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.

Entomopathogenic fungus disrupts the phloem-probing behavior of Diaphorina citri and may be an important biological control tool in citrus.

Citrus is among the most important fruit crops worldwide; however, numerous pests and diseases affect the orchards, increasing production costs. The psyllid Diaphorina citri, is a vector of the phloem-limited bacteria 'Candidatus Liberibacter spp.', the causal agent of Huanglongbing (HLB) disease. The lack of a cure for HLB requires management of the vector, mainly by intensive use of chemical insecticides, leading to the selection of resistant populations. Our study determined the effects of the entomopathogenic fungus Cordyceps fumosorosea on the probing behavior of D. citri at different time points after the fungus was applied by spraying. The electrical penetration graph technique was used to monitor the stylet activities of D. citri after application of the microbiological product. The effects were more pronounced between 30 and 96 h after the insects were sprayed, with significant disruption of the stylet activities related to the phloem and directly associated with the transmission of HLB. Our study indicated that the microbiological product Challenger®, with the active ingredient C. fumosorosea fungus, can significantly change the probing behavior of D. citri, may be helpful in more-sustainable management of the vector, and can be used to reduce the spread of HLB.

Effect of Sugarcane Cultivars Infected with Sugarcane Yellow Leaf Virus (ScYLV) on Feeding Behavior and Biological Performance of Melanaphis sacchari (Hemiptera: Aphididae).

Sugarcane yellow leaf virus (ScYLV), Polerovirus, Luteoviridae, is one of the main viruses that infect sugarcane worldwide. The virus is transmitted by the aphid Melanaphis sacchari in a persistent, circulative manner. To better understand the interactions between ScYLV, sugarcane genotypes and M. sacchari, we explored the effect of sugarcane cultivars on the feeding behavior and biological performance of the vector. The number of nymphs, adults, winged, total number of aphids and dead aphids was assayed, and an electrical penetration graph (EPG) was used to monitor the stylet activities. Multivariate analysis showed changes in the vector's behavior and biology on cultivars, identifying specific groups of resistance. In the cultivar 7569, only 5.5% of the insects were able to stay longer on sustained phloem ingestion, while in the other seven cultivars these values varied from 20% to 60%. M. sacchari showed low phloem activities in cultivars 7569 and Bio266. Overall, cultivar 7569 showed the worst biological performance of aphids, with the insects presenting mechanical difficulties for feeding and a shorter duration of the phloem period, and thus being considered the most resistant. We conclude that ScYLV virus infection in different sugarcane cultivars induced specific changes in the host plant, modifying the behavior of its main vector, which may favor or impair virus transmission.

Foliar Spraying of Tomato Plants with Systemic Insecticides: Effects on Feeding Behavior, Mortality and Oviposition of Bemisia tabaci (Hemiptera: Aleyrodidae) and Inoculation Efficiency of Tomato Chlorosis Virus.

Tomato chlorosis virus (ToCV) is a phloem-limited crinivirus transmitted by whiteflies and seriously affects tomato crops worldwide. As with most vector-borne viral diseases, no cure is available, and the virus is managed primarily by the control of the vector. This study determined the effects of the foliar spraying with the insecticides, acetamiprid, flupyradifurone and cyantraniliprole, on the feeding behavior, mortality, oviposition and transmission efficiency of ToCV by B. tabaci MEAM1 in tomato plants. To evaluate mortality, oviposition and ToCV transmission in greenhouse conditions, viruliferous whiteflies were released on insecticide-treated plants at different time points (3, 24 and 72 h; 7 and 14 days) after spraying. Insect mortality was higher on plants treated with insecticides; however, only cyantraniliprole and flupyradifurone differed from them in all time points. The electrical penetration graph (DC-EPG) technique was used to monitor stylet activities of viruliferous B. tabaci in tomato plants 72 h after insecticide application. Only flupyradifurone affected the stylet activities of B. tabaci, reducing the number and duration of intracellular punctures (pd) and ingestion of phloem sap (E2), a behavior that possibly resulted in the lower percentage of ToCV transmission in this treatment (0-60%) in relation to the control treatment (60-90%) over the periods evaluated. Our results indicate that flupyradifurone may contribute to management of this pest and ToCV in tomato crops.

Gene silencing of Diaphorina citri candidate effectors promotes changes in feeding behaviors.

Insect effectors are mainly secreted by salivary glands, modulate plant physiology and favor the establishment and transmission of pathogens. Feeding is the principal vehicle of transmission of Candidatus Liberibacter asiaticus (Ca. Las) by the Asian citrus psyllid (ACP), Diaphorina citri. This study aimed to predict putative ACP effectors that may act on the Huanglongbing (HLB) pathosystem. Bioinformatics analysis led to the identification of 131 candidate effectors. Gene expression investigations were performed to select genes that were overexpressed in the ACP head and modulated by Ca. Las. To evaluate the actions of candidate effectors on D. citri feeding, six effectors were selected for gene silencing bioassays. Double-stranded RNAs (dsRNAs) of the target genes were delivered to D. citri adults via artificial diets for five days. RNAi silencing caused a reduction in the ACP lifespan and decreased the salivary sheath size and honeydew production. Moreover, after dsRNA delivery of the target genes using artificial diet, the feeding behaviors of the insects were evaluated on young leaves from citrus seedlings. These analyses proved that knockdown of D. citri effectors also interfered with ACP feeding abilities in planta, causing a decrease in honeydew production and reducing ACP survival. Electrical penetration graph (EPG) analysis confirmed the actions of the effectors on D. citri feeding behaviors. These results indicate that gene silencing of D. citri effectors may cause changes in D. citri feeding behaviors and could potentially be used for ACP control.

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.

Tomato Infection by Whitefly-Transmitted Circulative and Non-Circulative Viruses Induce Contrasting Changes in Plant Volatiles and Vector Behaviour.

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.