Is the endophyte-based plant protection against aphids mediated by changes in the insect microbiome?

Abstract

Aphids are important herbivores in natural and managed environments. We studied the response of aphids and their associated microbiota to the presence of the fungal endophyte Epichloë sp. LpTG-3 strain AR37, and the AR37-derived alkaloids in plants. We hypothesized that AR37 and/or AR37-derived alkaloids would reduce the aphid performance, and that this reduction would be associated with endophyte-mediated changes in the abundance, composition, and diversity of beneficial bacterial endosymbionts of aphids (e.g., Buchnera). Plants of Lolium perenne associated with AR37 variants able (wild type and ∆idtA) and unable (∆idtM) to produce indole diterpene alkaloids were challenged with Rhopalosiphum padi aphids. We measured aphid population size, plant biomass, and the abundance, composition and diversity of the aphid's bacterial microbiota. The presence of AR37 increased the resistance of plants against R. padi aphids via the production of indole diterpene alkaloids, and this effect was independent of the plant biomass. The endophyte-mediated reduction in aphid performance was not associated with changes in the abundance, composition and diversity of the insect's bacterial microbiota. However, we cannot rule out that the reduction in aphid performance could be associated with a putative endophyte effect on the bacterial provision of benefits to aphids. Our study highlighted the protective role of endophyte-derived indole diterpene alkaloids against aphids. Further investigations will be needed to determine if there is a link between the endophyte-mediated aphid resistance and the integrity of the insect's bacterial microbiota.

References

[1] Anderson, M.J. (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecology, 26, 32–46.

[2] Bastías, D.A., Bustos, L.B., Jáuregui, R., Barrera, A., Acuña-Rodríguez, I.S., Molina-Montenegro, M.A.et al. (2022) Epichloë fungal endophytes influence seed-associated bacterial communities. Frontiers in Microbiology, 12, 795354.

[3] Bastias, D.A., Ueno, A.C., Machado Assefh, C.R., Alvarez, A.E., Young, C.A. and Gundel, P.E. (2017) Metabolism or behavior: explaining the performance of aphids on alkaloid-producing fungal endophytes in annual ryegrass (Lolium multiflorum). Oecologia, 185, 245–256.

[4] Bowatte, S., Barrett, B., Luscombe, C., Hume, D.E., Luo, D., Theobald, P.et al. (2011) Effect of grass species and fungal endophyte on soil nitrification potential. New Zealand Journal of Agricultural Research, 54, 275–284.

[5] Braendle, C., Miura, T., Bickel, R., Shingleton, A.W., Kambhampati, S. and Stern, D.L. (2003) Developmental origin and evolution of bacteriocytes in the aphid–Buchnera symbiosis. PLoS Biology, 1, e21.

[6] Burger, N.F.V., Nicolis, V.F. and Botha, A.M. (2024) Host-specific co-evolution likely driven by diet in Buchnera aphidicola. BMC Genomics, 25, 153.

[7] Callahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A. and Holmes, S.P. (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nature Methods, 13, 581–583.

[8] Caradus, J.R. and Johnson, L.J. (2020) Epichloë fungal endophytes-From a biological curiosity in wild grasses to an essential component of resilient high performing ryegrass and fescue pastures. Journal of Fungi, 6, 322.

[9] Card, S.D., Bastías, D.A., Zhang, W., Hume, D.E. and Caradus, J.R. (2024) Epichloë—a key element of New Zealand's agricultural landscape. New Zealand Journal of Botany, 62, 519–547.

[10] Chaudhary, R., Atamian, H.S., Shen, Z., Briggs, S.P. and Kaloshian, I. (2014) GroEL from the endosymbiont Buchnera aphidicola betrays the aphid by triggering plant defense. Proceedings of the National Academy of Sciences USA, 111, 8919–8924.

[11] Darby, A.C. and Douglas, A.E. (2003) Elucidation of the transmission patterns of an insect-borne bacterium. Applied and Environmental Microbiology, 69, 4403–4407.

[12] Douglas, A.E. (1998) Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Annual Review of Entomology, 43, 17–37.

[13] Eisenhofer, R., Minich, J.J., Marotz, C., Cooper, A., Knight, R. and Weyrich, L.S. (2019) Contamination in low microbial biomass microbiome studies: issues and recommendations. Trends in Microbiology, 27, 105–117.

[14] Finch, S.C., Prinsep, M.R., Popay, A.J., Wilkins, A.L., Webb, N.G., Bhattarai, S.et al. (2020) Identification and structure elucidation of epoxyjanthitrems from Lolium perenne infected with the endophytic fungus Epichloë festucae var. lolii and determination of the tremorgenic and anti-insect activity of epoxyjanthitrem I. Toxins, 12, 526.

[15] Fuchs, B., Krischke, M., Mueller, M.J. and Krauss, J. (2013) Peramine and lolitrem B from endophyte-grass associations cascade up the food chain. Journal of Chemical Ecology, 39, 1385–1389.

[16] Goggin, F.L. (2007) Plant–aphid interactions: molecular and ecological perspectives. Current Opinion in Plant Biology, 10, 399–408.

[17] Guo, J., Liu, X., Poncelet, N., He, K., Francis, F. and Wang, Z. (2019) Detection and geographic distribution of seven facultative endosymbionts in two Rhopalosiphum aphid species. MicrobiologyOpen, 8, e00817.

[18] Hall, B.G. (2013) Building phylogenetic trees from molecular data with MEGA. Molecular Biology and Evolution, 30, 1229–1235.

[19] Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S.et al. (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649.

[20] Koga, R., Meng, X.Y., Tsuchida, T. and Fukatsu, T. (2012) Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte–embryo interface. Proceedings of the National Academy of Sciences USA, 109, E1230–E1237.

[21] Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549.

[22] Latch, G.C.M. and Christensen, M.J. (1985) Artificial infection of grasses with endophytes. Annals of Applied Biology, 107, 17–24.

[23] Leeuw, J.D. and Mair, P. (2015) Shepard diagram. In Wiley StatsRef: Statistics Reference Online (eds. N. Balakrishnan, T. Colton, B. Everitt, W. Piegorsch, F. Ruggeri & J.L. Teugels), pp. 1–3. American Cancer Society.

[24] Lenth, R.V. (2016) Least-squares means: the R package lsmeans. Journal of Statistical Software, 69, 1–33.

[25] Leybourne, D.J., Bos, J.I.B., Valentine, T.A. and Karley, A.J. (2020) The price of protection: a defensive endosymbiont impairs nymph growth in the bird cherry-oat aphid, Rhopalosiphum padi. Insect Science, 27, 69–85.

[26] Liu, B., Ju, Y., Xia, C., Zhong, R., Christensen, M.J., Zhang, X.et al. (2022) The effect of Epichloë endophyte on phyllosphere microbes and leaf metabolites in Achnatherum inebrians. iScience, 25, 104144.

[27] Love, M.I., Huber, W. and Anders, S. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.

[28] Machado-Assefh, C.R., Lopez-Isasmendi, G., Tjallingii, W.F., Jander, G. and Alvarez, A.E. (2015) Disrupting Buchnera aphidicola, the endosymbiotic bacteria of Myzus persicae, delays host plant acceptance. Arthropod-Plant Interactions, 9, 529–541.

[29] Meister, B., Krauss, J., Härri, S.A., Schneider, M.V. and Müller, C.B. (2006) Fungal endosymbionts affect aphid population size by reduction of adult life span and fecundity. Basic and Applied Ecology, 7, 244–252.

[30] Miller, T.A., Hudson, D.A., Johnson, R.D., Singh, J.S., Mace, W.J., Forester, N.T.et al. (2022) Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing. Frontiers in Fungal Biology, 3, 944234.

[31] Moran, N.A. (2021) Microbe Profile: Buchnera aphidicola: ancient aphid accomplice and endosymbiont exemplar. Microbiology (Reading, England), 167, 001127.

[32] Moran, N.A., McCutcheon, J.P. and Nakabachi, A. (2008) Genomics and evolution of heritable bacterial symbionts. Annual Review of Genetics, 42, 165–190.

[33] Morella, N.M., Weng, F.C.H., Joubert, P.M., Metcalf, C.J.E., Lindow, S. and Koskella, B. (2020) Successive passaging of a plant-associated microbiome reveals robust habitat and host genotype-dependent selection. Proceedings of the National Academy of Sciences USA, 117, 1148–1159.

[34] Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D.et al. (2020) Vegan: community ecology package. R package Version 2.5-7.

[35] Pineda, A., Zheng, S.J., van Loon, J.J.A., Pieterse, C.M.J. and Dicke, M. (2010) Helping plants to deal with insects: the role of beneficial soil-borne microbes. Trends in Plant Science, 15, 507–514.

[36] Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. and R Core Team. (2009) Nlme: linear and nonlinear mixed effects models. R package.

[37] Popay, A.J., Hume, D.E., Mace, W.J., Faville, M.J., Finch, S.C. and Cave, V. (2021) A root aphid Aploneura lentisci is affected by Epichloë endophyte strain and impacts perennial ryegrass growth in the field. Crop and Pasture Science, 72, 155–164.

[38] R Core Team. (2014) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.

[39] Rasmussen, S., Parsons, A.J., Russell, J., Bastías, D.A. and Liu, Q. (2023) Plant species, nitrogen status and endophytes are drivers of soil microbial communities in grasslands. Crop and Pasture Science, 75, CP23149.

[40] Roberts, E. and Lindow, S. (2014) Loline alkaloid production by fungal endophytes of fescue species select for particular epiphytic bacterial microflora. The ISME Journal, 8, 359–368.

[41] Sandström, J.P. and Moran, N.A. (2001) Amino acid budgets in three aphid species using the same host plant. Physiological Entomology, 26, 202–211.

[42] Schardl, C.L., Afkhami, M.E., Gundel, P.E., Iannone, L.J., Young, C.A., Creamer, R. et al. (2023) Diversity of seed endophytes: causes and implications. In Plant Relationships: Fungal-Plant Interactions (eds. B. Scott & C. Mesarich), pp. 83–132. Springer International Publishing, Cham.

[43] Schardl, C.L., Leuchtmann, A. and Spiering, M.J. (2004) Symbioses of grasses with seedborne fungal endophytes. Annual Review of Plant Biology, 55, 315–340.

[44] Shi, S., Kumar, S., Young, S., Maclean, P. and Jauregui, R. (2023) Evaluation of 16S rRNA gene primer pairs for bacterial community profiling in an across soil and ryegrass plant study. Journal of Sustainable Agriculture and Environment, 2, 500–512.

[45] Siegel, M.R., Latch, G.C.M., Bush, L.P., Fannin, F.F., Rowan, D.D., Tapper, B.A.et al. (1990) Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response. Journal of Chemical Ecology, 16, 3301–3315.

[46] 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.

[47] Smith, T.E. and Moran, N.A. (2020) Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera. Proceedings of the National Academy of Sciences USA, 117, 2113–2121.

[48] Wilkinson, H.H., Siegel, M.R., Blankenship, J.D., Mallory, A.C., Bush, L.P. and Schardl, C.L. (2000) Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism. Molecular Plant-Microbe Interactions, 13, 1027–1033.

[49] Wood, D.E., Lu, J. and Langmead, B. (2019) Improved metagenomic analysis with Kraken 2. Genome Biology, 20, 257.

[50] Wooley, J.C., Godzik, A. and Friedberg, I. (2010) A primer on metagenomics. PLoS Computational Biology, 6, e1000667.

[51] Xu, T.T., Chen, J., Jiang, L.Y. and Qiao, G.X. (2021) Diversity of bacteria associated with Hormaphidinae aphids (Hemiptera: Aphididae). Insect Science, 28, 165–179.

[52] Zepeda-Paulo, F., Ortiz-Martínez, S., Silva, A.X. and Lavandero, B. (2018) Low bacterial community diversity in two introduced aphid pests revealed with 16S rRNA amplicon sequencing. PeerJ, 6, e4725.

[53] Zhang, W., Forester, N.T., Applegate, E.R., Liu, X. and Johnson, L.J. (2023a) High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission. Molecular Plant Pathology, 24, 1430–1442.

[54] Zhang, W., Forester, N.T., Chettri, P., Heilijgers, M., Mace, W.J., Maes, E.et al. (2023b) Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide epichloëcyclins in Epichloë festucae. Journal of Agricultural and Food Chemistry, 71, 13965–13978.

[55] Zhang, W., Gundel, P.E., Jáuregui, R., Card, S.D., Mace, W.J., Johnson, R.D., et al. (2024) The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota. Plant, Cell & Environment, 47, 2865–2878.

[56] Züst, T. and Agrawal, A.A. (2016) Mechanisms and evolution of plant resistance to aphids. Nature Plants, 2, 15206.

[57] Zuur, A.F., Ieno, E.N., Walker, N.J., Saveliev, A.A. and Smit, G.M. (2009) Mixed Effects Models and Extensions in Ecology with R, 1st edn. Springer Sciences, USA.

Rights & permissions

2025 The Author(s). Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.