Epidemiological role of Orientus ishidae in Apple Proliferation and implications for sustainable orchard management

Phytoplasmas are obligate, phloem-limited plant pathogenic bacteria responsible for numerous economically damaging diseases worldwide. Among phytoplasma-associated diseases, Apple Proliferation (AP), caused by 'Candidatus Phytoplasma mali' (16SrX-A), represents a major threat to apple production in Europe, particularly in Trentino (Northern Italy), where it induces typical symptoms, such as witches' broom formation, enlarged stipules and the production of small fruits, leading to severe losses in both yield and income. Since 2019, a renewed AP outbreak has been recorded in Trentino, despite the fact that monitored populations of the confirmed psyllid vectors Cacopsylla picta and C. melanoneura remained low and unevenly distributed across the region, population levels that, based on historical epidemiological data, would not be expected to sustain the observed disease incidence. This discrepancy raised the hypothesis that one or more additional vector species might be contributing to AP spread. Concurrently, unusually high populations of the invasive mosaic leafhopper Orientus ishidae were detected in apple orchards throughout the region. This East Asian species, now widespread in Europe, is a polyphagous phloem-feeder and a confirmed vector of the 16SrV-C and D phytoplasmas, causal agents of Flavescence dorée (FD) disease. While the molecular detection of 'Ca. P. mali' in field-collected specimens, coupled with the concurrent increase in both AP incidence and O. ishidae population levels, strengthened the hypothesis of its involvement in AP epidemiology, no experimental evidence on its actual transmission competence existed, leaving its AP epidemiological role entirely unresolved. Beyond the transmission question, several fundamental aspects of the biology and landscape ecology of O. ishidae in northern Italian agroecosystems remained unaddressed: its diffusion and population dynamics in apple orchards, its ability to complete development on apple, and whether orchard populations could function as epidemiological bridges toward adjacent vineyards. Notably, the suitability of cultivated grapevine as a developmental host had never been experimentally assessed, a gap that left unexplained the consistently low abundance of O. ishidae in vineyards despite its confirmed FD vector status, and that carries direct implications for FD risk assessment in mixed apple–vineyard landscapes. Monitoring conducted across northern Italy (2022–2023) revealed that O. ishidae is widespread and abundant in apple orchards, particularly under organic management, reaching mean weekly captures of up to 8.4 individuals per trap. Field-collected individuals were screened by PCR, with 16–18% testing positive for ‘Ca. P. mali’, confirming natural acquisition. Laboratory and semi-field trials demonstrated that apple is a suitable reproductive host: nymphs completed development within 25–30 days and adults survived 23–28 days, while feeding activity induced characteristic yellowing symptoms affecting up to 23% of leaves. Transmission experiments (2022–2024) were conducted using different phytoplasma strains (AT1, AT2), acquisition access periods, and inoculum sources. Although 4–22% of insects tested positive after exposure, qPCR quantification revealed extremely low phytoplasma titers, and no transmission to healthy test plants occurred. Infection dynamics experiments showed a progressive decline in phytoplasma detection following acquisition, with positivity decreasing from 85% immediately after a 5-day acquisition period to 0% after 15 latent days, indicating transient passive acquisition rather than propagative infection. These results demonstrate that O. ishidae is not a competent AP vector and functions as a dead-end host for ‘Ca. P. mali’. Given the spatial interspersion of apple orchards and vineyards in Trentino, additional experiments evaluated the species’ potential role in FD epidemiology. Survival assays testing nymphs development and adult survival on different host plant revealed that cultivated grapevine is an unsuitable host: no nymphs completed development, and adult median survival ranged from 1–3 days across cultivars, compared to 23–28 days on apple. Field transect studies at apple–vineyard interfaces showed that adult abundance declined by an order of magnitude beyond orchard boundaries, indicating limited spillover. Collectively, these findings identify apple orchards as demographic sources while demonstrating that host plant constraints restrict establishment in vineyards, thereby limiting the epidemiological relevance of O. ishidae in mixed agricultural landscapes. Since O. ishidae was excluded as an AP vector, attention was redirected to quantifying the economic implications of the epidemic and evaluating management strategies for confirmed epidemiological drivers. To quantify the practical implications of these findings, a 20-year partial budget analysis was conducted to estimate the economic impact of AP and evaluate alternative management strategies. Results demonstrated that the unmanaged scenario caused the greatest economic damage, with gross return reductions approaching 30%. Removal of symptomatic trees followed by systematic replacing proved most effective despite higher short-term costs, reducing net income losses to -2.1% (IPM) and -4.0% (organic) compared to -2.9% and -6.0% for removal without replacing. Sensitivity analyses revealed that profitability was more responsive to epidemiological than economic parameters, emphasizing that improved disease monitoring and modeling would enhance decision-making more effectively than refined cost estimates. In conclusion, this work demonstrates that O. ishidae, despite its abundance in apple orchards and ability to acquire 'Ca. P. mali', does not function as an AP vector and plays no significant role in AP epidemiology in Trentino. Therefore, further research should focus on its FD transmission risk, while AP management remains centered on vector control, tree removal, and the long-term research objective of developing resistant rootstocks.