DC-electrical penetration graph waveforms for Dalbulus maidis (Hemiptera: Cicadellidae) and the effects of entomopathogenic fungi on its probing behavior.

The corn leafhopper Dalbulus maidis is an important transmitter of viruses and bacteria to maize plants. Data on the stylet probing and ingestion behavior of D. maidis, gathered using the DC-electrical penetration graph (DC-EPG) technique, are limited. The increasing prevalence of this pest and the diseases that it transmits in maize crops heighten the importance of studying how control tools affect the probing behavior of these insects, to reduce or prevent the chances of pathogen transmission and the damage from feeding. Our study recorded stylet activities of D. maidis, using a DC-EPG system and compared the appearances of waveforms with those from published AC and AC-DC EPG studies. All types of systems produced similar waveform appearances; therefore, we used the waveform nomenclature previously published. We also determined the effects of the entomopathogenic fungus Cordyceps javanica on the probing behavior of D. maidis at different time points after the fungus was applied by spraying. Forty-eight hours after the insects were sprayed, the effects were pronounced, with significant disruption of the stylet activities in phloem and non-phloem phases. Our study indicated that this commercial microbiological product, with the active ingredient C. javanica, can alter the probing behavior of D. maidis and may be helpful in management of the vector.

Yellow Leaf Disease Resistance and Melanaphis sacchari Preference in Commercial Sugarcane Cultivars.

Sugarcane yellow leaf disease (YLD) caused by sugarcane yellow leaf virus (ScYLV) is a major threat for the sugarcane industry worldwide, and the aphid Melanaphis sacchari is its main vector. Breeding programs in Brazil have provided cultivars with intermediate resistance to ScYLV, whereas the incidence of ScYLV has been underestimated partly due to the complexity of YLD symptom expression and identification. Here, we evaluated YLD symptoms in a field assay using eight sugarcane genotypes comprising six well-established commercial high-sucrose cultivars, one biomass yield cultivar, and a susceptible reference under greenhouse conditions, along with estimation of virus titer through RT-qPCR from leaf samples. Additionally, a free-choice bioassay was used to determine the number of aphids feeding on the SCYLV-infected cultivars. Most of the cultivars showed some degree of resistance to YLD, while also revealing positive RT-qPCR results for ScYLV and virus titers with non-significant correlation with YLD severity. The cultivars IACSP01-5503 and IACBIO-266 were similar in terms of aphid preference and ScYLV resistance traits, whereas the least preferred cultivar by M. sacchari, IACSP96-7569, showed intermediate symptoms but similar virus titer to the susceptible reference, SP71-6163. We conclude that current genetic resistance incorporated into sugarcane commercial cultivars does not effectively prevent the spread of ScYLV by its main aphid vector.

Genetic transformation of 'Hamlin' and 'Valencia' sweet orange plants expressing the cry11A gene of Bacillus thuringiensis as an additional tool for the management of Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter spp., the bacteria associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. HLB management has heavily relied on insecticide applications to control the ACP, although there are efforts towards more sustainable alternatives. In previous work, our group assessed the potential bioactivity of different strains of Bacillus thuringiensis (Eubacteriales: Bacillaceae) (Bt) containing cry/cyt genes as feasible tools to control ACP nymphs. Here, we report an attempt to use the cry11A gene from Bt to produce transgenic sweet orange plants using two promoters. For the genetic transformation, 'Hamlin' and 'Valencia' sweet orange seedlings were used as sources of explants. Transgenic plants were detected by polymerase chain reaction (PCR) with specific primers, and the transgene copy number was confirmed by Southern blot analyses. Transcript expression levels were determined by qPCR. Mortality assays of D. citri nymphs were carried out in a greenhouse, and the effect of the events tested ranged from 22% to 43% at the end of the five-day exposure period. To our knowledge, this is the first manuscript reporting the production of citrus plants expressing the Bt cry11A gene for the management of D. citri nymphs.

Development on Infected Citrus over Generations Increases Vector Infection by 'Candidatus Liberibacter Asiaticus in Diaphorina citri'.

'Candidatus Liberibacter asiaticus' (CLas) is a major causal agent of citrus Huanglongbing (HLB), which is transmitted by Asian citrus psyllid (ACP), Diaphorina citri, causing severe losses in various regions of the world. Vector efficiency is higher when acquisition occurs by ACP immature stages and over longer feeding periods. In this context, our goal was to evaluate the progression of CLas population and infection rate over four ACP generations that continuously developed on infected citrus plants. We showed that the frequency of CLas-positive adult samples increased from 42% in the parental generation to 100% in the fourth generation developing on CLas-infected citrus. The bacterial population in the vector also increased over generations. This information reinforces the importance of HLB management strategies, such as vector control and eradication of diseased citrus trees, to avoid the development of CLas-infected ACP generations with higher bacterial loads and, likely, a higher probability of spreading the pathogen in citrus orchards.

Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory.

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 and wide host range.

Stylet penetration activities of the whitefly Bemisia tabaci associated with inoculation of the crinivirus Tomato chlorosis virus.

Bemisiatabaci is an important vector of numerous plant viruses, including the emergent semi-persistently transmitted crinivirus Tomato chlorosis virus (ToCV). Its vector feeding behaviour is complex, with important implications for virus transmission, epidemiology and control. Thus, the objective of this study was to investigate the role of the stylet penetration activities of B. tabaci in the inoculation of ToCV in tomatoes by using the electrical penetration graph (EPG) technique. EPG recordings were classified into six categories depending on the waveforms observed. The results showed that ToCV inoculation is mainly associated with stylet activities in phloem sieve elements (E1 waveform), as there was a significant increase in the rate of transmission when whiteflies performed waveform E1. The precise stylet activities - either salivation or egestion - associated with virion release, presumably from retention sites in the foregut, need further investigation.