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 ofAstragulus (Molnár 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 ofPhytophora 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 ofPhytophora (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,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. 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). Medicago lupulina primarily
affiliates with two species of rhizobia, Ensifer meliloti andEnsifer medicae (Harrison et al. 2017).
Besides the main nodule-forming rhizobia species, nodules are often
colonized by a diversity of other bacteria. Work in relatedMedicago 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, includingOchrobactrum (Trujillo et al. 2005), Devosia (Rivas et al.
2002), Microvirga (Msaddak et al. 2017), andParaburkholderia (Elliott et al. 2007) are all capable of
nodulating plants and fixing nitrogen. We might find nodulating rhizobia
besides Ensifer that nodulate 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 Ensifer in
our collection we did recover a number of putative plant-growth
promoting bacteria, including rhizobia in the genus Microvirga.