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.