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Chestnuts bred for blight resistance depart nursery with distinct fungal rhizobiomes

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Abstract

Restoration of the American chestnut (Castanea dentata) is underway using backcross breeding that confers chestnut blight disease resistance from Asian chestnuts (most often Castanea mollissima) to the susceptible host. Successful restoration will depend on blight resistance and performance of hybrid seedlings, which can be impacted by below-ground fungal communities. We compared fungal communities in roots and rhizospheres (rhizobiomes) of nursery-grown, 1-year-old chestnut seedlings from different genetic families of American chestnut, Chinese chestnut, and hybrids from backcross breeding generations as well as those present in the nursery soil. We specifically focused on the ectomycorrhizal (EcM) fungi that may facilitate host performance in the nursery and aid in seedling establishment after outplanting. Seedling rhizobiomes and nursery soil communities were distinct and seedlings recruited heterogeneous communities from shared nursery soil. The rhizobiomes included EcM fungi as well as endophytes, putative pathogens, and likely saprobes, but their relative proportions varied widely within and among the chestnut families. Notably, hybrid seedlings that hosted few EcM fungi hosted a large proportion of potential pathogens and endophytes, with possible consequences in outplanting success. Our data show that chestnut seedlings recruit divergent rhizobiomes and depart nurseries with communities that may facilitate or compromise the seedling performance in the field.

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Acknowledgments

The authors are grateful to Highlands Biological Station Grant-In-Aide in 2013 that provided travel support and to Cornelia Pinchot (USDA-FS) for collecting soil samples from nursery in Indiana. We also thank Alina Akhunova and the Kansas State University Integrated Genomics Facility (http://www.k-state.edu/igenomics/index.html) for library quality control, preparation, and Illumina MiSeq sequencing. This project was partly supported by the USDA-NIFA capacity program KS495.

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  1. Division of Biology, Kansas State University, Manhattan, KS, 66506, USA

    Christopher Reazin & Ari Jumpponen

  2. BCH-EPP Department, Mississippi State University, Mississippi State, MS, 39762, USA

    Richard Baird

  3. Southern Research Station, USDA Forest Service, Knoxville, TN, 37996, USA

    Stacy Clark

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  1. Christopher Reazin
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Supplemental Table S1

Indicator taxon analyses (indicator values, mean ± St. Dev and associated P values) contrasting nursery soil and chestnut rhizosphere samples in the full dataset including all samples and OTUs. The Indicator Taxon Analyses (Dufrene and Legendre 1997) were performed in PC-ORD and indicate OTUs (taxa) that are disproportionately enriched under one treatment comparison compared to others. Column “Indicator” identifies if the OTU is an indicator (P < 0.05) for either soil or rhizosphere, or neither (Not Significant - NS). The analyses identify 171 indicators in total; 26 for chestnut rhizospheres and 145 for soils. The table also includes the taxon affinities for each of the observed OTUs (TXT 48 kb)

Supplemental Table S2

The 86 significant indicators identified in the indicator taxon analyses (indicator values, mean ± St. Dev and associated P values) of the rhizosphere samples representing different chestnut families in the full dataset including all samples and OTUs. The Indicator Taxon Analyses (Dufrene and Legendre 1997) were performed in PC-ORD and indicate OTUs (taxa) that are disproportionately enriched in one chestnut family in comparison to others. Column ‘Family’ represents chestnut family in which the OTU was most abundant and column ‘P Value’ identifies if the OTU is an indicator (P < 0.05). (TXT 9 kb)

Supplemental Table S3

Pairwise comparisons of the fungal communities associated with the rhizospheres of nine families. PerMANOVA analyses that included only core OTUs that occurred in at least half of the 36 rhizosphere samples and excluded soil samples distinguished fungal communities (F8,27 = 2.93, P = 0.001). These pairwise comparisons indicate that fungal communities of most families differ from each other. Shown are the t-test variables and associated P-values, those that do not differ are highlighted in bold. (DOCX 14 kb)

Supplemental Figure S1

Non-metric Multidimensional Scaling (NMS) ordination of fungal communities of the one-year-old American chestnut seedlings and the nursery soils in which they were grown. A three-dimensional ordination (k = 3) provided an optimal solution and represented 86.4% of the variation with a stress of 13.36, separating the soils and a majority of the chestnut breeding line rhizobiomes. a) Axis 1 and Axis 2 that represent 57.5% of the variation; b) Axis 1 and Axis 3 that represent 59.6% of the variation and distinguish nursery soils from the American chestnut rhizobiomes; and, c) Axis 2 and Axis 3 that represent 55.7% of the variation and also distinguish nursery soils from the American chestnut rhizobiomes. (PNG 179 kb)

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Supplemental Figure S2

Richness and diversity estimators of American chestnut rhizobiomes and the nursery soils they were grown: a) coverage; b) observed richness (SObs); c) extrapolative ChaoI that estimates the total OTU number; d) extrapolative Boneh that estimates the number of additional OTUs that would have been observed had the sampling been complete; e) Shannon diversity (H′); and, f) evenness based on Shannon’s diversity (EH) of nursery soil and chestnut hybrid roots. The asterisks show the results of Dunnett’s tests, which compare means of each chestnut rhizobiome estimates against the nursery soil: *** – P < 0.001; ** – 0.001 ≤ P < 0.01; * – 0.01 ≤ P < 0.05. The data indicate greater coverage and lower richness (Observed and extrapolated) in the roots than in the soils, whereas diversity and evenness did not differ. Further, none of the estimators differed among the American chestnut rhizobiomes. (PNG 114 kb)

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Supplemental Figure S3

Non-metric Multidimensional Scaling (NMS) ordination of fungal communities representing American chestnut rhizobiomes. The ordination was optimally resolved on three axes that represent 29.7%, 27.6%, and 28.5% of the variability, for a total of 85.6% with stress 0.14. Permutation-based MANOVA indicated that fungal communities differ among the nine analyzed chestnut rhizobiomes (F8,27 = 2.98, P < 0.001). a) Axis 1 and Axis 2 that represent 57.3% of the variation; b) Axis 1 and Axis 3 that represent 58.0% of the variation; and, c) Axis 2 and Axis 3 that represent 55.9% of the variation and also distinguish nursery soils from the American chestnut rhizobiomes. The ordination distinguishes most chestnut species and hybrid families (Table 2). (PNG 205 kb)

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Reazin, C., Baird, R., Clark, S. et al. Chestnuts bred for blight resistance depart nursery with distinct fungal rhizobiomes. Mycorrhiza 29, 313–324 (2019). https://doi.org/10.1007/s00572-019-00897-z

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