Insights

into the impact of geography and genetics on the pecan

microbiome
Deborah K. Ray, J. Nick Fisk, Kimberly Cervantes, Esteban Molina, Jaya R. Soneji, Jennifer J. Randall.
Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, NM 88003. Email: jrandall@nmsu.edu

ABSTRACT MATERIALS & METHODS CONTINUED

Carya illinoinensis, commonly known as pecan, is native to many Microbiome Analysis
Fig.3 Relative Abundance of
Fig.3 Relative Abundance of
regions across North America and has a lifespan that can extend over Quality control, chimera filtering, clustering, and analysis of the sequencing results was Microbial Genera
Microbial Genera
100 years. This long life span and broad center of origin, along with performed with mothur v.1.39.0. For operational taxonomic unit (OTU) clustering and
cross-pollination, results in genotype-specific differences in the identification, the full SILVA bacterial database (accessed January 2017) was used as
microbiome of this native nut tree. Recent innovations in technology reference. Conservative recommendations put forth by Schloss et. al (2009) for analysis
used to assess microbiome interactions have made it possible for us to of Illumina MiSeq data (accounting for the longer 2X250 read length) were followed.
gain insight into the microbial communities associated with pecan. In a Additional statistics and visualizations were performed using the program language and
previous experiment, we explored the nature of microbial software R.
communities of pecans through culturing and sequencing the
microorganisms present in tissue culture and greenhouse propagated Diversity of Microbial Populations
trees. The diversity of our findings led us to conduct further research

Methylobacterium
on the microbiomes of micropropagated pecans. In an attempt to

Western Genotype
identify the microorganisms present in unique pecan genotypes, total
DNA was isolated and enriched using the NEB Next Microbiome
Enrichment kit. Our samples included two genotypes and two
antibiotic treatments, with a control sample for each genotype. This
enriched DNA underwent Next Generation Sequencing to elucidate

no Methylobacterium
microbial populations present. Microbiome analysis revealed diverse
communities of microorganisms in both genotypes, which were
significantly impacted by antibiotic treatments.
MATERIALS & METHODS Fig. 4 Cluster Dendrogram
Fig.4 Cluster Dendrogramof Population
of
Plant Material and Treatments
Plant material was taken from six micropropagated pecan trees (Fig.
Similarity (OTU Level=0.03)
Population Similarity (OTU Level=0.03)

Methylobacterium
Riverside Genotype

1), three each from a Western genotype from Arizona and a Riverside S6

genotype from New Mexico. One sample per genotype was used as a S5
control and received no antibiotic treatment. The remaining two S3
samples from each genotype received antibiotic treatments, one at a S2
high level and one at a low level. Tissues were ground using liquid S1

no Methylobacterium
nitrogen in a mortar and pestle. Samples were then stored in a -20C
S4
freezer to avoid degradation until further processing.
Fig.3 and
Fig.3 4 S1-S3
and are are
4 S1-S3 a Western genotype
a Western and S4-S6
genotype and are a Riverside
S4-S6 genotype.
are a Riverside
DNA extraction S1genotype.
and S4 wereS1theand
controls
S4 with
wereno antibiotic treatment.
the controls with S2noandantibiotic
S5 had low
DNA was extracted using Qiagen DNeasy Plant Mini Kit (Qiagen, levels of antibiotic
treatment. S2 andtreatment.
S5 had lowS3levels
and of
S6antibiotic
had hightreatment.
levels of antibiotic
S3 and
Germany) and stored in a -20C freezer to preserve the DNA. treatment.
S6 had high levels of antibiotic treatment.
Microbiome Enrichment kit Fig.2 The most abundant genera (threshold=0.03) per sample as a proportion of total abundance in that
The genomic DNA was utilized for microbe enrichment using a NEB sample. Other refers to the cumulative total of all other genera. S1-S3 are a Western genotype and CONCLUSIONS
Next Microbiome DNA Enrichment Kit to identify microbial S4-S6 are a Riversisde genotype. S1 and S4 were the controls with no antibiotic treatment. S2 and S5 Antibiotic treatments led to shifts in microbiome compositions
populations. The DNA was then sent for next generation sequencing had low levels of antibiotic treatment. S3 and S6 had high levels of antibiotic treatment. and an overall decrease in microbial diversity. It is unknown what
using services provided by the University of Minnesota. influence this will have in a field environment.
RESULTS Methylobacterium is a keystone feature of the microbiome of
Plant Material Microbiome sequence analysis indicated that different microbial populations were these genotypes.
present in all six samples (Fig. 2). Next, we are comparing microbiomes from pecan trees of the
The most abundant microbe identified in four out of six samples was same variety in different geographical locations as well as
Methylobacterium. In two samples, S5 and S6, Cryobacterium was most abundant different varieties in the same location.
(Fig. 2). S5 and S6 are both a Riverside genotype and both received antibiotic
treatment. REFERENCES
High antibiotic treatments resulted in a loss of alpha diversity, meaning that it Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent,
decreased the total number of microbial species present (Fig. 3). community-supported software for describing and comparing microbial
communities. Appl Environ Microbiol, 2009. 75(23):7537-41
The microbial populations in all of our Western genotype samples were similar in
control group and both low and high antibiotic treatments(Fig. 4).
ACKNOWLEDGEMENTS
The microbial populations in both antibiotic treated Riverside genotype samples were The authors would like to thank NEB for providing us with the NEB Next
Fig.1 (A) Micropropagated pecan trees. (B) Transfers of micropropagated similar, while the control sample without antibiotic treatment had a highly unique Microbiome DNA Enrichment Kit. The authors would like to thank the New
pecan trees to greenhouse under controlled environment. microbial community (Fig. 4). Mexico State University Experimental Station for their support.