Publications

4431

Candidatus Rickettsia colombiensis is a new candidate species of Rickettsiae spotted fever group that have been isolated only from ticks. The pathogenicity of Ca. R. colombiensis to human and animals is unknown. This study evaluated the pathogenic potential of Ca. R. colombiensis in Syrian hamsters and assessed the cross-reactivity between Ca. R. colombiensis and other Rickettsia in human and hamster sera. Shell vial technique was employed to isolate Ca. R. colombiensis. Subsequently, five male Syrian hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 106 Vero cells infected with Ca. R. colombiensis. One control hamster was used in each group. The health status was assessed daily, and necropsies were performed. Serum samples were tested by indirect immunofluorescence and tissues were processed by qPCR and histological stains. All Syrian hamsters remained healthy during the trial. No histopathological damages associated with rickettsial infection were observed. No Rickettsial DNA was detected in tissues. Syrian hamsters showed IgG antibody titers ranging from 1:64 to 1:1024. Control hamsters were negative. Regarding human sera, 56% (84/150) had IgG cross-reactivity antibodies against Ca. R. colombiensis. Subsequently, in a selected subset of 30 sera with moderate to high titers, all samples reacted with Ca. R. colombiensis antigen. Under specific conditions of this study, Ca. R. colombiensis did not behave as a highly virulent pathogen in the hamster model, although all infected Syrian hamsters developed IgG antibodies responses. Regarding cross-reactivity, it is possible to serologically diagnose rickettsial infection using Ca. R. colombiensis as an antigen.

Abstract Comparing laboratory and field experiments is crucial to understand the ecological relevance of insect behaviour. In particular the use of reared and wild insects in behavioural experiments should be carefully considered and decided upon. In this study, we investigated colour preferences of the pear psyllid Cacopsylla pyri for different green colours, using both reared and wild-caught individuals. C. pyri is the main vector for ‘ Candidatus Phytoplasma pyri’, which is a phloem-limited bacterium that causes pear decline, a disease that damages the fruit and negatively affects the tree’s fitness. To investigate, whether the colour of non-symptomatic pear leaves may differ in relation to infection, spectral reflectance measurements were conducted, revealing no consistent differences. Based on these measurements, sticky traps were designed to match the natural reflectance of leaves in three different green colours. Laboratory experiments with reared C. pyri showed no clear preference for any of the three colours, though males exhibited a slight tendency towards light green (532 nm). In contrast, wild-caught psyllids tested under identical laboratory conditions strongly preferred light green traps, regardless of sex. Field experiments did not confirm these preferences. Non-target arthropods exhibited different responses: medium green (541 nm) and dark green (551 nm) traps captured higher numbers, highlighting that trap colour influences bycatch too. Over time, seasonal dynamics showed a moderate shift in psyllid preference towards medium green over time, whereas other arthropods increasingly preferred light green. Our findings demonstrate that the visual behaviour of psyllids is finely tuned to specific green colours, but that individuals reared in a laboratory may not fully represent behaviour of wild animals. The strong agreement between wild-caught laboratory assays and field experiments underscores the ecological validity of a green colour with a wavelength peak of 532 nm as an attractive trap colour. This emphasises the importance of validating laboratory results under natural conditions.

Pear decline (PD), associated with ‘Candidatus Phytoplasma pyri’, is one of the most severe diseases affecting pear cultivation in Europe and the United States. Several psyllid species act as vectors of phytoplasmas belonging to the 16SrX group and play a key role in the epidemiology of the disease. This study aimed to characterize the epidemiology of pear decline in Sicily using integrated field, molecular, vector, and remote sensing approaches, four years after the first detection of PD in the region. Visual surveys and molecular analyses were conducted over two years in eight pear orchards. A total of 115 plant samples and 101 Cacopsylla spp. specimens, selected from 1435 collected individuals, were analysed, confirming the presence of ‘Ca. P. pyri’ in 69% of symptomatic plants and in 4.6% of C. pyri individuals. Genetic characterization revealed a high degree of similarity among the phytoplasma isolates analysed. Remote sensing analyses conducted since 2018, combined with vector population monitoring, confirmed the epidemic nature of PD and indicated the persistence of a risk of further pathogen spread within the region, supporting the use of remote sensing as a complementary tool for large-scale disease monitoring.

‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing, is transmitted by the Asian citrus psyllid Diaphorina citri. While CLas-positive (CLas+) females exhibit increased fecundity and metabolic demands, their neuroendocrine regulation remains unclear. We propose CLas manipulates dopamine (DA) signaling to enhance psyllid fecundity and CLas proliferation. Metabolomics revealed elevated DA in CLas+ females. Silencing DA synthesis genes and receptor DcDop2 via RNAi reduced lipid reserves, fecundity, and ovarian CLas titers. Through combined in vivo and in vitro experiments, we demonstrated that the microRNA miR-31a suppresses DcDop2 expression by binding to its 3’ untranslated region. Overexpression of miR-31a resulted in decreased DcDop2 expression and CLas titers in the ovaries, eliciting phenotypic defects akin to DcDop2 knockdown. Furthermore, DcDop2 knockdown and miR-31a overexpression reduced juvenile hormone (JH) levels and adipokinetic hormone (AKH) signaling in fat bodies and ovaries. Consequently, CLas hijacks the DA/DcDop2-miR-31a-AKH-JH signaling cascade to improve D. citri lipid metabolism and fecundity, while simultaneously promoting its replication. These findings suggest a mutualistic interaction between CLas proliferation and ovarian development within the insect host that enrich our understanding of the molecular interplay between plant pathogens and vector insects and offer novel targets and strategies for the field management of HLB.

‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing, is transmitted by the Asian citrus psyllid Diaphorina citri. While CLas-positive (CLas+) females exhibit increased fecundity and metabolic demands, their neuroendocrine regulation remains unclear. We propose CLas manipulates dopamine (DA) signaling to enhance psyllid fecundity and CLas proliferation. Metabolomics revealed elevated DA in CLas+ females. Silencing DA synthesis genes and receptor DcDop2 via RNAi reduced lipid reserves, fecundity, and ovarian CLas titers. Through combined in vivo and in vitro experiments, we demonstrated that the microRNA miR-31a suppresses DcDop2 expression by binding to its 3’ untranslated region. Overexpression of miR-31a resulted in decreased DcDop2 expression and CLas titers in the ovaries, eliciting phenotypic defects akin to DcDop2 knockdown. Furthermore, DcDop2 knockdown and miR-31a overexpression reduced juvenile hormone (JH) levels and adipokinetic hormone (AKH) signaling in fat bodies and ovaries. Consequently, CLas hijacks the DA/DcDop2-miR-31a-AKH-JH signaling cascade to improve D. citri lipid metabolism and fecundity, while simultaneously promoting its replication. These findings suggest a mutualistic interaction between CLas proliferation and ovarian development within the insect host that enrich our understanding of the molecular interplay between plant pathogens and vector insects and offer novel targets and strategies for the field management of HLB.

Candidatus Liberibacter spp. can infect most citrus plants and rely entirely on phloem sieve tube cells of the host plant for survival. Candidatus Liberibacter primarily contains Ca. L. asiaticus (CLas), Ca. L. africanus (CLaf), and Ca. L. americanus (CLam). Among these, CLas is the most harmful and widely distributed and is the primary pathogen of the devastating citrus disease Huanglongbing (HLB). Effectors are among the core weapons secreted by pathogens into plant cells to attack the plant immune system. In this study, we focused on CLas-specific effectors and those that are highly expressed during the infection stage to identify essential virulence effectors. Using secretion signal peptide prediction analysis, 40 candidate effectors with potential secretory capabilities were identified. Transient expression of these candidate effectors in Nicotiana benthamiana revealed their impact on pattern-triggered immunity, including INF-induced cell death and microbial pattern-induced reactive oxygen species (ROS) bursts, and the resistance of N. benthamiana to the bacterial pathogen Pst DC3000. 10 candidate effectors capable of suppressing plant immunity were identified. The stable expression of these candidate effectors in Arabidopsis showed that several candidate effectors enhanced plant susceptibility to Pst DC3000 and inhibited flg22-induced ROS production and MAPK activation. Among the three candidate effectors that significantly suppressed ROS burst, one effector, E3 (CLIBASIA_03085), interacts with the plant NADPH oxidase RbohD, a key enzyme responsible for ROS production. This suggests that E3 likely inhibits ROS accumulation by directly targeting RbohD. Here, we identified multiple candidate effectors capable of suppressing microbial pattern-triggered immunity that may be essential virulence factors for CLas infection, enhancing our understanding of CLas pathogenesis.