in natural areas
Phytoplasmas are a highly diverse group of plant pathogens and new
strains continue to be discovered at a steady pace worldwide but most
such discoveries still mainly result from screening of plants showing
“typical” phytoplasma disease symptoms in human-managed ecosystems.
In our study we unveiled 6 new associations between phytoplasmas and
their insect hosts, recording new phytoplasma group records for 3
countries, and we detected new phytoplasma strains that will be further
characterized and described separately (Wei et. al., in review). Our
screening also highlights that potential vectors collected in natural
areas worldwide are poorly studied (as suggested by Trivellone &
Dietrich, 2020) and potentially harbor phytoplasma species not yet
discovered and described. Our discovery of new phytoplasma subgroup
strains in natural areas worldwide is not surprising, given the
>300-million-year history of co-evolution between
phytoplasmas, their plant hosts, and insect vectors and the lack of
previous attention to phytoplasmas in non-managed ecosystems (Cao et
al., 2020; Trivellone & Dietrich 2020). According to a recent molecular
timetree for phytoplasmas (Cao et al., 2020), the earliest divergences
of phytoplasmas approximately coincided with those of their vascular
plant hosts and some phytoplasma lineages are associated with particular
major lineages of plants and hemipteran insects. If many such
associations are evolutionarily conservative, then phylogenies may be
useful tools for predicting undocumented associations between
phytoplasmas, insects and plants. Also, because coevolutionary theory
suggests that associations between parasites and their hosts should
evolve toward commensalism over time (i.e., virulence should decrease;
Alizon et al., 2009; Jansen et al., 2015), plants naturally infected by
phytoplasmas in natural areas may not exhibit the classical symptoms of
phytoplasma disease found in crop plants. Thus, many naturally occurring
plant-phytoplasma associations may be asymptomatic, so screening of
potential vectors and/or asymptomatic plants, may be necessary to reveal
the true diversity of unknown phytoplasma strains in native ecosystems.
Collections of leafhoppers used in our study were obtained in natural
habitats. Although no evidence of diseased plant hosts was reported from
the investigated sites, the collections were originally made for the
purpose of documenting insect biodiversity, rather than within the
context of plant pathogen surveys. For this reason, we cannot speculate
on the disease epidemiology of phytoplasmas associated with leafhoppers
tested for the present study. Further investigations are needed to
document the host plants and phenotypic effects of phytoplasma
infections for the newly documented strains.
Interestingly, 5 of the 6 leafhopper genera recorded here as phytoplasma
hosts have not been previously reported as potential or competent
vectors of phytoplasmas but all belong to tribes that include known
phytoplasma vectors. Cophylogenetic studies may be used to predict new
pathogen-host associations and emerging diseases (reviewed in Brooks et
al., 2019). In our study 4 species in the tribe Paralimnini from 3
different countries were found to be associated with new phytoplasma
strains related to the 16SrXI/16SrXIV and 16SrII/16SrXV groups. Previous
studies have associated species of this tribe with groups 16SrI,
16SrXII, and 16SrIX (Trivellone, 2018). A previous phylogeny of
Deltocephalinae recovered Paralimnini as a monophyletic group within a
larger clade of leafhoppers that includes notorious groups of competent
vectors of phytoplasma groups 16SrII, 16SrXI and 16SrXIV in the tribe
Opsiini (Zahniser & Dietrich, 2013; Trivellone, 2018). Preliminary
cophylogenetic analysis (Trivellone unpublished) suggests that potential
host shifts of phytoplasma strains in the 16SrII/16SrXV, 16SrXI/16SrXIV
groups may have occurred among species of deltocephaline leafhoppers.
The newly documented associations reported here provide evidence to
support this hypothesis. These associations remained undetected in
natural areas until now.
Previous research showed that integrating different sources of knowledge
is of paramount importance for discovering potentially emergent
pathogens. Studies on zoonotic diseases showed that museum
biorepositories represent an invaluable but still poorly utilized
resource for pathogen discovery, due to the wealth of species
represented and prevalent best practices of specimen preservation,
identification and collecting-event description (Dunnum et al., 2017).
Furthermore, existing databases and traditional ecological knowledge can
contribute to discovery of the location and timing of potential
spillover of pathogens into human-managed systems worldwide (Brook et
al., 2009; Kutz et al., 2009).
Given that most previous research on phytoplasmas has been performed
within the relatively narrow context of plant disease epidemiology in
agroecosystems, we suggest that the diversity of phytoplasmas is
severely underestimated and that natural areas worldwide should harbor a
rich undiscovered diversity of phytoplasmas and their actual or
potential insect vectors.
Similar
phytoplasma infection prevalence in agroecosystems and natural grassland
was previously reported in the literature; however, knowledge of the
entire range of hosts (plants and insects) and symptoms caused by
phytoplasmas in natural habitats remains inadequate (for a review see
Trivellone & Dietrich,
2020).
Museum
biorepository as source of unknown phytoplasmas
Plant, fungal and animal specimens deposited in natural history museums
and public or private collections are becoming increasingly accessible
due to web-based interfaces. Recently it was pointed out that the
hundreds of millions of samples preserved in collections are useful for
many purposes beyond their traditional uses in comparative morphology,
taxonomy and biogeography (Meineke et al., 2019). Because they provide
broad taxonomic, spatial and temporal coverage of Earth’s biodiversity,
such collections provide opportunities to analyze global changes and
under-investigated areas. Species interactions documented by collections
have mainly been investigated using metadata (e.g., Bartomeus et al.,
2019; Meineke & Davies, 2019). The advent of increasingly sensitive
molecular methods has recently allowed more cryptic symbiotic
associations to be explored directly by the testing preserved tissues of
potential hosts for presence of microbes and other symbionts (e.g., Daru
et al., 2019). To our knowledge, this is the first time that
phytoplasma-insect associations have been documented using museum
specimens. Our screening confirmed presence of phytoplasmas in 6
leafhopper specimens (accounting for ~ 3% of the subset
of 227 leafhopper analyzed). Most studies conducted until now in
agroecosystems reported prevalence of infection on local or regional
scales, mainly focusing on epidemiological cycles. Major examples from
European crop fields (one or few plots) revealed prevalence of infection
in single insect species hosts ranging from 0 to 52% for a single
phytoplasma strain (as an example, 33-37.5% Acs et al., 2011; 7-13%
Lessio et al., 2016; 0-52% Mitrovic et al., 2012; 0-35% Sforza et al.
1998; 6-50% Trivellone et al., 2005). Such studies employ extensive
resources and sampling over a prolonged timespan during the growing
season. Also, because these studies usually aim to document ongoing
disease outbreaks, they are not directly comparable to our study, in
which samples were collected in the absence of any prior evidence of
phytoplasma infection at locations where the samples were collected.
Because we mostly tested single specimens from collecting events spread
over 20 years on multiple continents, it is not surprising that most of
our samples tested negative for presence of phytoplasmas. Our data do
not allow us to speculate on local infection rates of the new strains
detected. However, considering the spatial, temporal and taxonomic scale
of the samples available in museum biorepositories, our results can be
taken as a very rough, preliminary estimate of phytoplasma prevalence in
natural areas worldwide and suggest that the undiscovered diversity of
phytoplasmas in natural areas worldwide is substantial.
The present study provides strong evidence that both phytoplasma
diversity and potential insect vector diversity are underestimated in
natural habitats. Larger-scale studies of museum biorepositories will
likely fill major gaps in our knowledge of this diversity, the evolution
of phytoplasma-plant-vector associations and the potential for emergence
of new pathogens of agricultural importance.