Tools and knowledge to mitigate the potential spread of Xylella fastidiosa (Xylella) in Australia and New Zealand by understanding its potential vectors

Abstract

Xylella and its primary sectors are absent from Australia. To understand which Australian insects might be able to vector Xylella this project focused on five activities: multi-year insect field surveillance in southeastern Australian crops, examination of Australian insect reference collections, genetic analysis of insect diversity, genetic analysis of bacterial communities in insects, and feeding physiology experiments on plants. Five types of spittlebug insect were found to be widespread in Australia feeding on multiple plant families, possibly making them vector risks. One, Bathyllus, exhibited feeding behaviour indicating it would likely be able to spread this plant pathogen should Xylella arrive.

Summary

Spittlebugs and froghoppers (Hemiptera: Cercopoidea), and some leafhoppers (Hemiptera: Cicadellidae) are xylem-feeding insects which worldwide can cause economic damage through vectoring plant pathogenic bacteria such as Xylella fastidiosa. Australia does not have X. fastidiosa nor its main spittlebug and sharpshooter vectors, but it is home to at least forty species of native and endemic spittlebug and froghopper (superfamily Cercopoidea), which could potentially vector bacteria

The main objectives of this project were to provide the Australian plant industry with robust tools and knowledge to effectively respond to Xylella incursion, or mitigate the potential spread and severity of Xylella, if this pathogen were to arrive in Australia. This project focussed on facilitating improved understanding of the biology and population dynamics of potential insect vectors of Xylella in and around host crops in multiple regions across southeastern Australia.

The project focused on five main activities: (1) multi-year insect field surveillance in southeastern Australian crops, (2) examination of Australian insect reference collections, (3) genetic analysis of insect diversity, (4) genetic analysis of bacterial communities within insects, and (5) feeding physiology experiments on key crop and Xylella-susceptible plants. 

1. Project field surveillance was conducted using multiple collection methods over three years in commercial crops (including grape, citrus, cherry and olive) in southeastern Australia, revealing variation in the abundance of insect communities in different geographic locations and on different plant taxa (with >20,000 insect specimens collected). Many leafhoppers were collected, but only two species of potential Xylella vectors, both spittlebugs, were detected, with no other potential native vectors (i.e., sharpshooters) found. The pattern of insect abundance in and around crops suggested that effective surveillance of potential Xylella vectors should be conducted during late spring and early summer, preferably using sweep nets or light trapping to capture spittlebugs.
2. We investigated spittlebug specimens in reference collections across Australian museums and state agricultural departments. Specimen metadata (e.g., date, location, host plant, collection method) from these collections was used to examine spatiotemporal patterns, common collection methods, and plants associated with Australia’s spittlebug community. This assessment, of more than 5000 specimens, revealed that the most common genera in Australia are Anyllis, Bathyllus, Chaetophyes, Pectinariophyes, and Philagra, which represent more than three-quarters of the Australian reference specimens analysed. These genera are geographically distributed across multiple Australian states and found on numerous plant families. The genera Bathyllus, Anyllis and Philagra may have the greatest potential for vectoring of X. fastidiosa, if it was introduced to Australia, due to their broad geographic distributions, relatively wide putative host ranges, and high abundances during spring and summer. Bathyllus was found to have the widest host range of any recorded Australian spittlebug, with Bathyllus albicinctus, the only species in the genus, recorded on 44 plant genera spread across 27 plant families.
3. The insect genetic analysis conducted here generated 135 new COI DNA sequences for 22 Australian spittlebug taxa, across three families and 10 genera, including some scientifically undescribed species. We also assessed genetic diversity within Bathyllus albicinctus, which was shown to be a single species, despite substantial morphological variation between individuals. This new dataset forms the first comprehensive work on the genetic diversity of the Australian spittlebugs and froghoppers and, together with the high-resolution morphological images of each taxon that we have generated, now provides an invaluable tool for improving diagnostic identification of the Australian spittlebugs.
4. Diagnostic testing for presence / absence of plant pathogenic bacteria (Xylella, Ralstonia) was conducted on spittlebug adults and nymphs collected from Victoria. Standard molecular assays from the recently updated Xylella National Diagnostic Protocol (NDP) were used on field-collected specimens in the first instance of the NDP assays being applied to insect extracts, with no crossreactivity observed, validating these for use on insects. Furthermore, bacterial communities (microbiomes) present in nymphs of two common spittlebug species were assessed using high throughput sequencing (HTS) DNA metabarcoding of the bacterial DNA. This technique was applied to two native species, Bathyllus albicinctus and Philagra parva, and revealed significant divergence in the composition of microbiomes harboured by insect nymphs feeding on different plants, even when these plants were within a few meters of each other.
5. Detailed studies investigating the feeding physiology of the common native Australian spittlebug, Bathyllus albicinctus, to assess its ability to serve as a potential Xylella vector were conducted. Across multiple experiments, Westringia, olives, and, to a lesser extent grapevines and oranges, were found to potentially be able to support reproductively viable B. albicinctus populations. We also monitored feeding behaviour of B. albicinctus on susceptible plants using the electrical penetration graph (EPG) technique. These experiments showed that B. albicinctus adults continuously feed on grapevine, olive and orange plants and the feeding patterns obtained match those previously described for exotic xylem feeding spittlebugs known to vector Xylella. In addition, Xe (xylem egestion) patterns were observed in insects feeding on all of these susceptible plants, suggesting that B. albicinctus should be considered a likely vector of Xylella if this devastating bacterial disease was to be introduced in Australia.

Project outcomes were formally communicated at more than ten industry publications/conferences/workshops, including the Hemipteran Plant Interactions Symposium (HPIS) in Melbourne in December 2022 to a local and international audience. At the HPIS, formal and informal meetings took place with collaborators from Australia, New Zealand and Europe.