Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.

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Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC-and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

EFSA was asked to update the 2015 EFSA risk assessment on Xylella fastidiosa for the territory of the EU. In particular, EFSA was asked to focus on potential establishment, short-and long-range spread, the length of the asymptomatic period, the impact of X. fastidiosa and an update on risk reduction options. EFSA was asked to take into account the different subspecies and Sequence Types of X. fastidiosa. This was attempted throughout the scientific opinion but several issues with data availability meant that this could only be partially achieved. Models for risk of establishment showed most of the EU territory may be potentially suitable for X. fastidiosa although southern EU is most at risk. Differences in estimated areas of potential establishment were evident among X. fastidiosa subspecies, particularly X. fastidiosa subsp. multiplex which demonstrated areas of potential establishment further north in the EU. The model of establishment could be used to develop targeted surveys by Member States. The asymptomatic period of X. fastidiosa varied significantly for different host and pathogen subspecies combinations, for example from a median of approximately 1 month in ornamental plants and up to 10 months in olive, for pauca. This variable and long asymptomatic period is a considerable limitation to successful detection and control, particularly where surveillance is based on visual inspection. Modelling suggested that local eradication (e.g. within orchards) is possible, providing sampling intensity is sufficient for early detection and effective control measures are implemented swiftly (e.g. within 30 days). Modelling of long-range spread (e.g. regional scale) demonstrated the important role of long-range dispersal and the need to better understand this. Reducing buffer zone width in both containment and eradication scenarios increased the area infected. Intensive surveillance for early detection, and consequent plant removal, of new outbreaks is crucial for both successful eradication and containment at the regional scale, in addition to effective vector control. The assessment of impacts indicated that almond and Citrus spp. were at lower impact on yield compared to olive. Although the lowest impact was estimated for grapevine, and the highest for olive, this was based on several assumptions including that the assessment considered only Philaenus spumarius as a vector. If other xylem-feeding insects act as vectors the impact could be different. Since the Scientific Opinion published in 2015, there are still no risk reduction options that can remove the bacterium from the plant in open field conditions. Short-and long-range spread modelling showed that an early detection and rapid application of phytosanitary measures, consisting among others of plant removal and vector control, are essential to prevent further spread of the pathogen to new areas. Further data collection will allow a reduction in uncertainty and facilitate more tailored and effective control given the intraspecific diversity of X. fastidiosa an...

Virus infection frequently modifies plant phenotypes, leading to changes in behaviour and performance of their insect vectors in a way that transmission is enhanced, although this may not always be the case. Here, we investigated Bemisia tabaci response to tomato plants infected by Tomato chlorosis virus (ToCV), a non-circulative-transmitted crinivirus, and Tomato severe rugose virus (ToSRV), a circulative-transmitted begomovirus. Moreover, we examined the role of visual and olfactory cues in host plant selection by both viruliferous and non-viruliferous B. tabaci. Visual cues alone were assessed as targets for whitefly landing by placing leaves underneath a Plexiglas plate. A dual-choice arena was used to assess whitefly response to virus-infected and mock-inoculated tomato leaves under light and dark conditions. Thereafter, we tested the whitefly response to volatiles using an active air-flow Y-tube olfactometer, and chemically characterized the blends using gas chromatography coupled to mass spectrometry. Visual stimuli tests showed that whiteflies, irrespective of their infectious status, always preferred to land on virus-infected rather than on mock-inoculated leaves. Furthermore, whiteflies had no preference for either virus-infected or mock-inoculated leaves under dark conditions, but preferred virus-infected leaves in the presence of light. ToSRV-infection promoted a sharp decline in the concentration of some tomato volatiles, while an increase in the emission of some terpenes after ToCV infection was found. ToSRV-viruliferous whiteflies preferred volatiles emitted from mock-inoculated plants, a conducive behaviour to enhance virus spread, while volatiles from ToCV-infected plants were avoided by non-viruliferous whiteflies, a behaviour that is likely detrimental to the secondary spread of the virus. In conclusion, the circulative persistent begomovirus, ToSRV, seems to have evolved together with its vector B. tabaci to optimise its own spread. However, this type of virus-induced manipulation of vector behaviour was not observed for the semi persistent crinivirus, ToCV, which is not specifically transmitted by B. tabaci and has a much less intimate virus-vector relationship.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

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