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New Bacterial Pathogens and Endosymbionts of the Olive Fly, Bactrocera oleae, for the Development of Innovative Fruit Fly Biocontrol Strategies

Periodic Reporting for period 1 - OliveFlyBacteria (New Bacterial Pathogens and Endosymbionts of the Olive Fly, Bactrocera oleae, for the Development of Innovative Fruit Fly Biocontrol Strategies)

Reporting period: 2019-01-02 to 2021-01-01

Fruit flies cause severe economic losses in fruti- and viticulture in the Mediterranean basin. Triggered by ongoing climate change, the spread of invasive fruit fly species from Northern Africa to Mediterranean EU and Central European countries potentiates the threat to farmers. One highly specialized fruit fly of particular economically relevance for Mediterranean countries is the Olive Fly, Bactrocera oleae. The pest deposits its eggs into unripe olives, larvae develop within and feed on the ripening fruit, holding potential to infest entire harvests if uncontrolled. Infested fruit is no more suitable for consumption as table olive or for olive oil production.

Olive Fly management by chemical insecticides is both technically challenging due to the protected location of the larvae and problematic due to the enrichment of insecticide residues in olive oil. Therefore, microbial fruit fly pathogens with innovative modes of action are increasingly solicited to be developed into new biological control products. Fruit flies are generally associated with a diverse bacterial microbiome. The Olive Fly is stably associated with an obligatory symbiont, Candidatus Erwinia dacicola, and secondary symbionts of varying number, diversity and functional relationships.

During the past decade, bacteria from different species of the genus Providencia have been found associated with and pathogenic to fruit flies as the common vinegar fly, Drosophila melanogaster, the Mexican fruit fly, Anastrepha ludens, and the Mediterranean fruit fly, Ceratitis capitata. Providencia bacteria have properties potentially facilitating the development of Biological Control or Integrated Pest Management strategies as, e.g. being the insecticidal agent associated with entomoparasitic nematodes or attracting both male and female fruit flies from several Bactrocera species. In 2018, the new bacterial species Providencia entomophila has been identified in both olive flies and olive fly larvae from Tunisian olive groves.

In front of this scientific background, it has been the overall objective of the OliveFlyBacteria project:
• to identify Providencia bacteria - and especially Providencia entomophila - from Olive Fly populations across Southern Italy,
• to evaluate the biocontrol potential of the new Olive Fly associated Providencia isolates, and
• to lay a methodological basis for studies of the molecular interaction of Providencia bacteria and their Olive Fly host.
During the late summer and autumn period of 2019, olive orchards in Apulia, Calabria, Campania, Sardinia and Sicily regions were monitored for the presence of Olive Flies. In 2020, due to COVID-related mobility restrictions, screenings were limited to Campania region. Sampled infested olives and olive fly larvae, pupae and adults extracted or developed from these were screened for the presence of Providencia and further potentially insect-associated bacteria, both presumed symbionts and pathogens.

Cultivation-dependent screenings were supported by a Providencia-specific diagnostic PCR approach developed from comparative genome analyses and by bacterial microbiome pyrosequencing. In total, insects and plant material from over 500 olive samples were screened, and Providencia bacteria, including P. entomophila, were identified in Olive Flies from Calabria, Campania and Sicily, but not from Apulia and Sardinia.

Virulence bioassays revealed pronounced virulence of these bacteria against male and female adults of the Olive Fly upon hemocoelic injection, whereas ingested bacteria triggered changes in the oviposition behavior of Olive Fly females.
A protocol for a transposon-mediated mutagenesis approach for isolated Providencia bacteria has been developed to pave the way for functional genetics studies of the host-pathogen interaction. These studies will provide crucial data for the risk assessment of Providencia as potential biocontrol agent. A possible Olive Fly biocontrol strategy including Providencia bacteria has been drafted.

In order to exploit and disseminate project results beyond the funding period, further fundamental and applied research will be performed to bring about both Providencia-based bioinsecticide prototypes and the registration-relevant risk assessment. Results will be made public within the scientific community by means of scientific publications and congress presentations, to the relevant sectors of the society by means of participations in olive cultivator fairs and out-reach actions to schools and further education institutions.
Progress and results beyond the (scientific) state of the art
Fruit fly associated Providencia entomophila bacteria have for the first time been identified in Europe. This is important as the use of endemic microorganisms for microbial control purposes is under risk assessment aspects strongly preferred over the use of exotic micro-organisms as, e.g. those identified previously in Tunisia. Moreover, new Providencia isolates from Italy are not subject to third party property rights.
Moreover, at the fundamental research level, identification of new Providencia strains has given rise to a modification of the systematics of Providencia bacteria.
The first diagnostic protocol for the specific identification of Providencia entomophila bacteria in environmental samples has been developed and has fostered new specimen isolation.
For the first time, the effects of P. entomophila infection upon mortality and fecundity of Olive Flies have been consistently determined.
Innovative tools for genetic modification of Providencia bacteria have been established and tested with P. entomophila, enabling subsequent functional genetics and infection biology studies.

Socio-economic Impact:
The socio-economic impact of the project is mediated by the development of microbial control and integrated pest management strategies for the olive fly, i.e. a major economically relevant pest across Southern Europe, and potentially further fruit fly species including invasive and emerging pests.
Figure 1. Adult Olive Fly, Bactrocera oleae (A) and isolated Providencia bacterium (B).