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\begin{document}

\title{Back to the future: Using herbarium specimens to isolate nodule
associated bacteria}



\author[1]{Renee Helen Petipas}%
\author[2]{Amanda A.  Antoch}%
\author[1]{A.N.  Eaker}%
\author[1]{Hanna  Kehlet-Delgado}%
\author[1]{Maren Friesen}%
\affil[1]{Washington State University}%
\affil[2]{University of Washington}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Herbarium specimens are increasingly being used as sources of
information to understand the ecology and evolution of plants and their
associated microbes. Most studies have used specimens as a source of
genetic material using culture independent approaches. We demonstrate
that herbarium specimens can also be used to culture nodule-associated
bacteria, opening the possibility of using specimens to understand
plant-microbe interactions at new spatiotemporal scales. We used
historic and contemporary nodules of a common legume, Medicago lupulina,
to create a culture collection. We were able to recover historic
bacteria in 15 genera from three specimens (collected in 1950, 2004, and
2015). This work is the first of its kind to isolate historic bacteria
from herbarium specimens. Future work should include inoculating plants
with historic strains to see if they produce nodules and if they affect
plant phenotype and fitness. Although we were unable to recover any
Ensifer, the main symbiont of Medicago lupulina, we recovered some other
potential nodulating species, as well as many putative growth-promoting
bacteria.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION:}

Herbaria, historically used to answer questions in taxonomy and
systematics, are increasingly being used as a resource to investigate
questions in genetics/genomics, ecology, biogeography, and evolution.
Herbaria have been touted as a potential source of extinct plant
germplasm, (Albani Rocchetti et al. 2021, 2022, Wolkis et al. 2022),
even in one case allowing researchers to resurrect an extinct species
of\emph{Astragulus} (Moln\selectlanguage{ngerman}ár et al. 2015). With the advent of more
sophisticated and increasingly inexpensive molecular methods and a
burgeoning interest in microbiomes and plant-microbe interactions
researchers are increasing taking culture-independent approaches to
study plant-associated microbes in botanical collections (Li et al.
2007, Daru et al. 2018, Heberling and Burke 2019, Bieker et al. 2020,
Campos et al. 2021, 2023).

The power of herbarium samples for discovery in plant adjacent fields
was evident in a 2001 paper that overturned preexisting beliefs about
the origin of the blight that led to the Irish potato famine (Ristaino
et al. 2001). Follow-up work established that historic strains
of\emph{Phytophora infestans} found in herbarium specimens were from an
extinct lineage (Yoshida et al. 2013), highlighting that herbaria are a
source to discover unknown microbes. Most recent work explored in depth
the genomics that lead to the total displacement of historic strains
of\emph{Phytophora} (Martin et al. 2013). Work in wild yam demonstrated,
through the generation of 17 historical genomes, a much earlier origin
of the obligate symbiont than previously assumed. It also produced
evidence of horizontal transmission and gene transfer, which overthrew
pre-existing beliefs in exclusive mode of vertical transmission
(Danneels et al. 2021). Even more recently, authors took a metagenomic
approach to describe temporal shifts in a common plant
species,\emph{Ambrosia artemisiifolia} , this type of work could be
critical in expanding our temporal window to understand
microbiome-shifts in response to anthropogenic activities including
climate change (Burbano and Gutaker 2023).

Less commonly, researchers have taken culture dependent approaches. In a
recent proof-of-concept paper, authors demonstrated that leaf fungal
endophytes could be successfully cultured from herbarium specimens (Daru
et al. 2018). Similarly, historic tobamoviruses, a stable virion that
remains viable for long periods, was used to infect contemporary tobacco
plants. Astonishingly, authors were able to infect plants with virus
from specimens collected in 1899 and they found no correlation between
the age of the specimen and the ability to infect contemporary specimens
(Fraile et al. 1997).

To our knowledge no one has cultured root associated bacteria from
herbarium specimens, despite their importance to plant phenotype and
fitness (Friesen et al. 2011, Petipas et al. 2020, 2021). Here we work
with a common legume species to isolate bacteria associated with
contemporary and historic nodules. \emph{Medicago lupulina} is a largely
selfing, annual, biennial, or occasionally perennial plant species (Yan
et al. 2009) with a wide distribution throughout North America. It was
introduced to the continent in the late 1700s (Turkington and Cavers
1979), and by the early 1800s copious specimens could be found in
biological collections throughout North America. Specimens are often
mounted with roots intact and nodules can still be found on the roots of
even the oldest specimens (Fig. 1). \emph{Medicago lupulina} primarily
affiliates with two species of rhizobia, \emph{Ensifer meliloti}
and\emph{Ensifer medicae} (Harrison et al. 2017).

Besides the main nodule-forming rhizobia species, nodules are often
colonized by a diversity of other bacteria. Work in
related\emph{Medicago} species revealed taxa from at least 10 bacterial
genera colonizing nodules (Martínez-Hidalgo et al. 2022). Understanding
nodule associated bacteria is important because they can perform
important functions in the rhizosphere critical to plant development,
growth, and the functioning of the legume rhizobia symbiosis. For
example, nodule-associated bacteria can produce siderophores, solubilize
phosphate, change root morphology, enhance nodulation, all of which can
lead to enhanced growth and survival in legumes (Martínez-Hidalgo and
Hirsch 2017). In addition, rhizobia are a phylogenetically diverse group
of bacteria, taxa from a diverse group of genera,
including\emph{Ochrobactrum} (Trujillo et al. 2005), \emph{Devosia}
(Rivas et al. 2002), \emph{Microvirga} (Msaddak et al. 2017),
and\emph{Paraburkholderia} (Elliott et al. 2007) are all capable of
nodulating plants and fixing nitrogen. We might find nodulating rhizobia
besides \emph{Ensifer} that nodulate \emph{Medicago} .

Here we used seven media types to isolate bacteria from historic nodules
taken from herbarium specimens around Washington state. We were able to
culture isolates from three historical specimens, including 13 isolates
from a specimen from 1950, 62 isolates from 2004, and 26 isolates from
2015. We sent a subset of sequences for Sanger sequencing and found that
we were able to recover a diversity of bacteria including representation
from 15 genera. While we were unable to capture any \emph{Ensifer} in
our collection we did recover a number of putative plant-growth
promoting bacteria, including rhizobia in the genus \emph{Microvirga.}

\textbf{MATERIALS AND METHODS:}

Herbarium sampling-

We used nodules collected from herbarium specimens of \emph{Medicago
lupulina} housed at the Marion Ownbey Herbarium at Washington State
University. Marion Ownbey houses 400,000 vascular and non-vascular plant
specimens, including 122 specimens of \emph{Medicago lupulina} collected
primarily from North America (109 specimens). For our experiments we
used nodules from historic and contemporary specimens collected around
Washington State from Skagit County (1 nodule), Klickitat County (2
nodules), Grant County (1 nodule), Yakima County (1 nodule), Ferry
County (1 nodule) and Whitman County (2 nodules) from 1925-2019 (Table
1). For each specimen we use a clean, autoclaved razor blade to excise
nodules and a similarly sized piece of leaf material to be used as a
control for environmental contamination (Heberling and Burke 2019). We
used gloves and a clean surface (ethanol sterilized, autoclaved trays)
to collect material. Nodules and leaf material were stored in sterile
Eppendorf tubes at room temperature until use in the culturing
experiment. We used contemporary nodules collected from the Washington
State University campus in Pullman, Washington. Contemporary nodules
included one air dried nodule, stored at room temp in an Eppendorf tube
for one year (2018), a fresh collected nodule (2019), and a lab strain
of \emph{Ensifer medicae} (WSM-419).

Culture collection -

Seven different media types were prepared: Luria-Bertani (LB),
Tryptone-Yeast (TY), Yeast Mannitol (YM), diluted YM, YM plus calcium,
YM plus sodium chloride (NaCl), and YM plus pyruvate. The diluted YM was
prepared as a ½X dilution of YM media for all ingredients except agar.
The YM plus calcium was prepared according to the YM recipe with the
addition of 2 grams calcium carbonate (CaCO\textsubscript{3}) per liter,
instead of 1 gram per liter. Similarly, the YM plus NaCl was prepared
with 1 gram of NaCl per liter, which is 10 times the amount in the
protocol. The YM plus pyruvate was prepared as indicated in the
protocol, with the addition of 600ul of filter-sterilized (Acrodisc
syringe filter, 0.2\selectlanguage{greek}µ\selectlanguage{english}M, VWR, Atlanta, GA, USA) 2M sodium pyruvate to the
media post-autoclaving to produce a final concentration of 2mM sodium
pyruvate. Media recipes can be found in Table S1. Luria-Bertani (LB),
Tryptone-Yeast (TY), Yeast Mannitol (YM) were all chosen for this study
because they are commonly used to isolate rhizobia and non-rhizobia
species in plants. We used a dilute YM in the hopes that by diluting
nutrient rich media, we might discourage the growth of fast-growing
species such as \emph{Bacillus} , allowing slower-growing species to
grow, as well to lower the presence of free-radicals that can inhibit
cell growth (Oliver 2005). Salt was added to retain osmotic balance and
regulate membrane potential (Cin\selectlanguage{ngerman}átl 1969). Calcium was added because it
has been demonstrated to increase viability of rhizobia, by
strengthening the cell wall (Vincent 1962). Finally, pyruvate was added
to prevent oxidative damage (Giandomenico et al. 1997) by eliminating
metabolic by-products and free-radicals (Stevenson et al. 2004).

Nodules and leaves were rehydrated in filter-sterilized (Acrodisc
syringe filter, 0.2\selectlanguage{greek}µ\selectlanguage{english}M, VWR, Atlanta, GA, USA) 10\% sucrose solution for
48 hours. After rehydration, nodules were rinsed in autoclaved, filtered
water Milli-Q\selectlanguage{ngerman}\textsuperscript{®} seven times before transfer to 200ul ½X
phosphate buffered saline (PBS) for an additional 24 hours. Nodules (and
leaves) were crushed then streaked onto ten plates of each media type
for the 1950 specimen, but three plates of each media type for years
1974-2019. After completing the nodule and leaf control for the 1950
sample it seemed the amount of liquid was not enough to adequately
streak 70 plates. An autoclaved, filtered
Milli-Q\textsuperscript{®}water blank and an autoclaved PBS blank were
used as negative controls and were streaked onto five plates of each
media type. Plates were sealed with parafilm then incubated at 30°C for
12 hours before being observed for growth.

Plates were observed for growth every 24 hours. Plates with growth after
five days were re-streaked for isolation onto the same media type using
the quadrant streak method. Each sample underwent at least two isolation
re-streaks onto the same media type. If a plate had a single isolated
strain after two sets of re-streaks, cells were harvested for glycerol
stocks and pellets for DNA extractions. If the plate was not a single
isolated strain (\textgreater{}1 colony morphology on the plate),
isolation restreaks continued for each morphologically unique colony on
the plate until they were isolated. This took up to four sets of
re-streaks for some isolates. We described the isolates using criteria
from the American Society for Microbiology (Breakwell et al. 2007),
describing the pigmentation, form, margin, elevation, and texture (Table
S2). Glycerol stocks were made by flooding the plates with 3ml of
sterile ½X PBS. Cells were lifted from the surface of media and mixed
into the buffer using sterile plastic pestles. We added 750\selectlanguage{greek}µ\selectlanguage{english}l of
cell/buffer slurry to each 2 ml stock tube each with 750\selectlanguage{greek}µ\selectlanguage{english}l of 80\%
glycerol. Stock tubes were vortexed then stored in the -80 \selectlanguage{ngerman}°C freezer.
Remaining cell/buffer slurry was added to a 2ml Eppendorf tube and
centrifuged at 10,000rpm for 3 minutes or until a pellet formed. Once
pelleted, all supernatant was removed. Pellets were stored at -80 °C
prior to use for DNA extractions.

Sanger sequencing-

We used isolate descriptions to select morphologically diverse isolates
to send for Sanger sequencing because we wanted to describe the
diversity within our collection. We identified 52 isolates for Sanger
sequencing (\textasciitilde{}48\% of our collection) and we performed
DNA extractions using the Qiagen PowerLyzer Microbial DNA Kit
(Germantown, MD, USA). We then amplified the 16S rRNA region with
primers 27F and 1492R, which span nearly the entire length of the gene
(Weisburg et al. 1991). Amplicons were prepared with 2\selectlanguage{greek}µ\selectlanguage{english}l of template,
39.75\selectlanguage{greek}µ\selectlanguage{english}l of molecular grade water, 5\selectlanguage{greek}µ\selectlanguage{english}l of DreamTaq\selectlanguage{ngerman}® buffer, 1\selectlanguage{greek}µ\selectlanguage{english}l of
10xDNTP mix, 1\selectlanguage{greek}µ\selectlanguage{english}l forward primer (27F), 1\selectlanguage{greek}µ\selectlanguage{english}l reverse primer (1492R), and
0.25\selectlanguage{greek}µ\selectlanguage{english}l DreamTaq\selectlanguage{ngerman}®. Thermocycler conditions for bacterial PCR
amplification were 95°C for 5 minutes, followed by 33 cycles of 95°C for
30 seconds, 53°C for 30 seconds, 72°C for 2 minutes, with a final
extension of 72°C for 3 minutes. PCR products were purified using
Sera-Mag Speedbeads carboxylated magnetic beads (Thermo Fisher
Scientific, Waltham, MA, USA) and eluted in molecular grade water.
Samples were submitted to the Washington State University Laboratory for
Biotechnology and Bioanalysis (LBB) for Sanger sequencing using the ABI
3730 DNA Analyzer (ThermoFisher Scientific, Waltham, MA, USA). Isolates
that sequenced poorly were run twice. Forward and reverse sequences from
three isolates did not pass quality thresholds and were not included for
subsequent analyses.

Sanger sequences were trimmed to `max ambiguities'= 2 and consensus
sequences were made by combining forward and reverse reads with Geneious
Primer (v. 2023.2.1, Biomatters, Inc., CA, USA) with the ``high
sensitivity/slow'' method. For taxonomic identification, sequences were
used as BLAST queries to the NCBI 16S rRNA database with the following
settings: ``low complexity filter, max e-value =0.05, word size= 28, gap
cost= linear, program= megaBLAST.'' The species with the lowest E value
and highest percentage identity were selected as the identity of the
isolate. Here we present taxa to the genus level, but putative species
IDs can be found in Table S3.

\textbf{RESULTS:}

Culture collection-

To confirm that our isolates cultured from fresh and historic nodules
were not the result of contamination from the herbarium or the lab, we
compared our media blanks and herbarium leaf controls to growth on
plates that were streaked with either fresh or historic crushed nodules.
We didn't find any growth on our negative controls (Fig. 2A) or historic
leaf samples (Fig. 2B), compared to \textasciitilde{} 50\% probability
of growth on plates streaked with nodules from historic and contemporary
sources (\emph{X\textsuperscript{2}} =177.6, df=2,\emph{P}
\textgreater{}0.0001). The probability of growth was almost three times
higher for contemporary nodules compared to historic nodules
(contemporary probability= 0.968, historic probability=
0.359,\emph{X\textsuperscript{2}} =82.82, df=1, \emph{P}
\textgreater{}0.0001, Fig. 2). We also found that collection year
(historic nodules only) and media type interacted to determine the
probability of growth (\emph{X\textsuperscript{2}} =21.17, df=12,
\emph{P} =0.04795, Fig. 2D, G, H). Unfortunately, due to small samples
for each media type, planned contrasts could not detect differences
between media types, however it seems that isolates from 2004 did
markedly worse on LB media, whereas those from 1950 had a slight
preference for tryptone yeast.

Sanger sequencing-

In our sequenced samples we identified 15 unique bacterial genera. The
majority of isolates were in the genus
\emph{Bacillus}(\textasciitilde{}13\% of our sequenced isolates) but a
sizable amount belonged to other taxonomic groups including
\emph{Microvirga}(\textasciitilde{}6 \%), \emph{Micromonospora}
(\textasciitilde{}5 \%),\emph{Skermanella} (\textasciitilde{}5 \%), and
\emph{Paenibacillus}(\textasciitilde{}3 \%). Isolates from four genera
were isolated from our 1950 sample. We identified isolates from 12
genera in our two nodules taken from the 2004 specimen. We found
isolates from three genera from our nodule taken from a 2015 specimen
(Fig. 3).

\textbf{DISCUSSION:}

Here we demonstrate that bacteria from historic herbarium specimens are
culturable using microbiological media commonly used in microbiology
research. Herbaria are potentially a powerful tool to study questions
related to plant-microbe interactions, coevolution, and global change.

Potential nodule- forming bacteria-

We were not able to isolate \emph{Ensifer} from any of our historic
specimens, only from the contemporary \emph{Medicago} . Although
\emph{E. meliloti} has been described as desiccation tolerant,
\emph{Ensifer} do not generally produce any spores or cysts (Humann et
al. 2009), which potentially limited their ability to survive in the
extreme conditions of the herbaria. We were able to isolate potential
nodule-forming\emph{Microvirga} from the nodules of specimens from 2004
and 2015. Nodule forming \emph{Microvirga} form nodules in several
legume species, including \emph{Listia} , \emph{Lupinus} (Ardley et al.
2012, 2013), and\emph{Vigna} (Radl et al. 2014). Future work will
include sequencing the rest of our culture collection and increasing our
temporal resolution by sampling more specimens, from more dates,
allowing us to definitively say how long \emph{Ensifer} can survive in
herbaria. Additionally, we will investigate if historic
\emph{Microvirga} isolated from herbaria nodules can nodulate and
benefit \emph{Medicago lupulina} .

Non-nodule forming bacteria-

We were also able to recover a diversity of non-nodule forming species
of bacteria. The most abundant taxa in our collection are\emph{Bacillus}
and \emph{Micromonospora,} two genera that are commonly found associated
with nodules (Trujillo et al. 2010, De Meyer et al. 2015).
\emph{Bacillus} is potentially the most common nodule-associated
bacteria (Xu et al. 2014). Nodule-associated \emph{Bacillus} are
important constituents of the nodule microbiome, solubilizing phosphate
(Tapia-García et al. 2020), promoting nodulation (Chinnaswamy et al.
2018), and enhancing plant size (Bai et al. 2002).
Similarly,\emph{Micromonospora} is commonly found in the nodule
microbiome. In one study \emph{Micromonospora} was found in 50\% of
\emph{Trifolium} and\emph{Medicago} nodules examined (Benito et al.
2017)\emph{. Micromonospora} has been described as a rhizobia-helper
bacteria because of its ability to enhance nodulation and improve
nitrogen use by plants (Martínez-Hidalgo et al. 2014). In future work,
would like to use assays to check for siderophore production (Ahmad et
al. 2008), IAA production (Loper and Schroth 1986), and phosphate
solubilization(Ahmad et al. 2008). These functions are typically
correlated with plant growth promotion. Finally, we will inoculate
\emph{Medicago lupulina} to check plant-growth promotion with candidate
strains.

The interesting case of the 1950 specimen-

An outstanding question of this work is why we were able to culture from
a 73-year-old specimen but not from specimens from intervening years
(1974 and 1986). Because of our copious controls and rigorous
experimental conditions, we believe that these isolates are not from
environmental contamination. We suspect the reason we could culture from
the 1950 specimen was one of two factors: 1) We had much higher sample
sizes for the 1950 nodule. Initially we thought the historic crushed
nodule would provide enough material to streak 10 plates per media type,
however it seemed there was not enough liquid so after streaking 10
plates of each media type for the 1950 nodule and its associated leaf
control, we scaled the experiment back to 3 plates of each media type.
This resulted in over three times as many plates for our 1950 specimen,
this alone may have increased the probability that we could capture
historic microbes. Additionally, 2) historic specimens are handled very
differently depending on the collection conditions and/or the
collector/curator at the herbarium (Forrest et al. 2019). Specimens are
dried and frozen for variable amounts of time using different methods
resulting in increased probability of microbial survival. We don't have
information on how the 1950 specimen was handled but it is possible that
it was handled in a way that allowed spore forming bacteria such
as\emph{Bacillus} (the dominant genus found in our collection) to
persist.

Methodological/ethical considerations

We controlled for environmental contamination by rinsing nodules and
including leaf controls, which presumably were exposed to the same
sources of environmental contamination. However, post-harvest plants
have significantly different microbiomes than actively growing plants
both in taxonomic diversity and function. This ``post-mortem''
microbiome was found to be a significant portion of the microbes
identified on herbarium specimens (\emph{Alternaria alternata} comprised
up to 7\% of the total reads, Bieker et al. 2020). However, authors
found that non-bacterial microbes were the largest contributors of the
leaf post-mortem microbiome (Bieker et al. 2020). Potentially, some of
the microbes that we cultured are constituents of
\emph{Medicago's}post-mortem microbiome. We did isolate genera that are
known constituents of the \emph{Medicago} nodule microbiome
including\emph{Micromonospora} and \emph{Paenibaccillus}
(Martínez-Hidalgo et al. 2014, Lai et al. 2015, Hansen et al.
2020)\emph{.} Further work should compare historic and contemporary
nodule microbiomes of \emph{Medicago lupulina} to determine occurrence
of taxa found in our collection in contemporary populations.

General ethical issues that occur when using herbarium specimens are
also relevant for type of work. We took a highly conservative sampling
approach because our approach requires destructively sampling specimens.
We chose a common species that is abundant in herbaria across the
country. It would be impractical and unethical to work with rare
specimens that occur sporadically in herbaria. Fortunately, with the
digitization of collections all over the country and world access to
suitable specimens this kind of work is increasingly possible. Finally,
it is important to acknowledge that herbaria, like any biological
collection, benefitted from the period of overt colonialism spanning the
15\textsuperscript{th} to mid-20\textsuperscript{th} centuries. The
majority of plant specimens are held in the global north, despite being
collected globally, a pattern that has created an inverse relationship
between global biodiversity and where collections are housed (Park et
al. 2023). This trend in collections highlights not only a trend for the
colonizing countries to exploit foreign resources, but also wrests the
control of knowledge and biological resources away from countries of
origin, depriving resident explorers/scientists/researchers from local
resources and discoveries.

\textbf{CONCLUSIONS} : Herbarium collections contain specimens that
represent taxa from a wide range of locations collected over a timeframe
that is well beyond that of modern research. Our work demonstrates that
not only are herbarium specimens a source of genetic/genomic/metagenomic
information about plant species and their associated microorganisms, but
they are also the source of historic microbes. While microbial diversity
has been examined extensively, microbial function is only now being
explored. A source of data for understanding microbiome function are
growing culture collections. Enhancing culture collections through
herbarium collections will not only expand the source of culture
collection material but might help identify novel functions that are
relevant to pressing problems such as climate change (Rudgers et al.
2020). Additionally, herbarium microbial culture collections will allow
us to test hypotheses about plant-microbe interactions over larger
temporal and spatial scales. For example, previous work has suggested
that increasing soil nitrogen availability could cause the erosion of
interactions between nitrogen fixing microbial bacteria and plant hosts,
Although some support exists for this idea (Weese et al. 2015) ,
herbarium cultures could further the temporal window to test these
ideas.

\textbf{DATA AVAILABILITY:}

Data files and R code can be found on the Open Science Framework (OSF)
website: osfhttps://osf.io/qpykj/.

\textbf{ACKNOWLEDGEMENTS:}

We would like to thank Derek Pouchnik and Weiwei Du at the WSU
Laboratory for Biotechnology and Bioanalysis for their help with the
Sanger sequencing. We would also like to thank members of the Friesen
lab past and present for their help and advice on this project. This
work was supported by a National Science Foundation Postdoctoral
Research Fellowship in Biology (NSF award 1812184) awarded to R. H.
Petipas. We would also like to acknowledge that this work was done on
the traditional land of the first people of the Palouse. The Nimíipuu
(Nez Perce) Tribe and the Palus people continue to work, live, play, and
honor the traditions of their ancient heritage in Eastern Washington.
Additionally, our historical specimens were collected around Washington
State, which is home to 37 federally recognized tribes
(https://wsu.edu/about/land-acknowledgement/) and other non-federally
recognized tribes. The people of these tribes continue to thrive today
in Washington State and we acknowledge their careful stewardship of the
land over time immemorial to the present day.

\textbf{AUTHOR CONTRIBUTIONS:}

RHP, AAA, and MLF conceived of the study. RHP, AAA, and ANE performed
the study. RHP and HKD analyzed the data, and RHP and AAA wrote the
paper with input from all authors.

\textbf{DATA AVAILABILITY:} Sequence data will be archived at GenBank ®
the National Institute of Health's genetic sequence database. All other
data will be made publicly available upon acceptance for publication on
a public database such as Dryad.

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\textbf{TABLES:}

\textbf{Table 1:} Metadata for the herbarium specimens used in this
work, we used nodules from historic \emph{Medicago lupulina} collected
from Skagit County (1 nodule), Klickitat County (2 nodules), Grant
County (1 nodule), Ferry County (1 nodule), and Yakima County (1 nodule)
in Washington State from 1925-2015.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/356037/articles/684240-back-to-the-future-using-herbarium-specimens-to-isolate-nodule-associated-bacteria}

\textbf{FIGURE LEGENDS:}

Figure 1: Herbarium specimens are often mounted with roots intact and
nodules (inset) can be found on even the oldest specimens. The nodule
pictured here was take from specimen 324040 collected in 1962 from Walla
Walla, Washington. Photo credit: Water Fertig (plant specimen) and Layla
Taylor-Spriggs (inset)

Figure 2: We had no growth on any of our negative controls (A) and
historic leaf controls (B). We had leaf endophytes grow from our fresh
controls, however only for a subset of the media types (C). We had low
probability of growth for plates streaked with crushed nodules from 1950
(D) and no probability of growth on plates streaked with nodules from
1974 (E) and 1986 (F). Probability of growth was high for more recent
specimen nodules including 2004 (G) and 2015 (H). Probability of growth
was nearly 100\% with contemporary nodules (I).

Figure 3: Sanger sequencing (done on 42\% of our isolates) revealed
isolates from four genera isolated from our 1950 sample. We identified
isolates from 12 genera in our two nodules taken from the 2004 specimen.
We found isolates from three genera from our nodule taken from a 2015
specimen.

\textbf{Hosted file}

\verb`Collections_Figures_Oct162023_FIg1.docx`  available at \url{https://authorea.com/users/356037/articles/684240-back-to-the-future-using-herbarium-specimens-to-isolate-nodule-associated-bacteria}

\textbf{Hosted file}

\verb`Collections_Figures_Oct162023_FIg2.docx`  available at \url{https://authorea.com/users/356037/articles/684240-back-to-the-future-using-herbarium-specimens-to-isolate-nodule-associated-bacteria}

\textbf{Hosted file}

\verb`Collections_Figures_Oct162023_FIg3.docx`  available at \url{https://authorea.com/users/356037/articles/684240-back-to-the-future-using-herbarium-specimens-to-isolate-nodule-associated-bacteria}

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