Introduction
Phytoplasmas (Mollicutes , Acholeplasmatales ,Acholeplasmataceae ) are a large group of phloem-restricted, cell
wall-less, vector-borne bacteria that infect hundreds of plant species
and cause serious economic loss worldwide (Rao et al., 2018). In plants,
phytoplasma infection may induce a variety of typical symptoms including
virescence, phyllody, and witches’-broom, thereby altering plant
morphology, growth patterns and architecture (MacLean et al., 2011;
2014; Wei et al., 2013; 2019), although infections may also be
asymptomatic (Zwolinska et al., 2019).
Phytoplasmas are transmitted from plant to plant by phloem-feeding
hemipteran insect vectors, mainly leafhoppers, in a
persistent-propagative manner (Hogenhout et al., 2008; Lee et al., 2000;
Weintraub & Beanland, 2006). After acquisition of phytoplasmas from an
infected plant by a hemipteran insect, the phytoplasma cells must cross
the midgut epithelium, then multiply in the hemolymph in order to invade
the salivary glands before being inoculated into another host plant
(Hogenhout et al., 2008; Huang et al., 2020).
Attempts to culture phytoplasmas in vitro have, thus far, not
succeeded. Thus, phytoplasmas are currently assigned to the provisional
genus ‘Candidatus (Ca. ) Phytoplasma’, and 45 ‘Ca .
Phytoplasma’ species have been described (IRPCM, 2004; Kirdat et al.,
2020; Naderali et al., 2017; Rodrigues Jardim et al., 2020; Šafářová et
al., 2016; Zhao et al., 2021). Nevertheless, the phytoplasma lineage is
a highly diverse monophyletic group (Gupta et al., 2018; Zhao et al.,
2015), having been classified into 36 groups, and more than 150
subgroups based on distinct 16S rRNA gene restriction fragment length
polymorphism patterns (Lee et al., 1998; Naderali et al., 2017;
Rodrigues Jardim et al., 2020; Seemüller et al., 1998; Wei et al., 2007;
Zhao et al., 2009).
The intimate tri-trophic interaction among phytoplasmas, host plants,
and insect vectors defines a complex of multiple pathosystems worldwide
(Trivellone, 2019). Unfortunately, almost all phytoplasma-host
associations have been characterized by testing plants showing symptoms
of diseases in agroecosystems. However, because the association between
phytoplasmas, plants and insect vectors has been evolving for at least
300 million years (Cao et al., 2020), phytoplasmas and their vectors
should also be widespread and diverse in non-managed, native habitats
(Trivellone & Dietrich, 2020). Indeed, current theories of infectious
disease evolution suggest that most epidemic diseases afflicting humans,
livestock and crops emerge as a result of potentially pathogenic
organisms “jumping” from a native host to a new host following
anthropogenic disturbance of natural habitats (Brooks et al., 2019).
About 100 insect species have been recorded as competent vectors of
phytoplasmas; however, for most the of described ‘Ca.Phytoplasma’ species and 16S rRNA subgroups the suite of vectors is
still unknown (overview in Trivellone, 2019). Because insects are often
difficult to identify and individuals infected with phytoplasmas cannot
be distinguished from non-infected individuals except through
microscopy, molecular screening, or pathogen transmission trials,
efforts to identify competent phytoplasma vectors have lagged far behind
efforts to characterize phytoplasmas and their host plants. Due to the
mobility of insect vectors, spillovers of vector-borne phytoplasmas from
adjacent highly diverse natural habitats into agroecosystems were
hypothesized to play an important role in emergence of new phytoplasma
diseases (see Brooks et al., accepted). However, few attempts have been
made to study phytoplasma diversity in natural habitats. Therefore,
diversity, plant host range, and insect vector range of phytoplasmas are
probably significantly underestimated (Trivellone & Dietrich, 2020).
Due to increased awareness of the importance of wildlife as pathogen
reservoirs (Brooks et al., 2020), the use of museum biorepositories to
discover and track pathogens is a critical step for anticipating the
emergence and re-emergence of zoonotic diseases (DiEuliis et al., 2016;
Dunnum et al., 2017). The high levels of biodiversity and geographic
coverage represented in such repositories can also help unveil the
evolutionary history of pathogens and reveal previously unknown
interactions with actual or potential hosts.
In this study, we analyzed specimens of deltocephaline leafhoppers
(Hemiptera: Cicadellidae: Deltocephalinae) preserved in the collection
of the Illinois Natural History Survey (INHS)
(http://inhsinsectcollection.speciesfile.org/InsectCollection.aspx). The
INHS leafhopper collection is one of the largest in world with over
>400,000 specimens stored either pinned or in ethanol at
-20°C. In 2018, a subsample of ethanol-preserved leafhoppers collected
in natural habitats were tested for presence of phytoplasmas. The
results revealed that about 3% of tested insect specimens harbored
phytoplasmas. The newly discovered phytoplasmas belong to three distinct
taxonomic (16Sr) groups. Phytoplasmas were detected from a total of six
leafhopper species including five known and one recently described
species, all recorded for the first time as potential phytoplasma
vectors. These results indicated that phytoplasma diversity and
potential insect host range are indeed underestimated and further
large-scale investigation of leafhopper samples collected from natural
habitats is needed.