. Ecological Applications 1, 231–236.
Herbivory by Canada geese: diet selection and effect on lawns.Crossref | GoogleScholarGoogle Scholar |

Conover, M. R. (1998). Impact of consuming tall fescue leaves with the endophytic fungus, Acremonium coenophialum, on meadow voles. Journal of Mammalogy 79, 457–463.
Impact of consuming tall fescue leaves with the endophytic fungus, Acremonium coenophialum, on meadow voles.Crossref | GoogleScholarGoogle Scholar |

Conover, M. R., and Messmer, T. A. (1996). Feeding preferences and changes in mass of Canada geese grazing endophyte-infected tall fescue. The Condor 98, 859–862.
Feeding preferences and changes in mass of Canada geese grazing endophyte-infected tall fescue.Crossref | GoogleScholarGoogle Scholar |

Dolbeer, R. A., Wright, S. E., Weller, J., and Begier, M. J. (2012). Wildlife strikes to civil aircraft in the United States, 1990–2011. Federal Aviation Administration National Wildlife Strike Database Serial Report No 18, Washington, DC.

Durham, W. F., and Tannenbaum, M. G. (1998). Effects of endophyte consumption on food intake, growth, and reproduction in prairie voles. Canadian Journal of Zoology 76, 960–969.
Effects of endophyte consumption on food intake, growth, and reproduction in prairie voles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXmvFKisrY%3D&md5=4ebf2f301cf7f788f7e7fc2d563f4bc7CAS |

Easton, H. (2007). Grasses and Neotyphodium endophytes: co-adaptation and adaptive breeding. Euphytica 154, 295–306.
Grasses and Neotyphodium endophytes: co-adaptation and adaptive breeding.Crossref | GoogleScholarGoogle Scholar |

Federal Aviation Administration (2012). Wildlife hazard assessments and wildlife management plans at part 139 and GA Airports. Washington, USA.

Filipov, N., Thompson, F., Hill, N., Dawe, D., Stuedemann, J., Price, J., and Smith, C. (1998). Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed-based diets on rabbits. Journal of Animal Science 76, 2456–2463.
Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed-based diets on rabbits.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXmsV2jtLg%3D&md5=3fdcc59f4f8ad0243ea3851b255ae4d7CAS |

Finch, S. C., Pennell, C. G. L., Kerby, J. W. F., and Cave, V. M. (2016). Mice find endophyte-infected seed of tall fescue unpalatable – implications for the aviation industry. Grass and Forage Science 71, 659–666.
Mice find endophyte-infected seed of tall fescue unpalatable – implications for the aviation industry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC28Xhsl2hu7jJ&md5=1388bbb1e3b3db8088768b24387ba751CAS |

Forsyth, D. M., Scroggie, M. P., Arthur, A. D., Lindeman, M., Ramsey, D. S., McPhee, S. R., Bloomfield, T., and Stuart, I. G. (2015). Density-dependent effects of a widespread invasive herbivore on tree survival and biomass during reforestation. Ecosphere 6, art71.
Density-dependent effects of a widespread invasive herbivore on tree survival and biomass during reforestation.Crossref | GoogleScholarGoogle Scholar |

Gallagher, R. T., White, E. P., and Mortimer, P. H. (1981). Ryegrass staggers: isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures. New Zealand Veterinary Journal 29, 189–190.
Ryegrass staggers: isolation of potent neurotoxins lolitrem A and lolitrem B from staggers-producing pastures.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL387kvFemtA%3D%3D&md5=82a749560ec4ac08fd2b1d67793eb669CAS |

Gibb, J. A., and Williams, J. M. (1990). European rabbit. In ‘The Handbook of New Zealand Mammals’. 1st edn. (Ed. C. M. King.) pp. 138–160. (Oxford University Press: Auckland.)

Hoveland, C. S. (1993). Importance and economic significance of the Acremonium endophytes to performance of animals and grass plant. Agriculture, Ecosystems & Environment 44, 3–12.
Importance and economic significance of the Acremonium endophytes to performance of animals and grass plant.Crossref | GoogleScholarGoogle Scholar |

Huitu, O., Helander, M., Lehtonen, P., and Saikkonen, K. (2008). Consumption of grass endophytes alters the ultraviolet spectrum of vole urine. Oecologia 156, 333–340.
Consumption of grass endophytes alters the ultraviolet spectrum of vole urine.Crossref | GoogleScholarGoogle Scholar |

Ivlev, V. S. (1961). ‘Experimental Ecology of the Feeding of Fishes.’ (Yale University Press: New Haven, Ct.)

Jaksic, F. M., and Soriguer, R. C. (1981). Predation upon the European rabbit (Oryctolagus cuniculus) in Mediterranean habitats of Chile and Spain: a comparative analysis. Journal of Animal Ecology 50, 269–281.
Predation upon the European rabbit (Oryctolagus cuniculus) in Mediterranean habitats of Chile and Spain: a comparative analysis.Crossref | GoogleScholarGoogle Scholar |

Johnson, L., de Bonth, A., Briggs, L., Caradus, J., Finch, S., Fleetwood, D., Fletcher, L., Hume, D., Johnson, R., Popay, A., Tapper, B., Simpson, W., Voisey, C., and Card, S. (2013). The exploitation of epichloae endophytes for agricultural benefit. Fungal Diversity 60, 171–188.
The exploitation of epichloae endophytes for agricultural benefit.Crossref | GoogleScholarGoogle Scholar |

Korn, A., Gross, M., Usleber, E., Thom, N., Köhler, K., and Erhardt, G. (2014). Dietary ergot alkaloids as a possible cause of tail necrosis in rabbits. Mycotoxin Research 30, 241–250.
Dietary ergot alkaloids as a possible cause of tail necrosis in rabbits.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhs1emsr%2FL&md5=e867e0fd4c48e023e8485169825d19c7CAS |

Latch, G. C. M., and Vaughn, D. A. (1995). Search for seedborne endophytic fungi in rice. International Rice Research Notes 20, 4.

Latham, A. D. M., Nugent, G., and Warburton, B. (2012). Evaluation of camera traps for monitoring European rabbits before and after control operations in Otago, New Zealand. Wildlife Research 39, 621–628.
Evaluation of camera traps for monitoring European rabbits before and after control operations in Otago, New Zealand.Crossref | GoogleScholarGoogle Scholar |

Latham, A. D. M., Latham, M. C., Cieraad, E., Tompkins, D. M., and Warburton, B. (2015). Climate change turns up the heat on vertebrate pest control. Biological Invasions 17, 2821–2829.
Climate change turns up the heat on vertebrate pest control.Crossref | GoogleScholarGoogle Scholar |

Leuchtmann, A., Bacon, C. W., Schardl, C. L., White, J. F., and Tadych, M. (2014). Nomenclatural realignment of Neotyphodium species with genus Epichloë. Mycologia 106, 202–215.
Nomenclatural realignment of Neotyphodium species with genus Epichloë.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXpvFShsrk%3D&md5=f2d3eaa797c9580cd5bafe651ab83c06CAS |

Moore, J. R., Pratley, J. E., Mace, W. J., and Weston, L. A. (2015). Variation in alkaloid production from genetically diverse Lolium accessions infected with Epichloë species. Journal of Agricultural and Food Chemistry 63, 10355–10365.
Variation in alkaloid production from genetically diverse Lolium accessions infected with Epichloë species.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXhslykurvI&md5=851f9afa5b3bc17bf30c3c6777a09086CAS |

Norbury, G. I., and Reddiex, B. (2005). European rabbit. In ‘The Handbook of New Zealand Mammals’. 1st edn. (Ed. C. M. King.) pp. 131–151. (Oxford University Press: Melbourne.)

NPCA (2012). Pest rabbits: monitoring and good control guidelines. The National Pest Control Agencies, Wellington.

Panaccione, D., Cipoletti, J., Sedlock, A., Blemmings, K., Shardl, C., Machodo, C., and Seidel, G. (2006). Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne). Journal of Agricultural and Food Chemistry 54, 4582–4587.
Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XltVKgtb4%3D&md5=30f80491a0666d041d76f601a72aaf17CAS |

Pennell, C., and Rolston, M. (2003). The effect of grass–endophyte associations on feeding of Canada geese (Branta canadensis). Proceedings of the New Zealand Grasslands Association 65, 239–243.

Pennell, C., Rolston, M., de Bonth, A., Simpson, W., and Hume, D. (2010). Development of a bird-deterrent fungal endophyte in turf tall fescue. New Zealand Journal of Agricultural Research 53, 145–150.
Development of a bird-deterrent fungal endophyte in turf tall fescue.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXotl2is7s%3D&md5=e3a9698a62faf5595e105d527150af4aCAS |

Pennell, C. G. L., Popay, A. J., Rolston, M. P., Townsend, R. J., Lloyd-West, C. M., and Card, S. D. (2015). Avanex Unique Endophyte Technology – a biological deterrent for the aviation industries. Environmental Entomology 45, 101–108.
Avanex Unique Endophyte Technology – a biological deterrent for the aviation industries.Crossref | GoogleScholarGoogle Scholar |

Piper, E., Gadberry, M., Denard, T., Johnson, Z., and Flieger, M. (1997). Effect of feeding ergovaline and ergine on growing rats. In ‘Neotyphodium/Grass Interactions’. (Eds C. W. Bacon and N. S. Hill.) pp. 437–439. (Plenum Press: New York.)

Pownall, D. B., Familton, A. S., Field, R. J., Fletcher, L. R., and Lane, G. A. (1995). The effect of peramine ingestion in pen-fed lambs. Proceedings of the New Zealand Society of Animal Production 55, 186.

R Core Team (2015). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.

Sadler, K. (1980). Of rabbits and habitat, a long term look. Missouri Conservationist 41, 4–8.

SAS (2012). SAS Procedures Guide. SAS Institute Inc., Cary, NC.

Schardl, C. L. (2010). The epichloae, symbionts of the grass subfamily Poöideae. Annals of the Missouri Botanical Garden 97, 646–665.
The epichloae, symbionts of the grass subfamily Poöideae.Crossref | GoogleScholarGoogle Scholar |

Schardl, C. L., Young, C. A., Faulkner, J. R., Florea, S., and Pan, J. (2012). Chemotypic diversity of epichloae, fungal symbionts of grasses. Fungal Ecology 5, 331–344.
Chemotypic diversity of epichloae, fungal symbionts of grasses.Crossref | GoogleScholarGoogle Scholar |

Schardl, C. L., Florea, S., Pan, J., Nagabhyru, P., Bec, S., and Calie, P. J. (2013). The epichloae: alkaloid diversity and roles in symbiosis with grasses. Current Opinion in Plant Biology 16, 480–488.
The epichloae: alkaloid diversity and roles in symbiosis with grasses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtFSgsLbM&md5=73e187c3207e024787ea952bb622b1eaCAS |

Simpson, W. R., Schmid, J., Singh, J., Faville, M. J., and Johnson, R. D. (2012). A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne. Fungal Biology 116, 234–240.
A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC387ptVSisA%3D%3D&md5=fac5bc1aab30aff2451f730d27338a55CAS |

Spiering, M. J., Davies, E., Tapper, B. A., Schmid, J., and Lane, G. A. (2002). Simplified extraction of ergovaline and peramine for analysis of tissue distribution in endophyte-infected grass tillers. Journal of Agricultural and Food Chemistry 50, 5856–5862.
Simplified extraction of ergovaline and peramine for analysis of tissue distribution in endophyte-infected grass tillers.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XmvFKrsLg%3D&md5=f5760b87b019a075bcc5b731a8d9a05dCAS |

Swann, D. E., Hass, C. C., Dalton, D. C., and Wolf, S. A. (2004). Infrared-triggered cameras for detecting wildlife: an evaluation and review. Wildlife Society Bulletin 32, 357–365.
Infrared-triggered cameras for detecting wildlife: an evaluation and review.Crossref | GoogleScholarGoogle Scholar |

Thom, E., Popay, A., Waugh, C., and Minneé, E. (2014). Impact of novel endophytes in perennial ryegrass on herbage production and insect pests from pastures under dairy cow grazing in northern New Zealand. Grass and Forage Science 69, 191–204.
Impact of novel endophytes in perennial ryegrass on herbage production and insect pests from pastures under dairy cow grazing in northern New Zealand.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhtVKku7s%3D&md5=46de8bc1c6258836085b46f836ed936bCAS |

Tor-Agbidye, J., Blythe, L. L., and Craig, A. M. (2001). Correlation of endophyte toxins (ergovaline and lolitrem B) with clinical disease: fescue foot and perennial ryegrass staggers. Veterinary and Human Toxicology 43, 140–146.
| 1:CAS:528:DC%2BD3MXktFKit7g%3D&md5=78b326c22e5e37066725d3fc8b6ababbCAS |

Varney, D., Kappes, C., Jones, S., Newsome, R., Siegel, M., and Zavos, P. (1988). The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 89, 315–320.
The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1c3ntV2htQ%3D%3D&md5=57df32ad74184985177dedaa4c3b26e6CAS |

Washburn, B. E., and Seamans, T. W. (2004). Management of vegetation to reduce wildlife hazards at airports. Paper 396. USDA National Wildlife Research Center – Staff Publications.

Washburn, B. E., Barras, S. C., and Seamans, T. W. (2007a). Foraging preferences of captive Canada geese between tall fescue and perennial ryegrass mixtures for airfields. Federal Aviation Administration by U. S. Department of Agriculture, Animal Plant and Health Inspection Service, Wildlife Services, National Wildlife Research Center, Sandusky, OH.

Washburn, B. E., Loven, J. S., Begier, M. J., Sullivan, D. P., and Woods, H. A. (2007b). Evaluating commercially available tall fescue varieties for airfields. In ‘Proceedings of the FAA Worldwide Airport Technology Transfer Conference’. pp. 1–12. (Federal Aviation Administration: Atlantic City, NJ.)

Williams, K., Parer, I., Coman, B., Burley, J., and Braysher, M. (1995). ‘Managing Vertebrate Pests: Rabbits.’ (Australian Government Publishing Service: Canberra.)

Yates, S. G. (1962). Toxicity of tall fescue forage: a review. Economic Botany 16, 295–303.
Toxicity of tall fescue forage: a review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaF3sXmt1WitA%3D%3D&md5=da5ecceee65ce70da480a93a0e5ad009CAS |

Young, C. A., Hume, D. E., and McCulley, R. L. (2013). Forages and pastures symposium: fungal endophytes of tall fescue and perennial ryegrass: pasture friend or foe? Journal of Animal Science 91, 2379–2394.
Forages and pastures symposium: fungal endophytes of tall fescue and perennial ryegrass: pasture friend or foe?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXotl2gu7w%3D&md5=5533ae7f333dae2fc977119b6b31d7d7CAS |

Zavos, P., Salim, B., Jackson, J., Varney, D., Siegel, M., and Hemken, R. (1986). Effect of feeding tall fescue seed infected by endophytic fungus (Acremonium coenophialum) on reproductive performance in male rats. Theriogenology 25, 281–290.
Effect of feeding tall fescue seed infected by endophytic fungus (Acremonium coenophialum) on reproductive performance in male rats.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvVyrtA%3D%3D&md5=67d2a9a02816c40e10f0941739549d7eCAS |

Zavos, P., Varney, D., Jackson, J., Siegel, M., Bush, L., and Hemken, R. (1987). Effect of feeding fungal endophyte (Acremonium coenophialum)-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding. Theriogenology 27, 549–559.
Effect of feeding fungal endophyte (Acremonium coenophialum)-infected tall fescue seed on reproductive performance in CD-1 mice through continuous breeding.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvFShtw%3D%3D&md5=af0529fdd747329b8a61da9c0b893059CAS |