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 Although no evidence of diseased plant hosts was reported from the investigated sites, the collections were originally made for the purpose of documenting insect biodiversity, rather than within the context of plant pathogen surveys.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.