Book

of Abstracts

Organized by ICA-CSIC CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS INSTITUTO DE CIENCIAS AGRARIAS MADRID, SPAIN

Organizing Committee Alberto Fereres (Chairman, ICA-CSIC, Madrid, Spain) [email protected] Aránzazu Moreno (ICA-CSIC, Madrid, Spain) [email protected] Elisa Garzo (ICA-CSIC, Madrid, Spain) [email protected] María Plaza (ICA-CSIC, Madrid, Spain) [email protected] Francisco Ruiz-Raya (University of Granada, Spain) [email protected]

Scientific Committee Alberto Fereres (ICA-CSIC, Madrid, Spain) [email protected] Gregory Walker (University of California, Riverside, USA) [email protected] Saskia Hogenhout (John Innes Centre, Norwich, UK) [email protected] Joao Spotti Lopes (ESALQ/University of São Paulo, Brazil) [email protected] Phyllis Weintraub (ARO-Gilat Research Center, Israel) [email protected]

2 |

DONOR ACKNOLEDGEMENT

THANK YOUR FOR HPIS 2017 SUPPORT

HPIS 2017 Program 4 | PROGRAM

HPIS 2017 Program

The HPIS 2017 venue will be the CSIC main campus. The Oral Sessions will be held at the Auditorium located at the CSIC Main Building, Calle Serrano 117, 28006, Madrid, Spain.

Sunday 4th, June

• 7:00 PM - 10:00 PM: Registration & Welcome Reception at Residencia de Estudiantes. Calle del Pinar 21. Madrid. 2 min-walking distance from CSIC Main Building. https://goo.gl/maps/yj8NmRMZLGo

Monday 5th to Wednesday 7th, June - Scientific Sessions

• ORAL SESSIONS: CSIC Main Building Auditorium. C/ Serrano 117 • POSTERS & COFFEE BREAKS: CSIC Atrium located at the Instituto de Física Fundamental. C/Serrano 113b (2 min walk from Auditorium) • LUNCH: Divided in two groups: CSIC restaurant located in Main Building & Instituto de Química Orgánica (C/Serrano 150, 5 min walk from Auditorium)

Monday 5th, June

7:30-8:45 AM. Registration (continued)

8:45-9:00 OPEN CEREMONY Emilio Lora-Tamayo, President of the Consejo Superior de Investigaciones Científicas, CSIC; Dª Marina Villegas, Director of the National Research Agency (AEI) and Alberto Fereres, Chairman of the HPIS 2017 Organizing Committee.

09:00-10:45 ORAL PRESENTATIONS

Session 1. Hemipteran Systematics & Genomics

Session Chairs: Joao Spotti Lopes, Michael Wilson

9:00-9:30. Keynote Lecture Michael Wilson. Hemiptera and their vectors: the known and the unknowns

9:30-9:45. Arnold Gegechkori. Psyllids (Hemiptera, Psylloidea) as phloem feeding pests of agriculture crops of the Caucasus 9:45-10:00. Thomas Mathers. Sequencing two aphid genera: comparative genomics of Myzus and Brachycaudus to infer the genomic basis of generalism PROGRAM | 5

10:00-10:15. Shai Morin. Unravelling the role of Bemisia tabaci - plant associations in the species diversification 10:15-10:30. Jennifer Sjölund. The identification and detection of psyllid vectors of Candidatus Liberibacter solanacearum in suction traps 10:30-10:45. Anna Maria Botha. Is methylation one of the drivers of virulence in Diuraphis noxia (Kurd.) Hemiptera: Aphididae?

10:45-11:15. Coffee Break & Poster Exhibit

11:15-12:30 ORAL PRESENTATIONS

Session 2. Xylem Feeders

Session Chairs: Sandy Purcell, Rodrigo Almeida

11:15-11:45. Keynote Lecture Rodrigo Almeida. Is the role of vector choice on disease epidemiology as simple as it appears to be?

11:45-12:00. Adam Zeilinger. Epidemiology of novel defensive traits against Xylella fastidiosa in grapevines 12:00-12:15. Domenico Bosco. Host-plant association and host-shifting of nymphs and adults of Philaenus spumarius L. in Italian olive orchards 12:15-12:30. Miguel Ángel Miranda. Surveillance and identification of potential vectors of Xylella fastidiosa in Mallorca (Islas Baleares, Spain) after 2016 outbreak

12:30-14:30. Lunch and Poster Exhibit

14:30-16:00. ORAL PRESENTATIONS

Session 3. Phloem & Mesophyll Feeders

Session Chairs: Gregory Walker, Shu-Sheng Liu

14:30-15:00. Keynote Lecture Shu-Sheng Liu. Plant-mediated interspecific competition between whiteflies

15:00-15:15. Jaime Jiménez. Newly distinguished potential drops are related to phloem cell punctures associated to the inoculation of closterovirus by aphids 15:15-15:30. Freddy Tjallingii. Compatible/incompatible aphid-plant interactions 15:30-15:45. Gregory Walker. The role of sieve element occlusion proteins in aphid resistance 15:45-16:00. Jirapong Jairin. Loss of resistance to Nilaparvata lugens (Hemiptera: Delphacidae) may be due to low-level expression of Bph32 in rice panicles at heading stage

16:00-16:30. Coffee Break

16:30-18:30. ORAL PRESENTATIONS 6 | PROGRAM

Session 3. (continued) Phloem & Mesophyll Feeders

16:30-16:45. Kenneth Webster. Grapevine secondary metabolites involved in insect resistance 16:45-17:00. Taciana M.A. Kuhn. Settling and oviposition preferences of Russelliana solanicola Tuthill (Hemiptera: Psyllidae) among different cultivated and spontaneous plants 17:00-17:15. Elaine Backus. Chemical evidence of inducible tannins as plant defense response to Lygus lineolaris feeding on cotton squares: correlation with EPG waveforms 17:15-17:30. Zhiwei Kang. Infection of powdery mildew (Blumeria graminis) reduces the fitness of grain aphids (Sitobion avenae) through the restricted nutrition and induced defense response in wheat 17:30-17:45. Marcelo G. Santos. Leaf preference of the scale insects on fern species Asplenium serratum L.

Session 4. Hemiptera-Simbiont Interactions

Session Chairs: Murad Ghanim, David Martínez-Torres

17:45-18:00. David Martínez-Torres. Mutualism turns into aggressive mimicry in an aphid 18:00-18:15. Inès Pons Guillouard. Horizontal transmission of insect symbiont Serratia symbiotica via phloem and new insight into the acquisition of the symbiont 18:15-18:30. Jacqueline Morris. Microflora analyses of the Australian eggplant psyllid, Acizzia solanicola

18:30-19:30. POSTER SESSION 1 (odd numbers, w/presenters)

Tuesday 6th, June

8:30-10:30 ORAL PRESENTATIONS

Session 5. Hemiptera-Plant Pathogen Interactions

Session Chairs: Alberto Fereres, Jesús Navas-Castillo

8:30-9:00. Keynote Lecture Jesús Navas-Castillo. Differential transmission of criniviruses and begomoviruses by whiteflies of the Bemisia tabaci complex

9:00-9:15. Lilong Pan. Clathrin-mediated endocytosis is involved in Tomato yellow leaf curl virus transport across the midgut barrier of its whitefly vector 9:15-9:30. María Luisa Domingo-Calap. Host-vector-pathogen interactions in the context of mixed infections by hemipteran-transmitted viruses 9:30-9:45. Christiane Then. The N-terminus of the Cauliflower mosaic virus aphid transmission protein P2 is involved in both transmission and microtubule interaction PROGRAM | 7

9:45-10:00. Nilsa Bosque-Pérez. Drought stress facilitates conditional mutualism in a plant-virus- vector interaction 10:00-10:15. Veronique Brault. Is viral transmission by aphids enhanced upon manipulation of host Brassicaceae by Turnip yellows virus? 10:15-10:30. Torsten Will. Effects of Barley yellow dwarf virus infection on the interaction of virus susceptible and tolerant barley genotypes with different clones of the aphid species Rhopalosiphum padi

10:30-11:00. Coffee Break

11:00-12:30 ORAL PRESENTATIONS

11:00-11:15. Micky Eubanks. The effect of ant-aphid mutualisms and plant diversity on the spread of aphid-vectored plant viruses 11:15-11:30. Piotr Trebicki. Elevated CO2 impacts aphid and plant physiology, virus incidence and interactions between them 11:30-11:45. Xifeng Wang. Understanding the transmission mechanism of Rice stripe virus by its insect vector Laodelphax striatellus 11:45-12:00. Emmanuel Jacquot. Inoculation of Wheat dwarf virus strains to wheat and barley plants by Psammotettix alienus 12:00-12:15. Cristina Marzachì. Molecular characterization of phytoplasmas to trace the epidemiological routes of Flavescence dorée between Vitis spp. and the leafhopper vector 12:15-12:30. Adi Kliot. Do phytoplasma effectors target specific processes in insect vectors?

12:30-14:30. Lunch and Poster Exhibit

14:30-15:30. ORAL PRESENTATIONS

14:30-14:45. Simon Elliot. Invasive mutualisms between a plant pathogen and insect vectors in the Middle East and Brazil 14:45-15:00. Ofir Bahar. Genetic and metabolic analyses of Candidatus Liberibacter solanacearum infecting carrot 15:00-15:15. Murad Ghanim. New insights into psyllid-Liberibacter interactions 15:15-15:30. Judith Brown. Psyllid-Ca.Liberibacter interactions at molecular and cellular interfaces

15:30-16:00. Coffee Break

16:00-18:00. ORAL PRESENTATIONS

Session 6. Insect-Plant Dialog

Session Chairs: Saskia Hogenhout, Christine Coustau

16:00-16:30. Keynote Lecture Christine Coustau. Role of MIF immune regulators in plant-aphid interactions

16:30-16:45. Dganit Sadeh. Chemical and behavioral analysis of the whitefly Bemisia tabaci attraction to rosemary (Rosmarinus officinalis) 8 | PROGRAM

16:45-17:00. Grit Kunert. Modulation of legume defense signaling pathways by native and non- native pea aphid clones 17:00-17:15. Vered Tzin. Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar-induced aphid resistance in maize 17:15-17:30. Jia Fan. Identification and expression analysis of candidate odorant-receptors (ORs) by antennal transcriptome of Sitobion avenae 17:30-17:45. Teresa Vaello. Linking plant-soil feedbacks to several trophic levels in sweet pepper- aphid system 17:45-18:00. Sam Mugford. An aphid effector protein localizes to the cytoplasm of mesophyll cells and suppresses the first layer of the plant defence response

18:00-19:00. POSTER SESSION 2 (even numbers w/presenters)

Wednesday 7th, June

8:30-10:30 ORAL PRESENTATIONS

Session 6. (continued) Insect-Plant Dialog

8:30-8:45. Karen Kloth. On the verge of a hot, juicy dinner: A heat-inducible phloem protein that compromises aphid feeding 8:45-9:00. Agnieszka Woźniak. Nitric oxide as a signaling molecule in bioactive pea response to pea aphid infestation 9:00-9:15. Jorunn Bos. An aphid effector associates with a host trafficking protein to promote virulence 9:15-9:30. Lidia Blanco. Characterization and effect of glandular trichomes on plant aphid interactions in tomato 9:30-9:45. Colin Turnbull. Genetics of virulence in pea aphid on Medicago truncatula hosts differing in R gene complement 9:45-10:00. Eduard Venter. A wheat specific integrated-domain NLR gene as role player in the wheat-Diuraphis noxia interaction 10:00-10:15. Petra Bleeker. Small RNAs from whiteflies are transferred to tomato phloem during feeding 10:15-10:30. Osnat Malka. Metabolic responses to variable host plants play a key role in different degrees of polyphagy in the Bemisia tabaci species complex

10:30-11:00. Coffee Break

PROGRAM | 9

11:00-12:30 ORAL PRESENTATIONS

Session 7. Pest/Disease Management

Session Chairs: Phyllis Weintraub, Michael Rogers

11:00-11:30. Keynote Lecture Michael Rogers. Management of the Asian Citrus Psyllid and Citrus Greening Disease

11:30-11:45. Marcelo Miranda. Kaolin affecting the host plant finding ability of Diaphorina citri in field conditions 11:45-12:00. Jessica Dohmen-Vereijssen. Ecology and management of Bactericera cockerelli in potato crops 12:00-12:15. Silvia Rondón. Zebra chip in Oregon and Washington: a retrospective towards managing the disease 12:15-12:30. Yupa Hanboonsong. Evaluation of sugarcane varieties for differential resistance to insect vector of sugarcane white leaf disease

12:30-14:30. Lunch and Poster Exhibit (all Posters should be removed at 14:30)

14:30-16:00. ORAL PRESENTATIONS

14:30-14:45. Gary A. Thompson. Transcriptomics of altered aphid phenotype on Vat+ resistant plants 14:45-15:00. Adriana Álvarez. Searching for strategies to control the green peach aphid on potato crops 15:00-15:15. Rajagopalbabu Srinivasan. The effects of host resistance on whitefly-Tomato yellow leaf curl virus interactions: Implications for viral epidemics and management 15:15-15:30. Alvin Simmons. Whiteflies: Developing host plant resistance in watermelon from wild sources 15:30-15:45. Patricia Prade. Calophya terebinthifolii and Calophya lutea: Host specificity of two potential biological control agents of Brazilian peppertree in Florida, USA 15:45-16:00. Ikponmwosa Egbon. The genotype of Pereskia aculeata (Cactaceae) influences performance of its biological control agent, Catorhintha schaffneri (Coreidae) in South Africa

16:00-16:30. Coffee Break

16:30-18:00. ORAL PRESENTATIONS

16:30-16:45. Daniel Leybourne. Investigating plant-aphid-environment interactions: Rhopalosiphum padi –barley interactions under plant water stress 16:45-17:00. Imre Mezei. Isoclast Active as a new tool for managing virus vectors and virus transmission 17:00-17:15. Un Taek Lim. Laboratory evaluation of a mycoinsecticide (BotaniGard® ES) on Riptortus pedestris (Hemiptera: Alydidae) 10 | PROGRAM

17:15-17:30. Paola Riolo. Behavioural responses of Hyalesthes obsoletus to volatiles of plants sprayed with resistance inducers 17:30-17:45. Tatiane da Cunha. Bacillus thuringiensis translocation inside citrus plants and insecticidal activity against Diaphorina citri, vector of HLB causal agents 17:45-18:00. Ken Tabuchi. Effect of the amount of source habitat on the rice damage caused by a mirid pest Stenotus rubrovittatus: constructing a spatial predictive model of rice damage and the potential hazard map using land use data

20:30. CLOSING BANQUET

Thursday 8th to Saturday 10th (optional)

1. Satellite EPG Workshop. Venue: Instituto de Ciencias Agrarias. ICA-CSIC. Calle Serrano 115 duplicado. https://goo.gl/maps/na6nSmzvhv72

2. Optional Touristic tours to Toledo, Segovia, Cordoba and Granada are available at: http://www.hpis2017.csic.es/accommodationtours/

CSIC Campus & main building CSIC main auditorium Atrium at Physics Building (C/Serrano 117) (Oral Sessions) (Poster & Coffee Breaks)

11 | PROGRAM

HPIS 2017

Location of events

12 | LOCATION OF EVENTS

Welcome Opening Reception and Registration (Sunday June 4th, 7:00 PM). Registration and the Opening Reception will be held at the CSIC Residencia located in Calle Pinar 21, Madrid, just behind the CSIC Main Building. Registration will start at 7:00 pm and the Welcome Reception at 8:00 pm

Registration and Symposium (June 5th-7th, 2017). Registration (June 5th, 7:30 am – 8:45 pm) and Symposium talks (8:45 am – 6:30 pm) will be held in the CSIC Main Building Auditorium Calle Serrano 117.

LOCATION OF EVENTS | 13

Poster Sessions & Coffee Breaks (June 5th-7th, 2017). Poster sessions and coffee breaks will be located in CSIC Atrium located at the Instituto de Física Fundamental C/Serrano 113b (2 min walk from Auditorium).

Lunch & Poster Exhibit (June 5th-7th, 12:30 pm-2:30 pm). The lunch will be served in two different locations. Participants will be divided in two groups: CSIC main restaurant located in the Main Building just behind the CSIC Main Auditorium & Edificio de Química Orgánica CSIC (C/Serrano 150; located at 5 min walk from the Auditorium). Posters will be exhibited at the CSIC Atrium located at the Instituto de Física Fundamental, C/Serrano 113b.

14 | LOCATION OF EVENTS

Closing banquet (June 7th, 8:30 pm). The closing banquet will take place at the Restaurante Jai Alai, located at walking distance of the CSIC Main Campus (Calle de Balbina Valverde 2).

EPG Workshop (June 8th-10th). The EPG Workshop will be held from June 8th to June 10th at the Instituto de Ciencias Agrarias, located at the building just aside the CSIC Main Building (Calle Serrano 115 bis) from 9:00 am – 7:00 pm.

LOCATION OF EVENTS | 15

Street view location of events

Lunch will be served to participants with a yellow ticket at the Serrano 117 restaurant at 12:30 pm

Lunch will be served to participants with a blue ticket at the Serrano 150 restaurant at 12:40 pm

16 | INFORMATION

HPIS 2017 Information for Attendees

ORAL PRESENTATIONS Keynote speaker presentations will be 25 minutes long with an additional 5 minutes for questions. The rest of the oral presentations will be 12 minutes long with an additional 3 minutes for questions. Presentations should be prepared in PowerPoint for Windows (version 2013 or earlier) and delivered to local organizers at the Registration desk but not later than the day before the talk. Presentations for Monday 5th can be delivered at the Welcome reception on Sunday night. There will be a limited number of computers available to preview Powerpoint presentations at the Instituto de Ciencias Agrarias building but we recommend to bring your own laptop.

POSTER PRESENTATIONS Posters should be put up during the morning of the first day of the Symposium (June 5th) in the Atrium of the Instituto de Física Fundamental. Posters will be displayed for the entire conference and can be viewed over the coffee hours, lunch break and during the poster session from 6:00- 7:30 pm each day. Authors with odd numbers are requested to be present at their poster on Monday evening’s poster session (June 5). Authors with even numbers are requested to be present at their poster on Tuesday evening’s poster session (June 6). Posters should be displayed on Monday morning (June 5) and taken down by 5 pm Wednesday afternoon (June 7). Poster boards are 120 cm x 160 cm and will accommodate 90 cm wide x 120 cm high (portrait orientation) standard posters. Pins or tape for securing posters will be provided at the meeting.

EMAIL ACCESS ON THE CSIC CAMPUS There will be access to Eduroam on campus. To those participants that have no access to Eduroam we will provide an invitation with a password to connect to the CSIC WIFI "invitados" network. To connect to the “invitados” network during the meeting you should use the following information:

Username: [email protected]; Password: 6h4m8p5

EDUROAM (CONFIGURE THIS PRIOR TO ARRIVAL AT CSIC) CSIC supports Eduroam. Eduroam (education roaming) is the secure, world-wide roaming wireless access service developed for the international research and education community. If you are coming from an institution that also supports Eduroam you will be able to access the CSIC wifi network. Does your institution support Eduroam? Find out. You will want to configure Eduroam before you arrive at CSIC.

CERTIFICATE OF ATTENDANCE AND INVOICES Certificates and invoices will be available upon request. We will need an ID number, Tax Number and Address to prepare invoices of registration fees.

17 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Hemipteran Systematics

1| & Genomics

18 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Hemiptera and their vectors: the known and the unknowns

Wilson M*

National Museum of Wales, UK

*Corresponding author: [email protected] Keynote Lecture

The Hemiptera are a large and diverse order of exopterogote insects, which occur throughout the world. There are more than 50,000 known species in around 100 families and now divided into 3 suborders: Heteroptera (true bugs), Sternorrhyncha and Auchenorrhyncha. Species in each suborder are significant pests of crop plants and trees, either by direct feeding or more commonly as vectors of various plant diseases caused by phytoplasma, spiroplasmas, bacteria and viruses. Species that are known vectors are not evenly distributed throughout the families. An overview of the phylogeny of the Hemiptera will be presented together with an analysis of species that are known vectors, and comments on host plant associations.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 19

Psyllids (Hemiptera, Psylloidea) as phloem feeding pests of agriculture crops of the Caucasus

Gegechkori A*

Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia

*Corresponding author: [email protected] Oral Presentation

The psylloid fauna in the Caucasus comprising about 210 identified species. It is recognized one of the most comprehensively studied insect group of the suborder Sternorryncha in the North Hemisphere. Instead of 8-9 species known earlier from agriculture entomology of the Caucasus, 17 locally or largely economically important pest species found during our studies in 1963-2008. 17 species belong to four families, out of which fam. Liviidae includes – Euphyllura phillyraea (pest of introduced olive tree); fam. Psyllidae – nine species: Cyamophila medicaginis (pest of alfalafa), Cacopsylla pyri, C. pyricola, C. pyrisuga, C. permixta, C. bidens (pests of pears), C. mali, C. melanoneura f. taurica – pests of apples), C. crataegi (pests of domestic medlars), C. pruni (pests of plums); fam. Homotomidae includes Homotoma ficus (pest of fig trees); fam. Triozidae consist of four species: Trioza alacris (pest of Laurus nobilis), polyphagous Bactericera nigricornis causes damage to vegetable cultivars (potato, species of Petrocelinium, etc.), B. crithmi and B. apicalis (affecting carrots, pest in agriculture habitats), B. brassicae (pest of garlics and onions). So, seven mentioned species of Cacopsyla injure temperate horticulture crops; three species (E. phillyraea, H. ficus, T. alacris) are harmfull to subtropic cultures; forage crops (herb legumes) harbor C. medicaginis. Among 17 pest psyllids, found during our field observations, only two polyvoltine species in pear orchards – C. pyri, C. bidnes – may be actually considered as seriously noxious in the Caucasus. Nymps of T. alacris causes large galls on a leaf of L. nobilis, lowering its commercial value.

20 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Sequencing two aphid genera: comparative genomics of Myzus and Brachycaudus to infer the genomic basis of generalism

Mathers TC1,*, Mugford ST2, Oosterhout C3, Hogenhout SA2 and Swarbreck D1

1Earlham Institute, Norwich, UK 2John Innes Centre, Norwich, UK 3University of East Anglia, Norwich, UK

*Corresponding author: [email protected] Oral Presentation

Whilst most aphid species are specialists, limited to one or few closely related host species, generalism has evolved multiple times across diverse aphid genera. These generalist aphid species represent a significant threat to agriculture due to their large population sizes, broad host ranges, high prevalence of insecticide resistance and their potential to act as vectors for other plant pathogens. Understanding how generalist aphid species are able to circumvent the defences of diverse host plant species is essential to develop effective control measures and, more broadly, to understand genomic mechanisms that underlie the evolution of increased plasticity. To investigate the evolution of generalism in aphids we have developed the highly polyphagous aphid Myzus persicae and its close relatives as a comparative genomic model system. We sequenced, de novo assembled and annotated the genomes of eight species from Myzus and its sister genus Brachycaudus and compared them to the recently published genome of M. persicae. This genus- wide genomic dataset encompasses multiple transitions between specialism and generalism and allowed us to identify the genetic signatures of host range expansion by comparative analysis of rates of molecular evolution and gene duplication.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 21

Unravelling the role of Bemisia tabaci - plant associations in the species diversification

Morin S1 *, Feldmesser E2, Santos-Garcia D1, Seal S3, Colvin J3 and Malka O1

1 Hebrew University of Jerusalem, Rehovot 76100, Israel 2 Weizmann Institute of Science, Rehovot 76100, Israel 3 Natural Resources Institute, University of Greenwich, Kent, UK

*Corresponding author: [email protected] Oral Presentation

Bemisia tabaci is considered a generalist species complex containing at least 35 species. The understanding of the mechanisms driving this unusual diversification is limited, but largely focus on allopatric forces (separation by migration of the continents), and assume that most species share a similar broad host range. Recent meta-analysis of field sampling data suggests, however, that sympatric species within each continent, are more host-specific than commonly thought, with only few species showing true generalism. Therefore, it is quite likely that during evolution, various species acquired many genetic changes that relate to their ability to utilize plant hosts. These plant-related differences might have contributed to the speciation process that led to the establishment of multiple species at the same geographic range. In this study, we compared the expression pattern of a set of 298 P450s, GSTs, COEs, UGTs, sulfotransferases and ABC transporters detoxification genes, present in the B. tabaci transcriptome, using six sympatric and allopatric species and four plant hosts: eggplant (common non-toxic host) and pepper, cassava and kale (known to produce toxic plant chemicals). We also monitored the performance of the six species on the four plant hosts. These performance assays indicated that the six species can be divided into two groups, one with extended (generalists) and one with restricted host range. In parallel, we found the same grouping pattern when the species were clustered according to the expression pattern of their detoxification genes, despite the fact that the three generalist species (which clustered together) represent three different genetic (continental) groups. The observed putative correlation between the expression pattern of the detoxification system and the insect’s feeding habits, supports the possibility that plant hosts played an important role in the evolution of the species complex. The possible links between our findings and the oscillation hypothesis of speciation will be discussed.

22 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

The identification and detection of psyllid vectors of Candidatus Liberibacter solanacearum in suction traps

Sjölund MJ1*, Carnegie M1, Greenslade A2, Ouvrard D3, Highet F1, Kenyon DM1, Sigvald R4, Bell JR2

1 Science and Advice for Scottish Agriculture (SASA), UK 2 Rothamsted Research, UK 3 Department of Life Sciences, Natural History Museum, UK 4 Department of Ecology, Swedish University of Agricultural Sciences, Sweden

*Corresponding author: [email protected] Oral Presentation

Candidatus Liberibacter solanacearum (CaLsol) is an α-proteobacterium that infects several solanaceous and apiaceous crops. It is vectored by three species of psyllid – Bactericera cockerelli in the Americas and New Zealand; Bactericera trigonica in Europe and the Mediterranean, and Trioza apicalis in Northern Europe. CaLsol is the causal agent of zebra chip disease in potato that has led to huge economic losses in America and New Zealand. The European haplotypes are primarily associated with apiaceous crops, largely in carrot, although recent evidence suggests they can infect potato, albeit at low levels. There are approx. 3800 species of psyllid worldwide. Many psyllid species, including vectors, lack taxonomically useful characteristics and can be easily misidentified. With only three known vectors, their accurate identification is crucial to monitor known/potential vectors and gain an understanding of psyllid biology and ecology, building on our understanding of epidemiology and improving pest risk analyses. Information on the distribution of several important species remains incomplete, especially for Scotland - the second biggest producer of seed potatoes in Europe. We reviewed and surveyed psyllid populations using a network of 12.2m suction traps in England, Scotland and Sweden (Rothamsted Insect Survey and EXAMINE networks). We found new species for all three countries, with several species displaying a summer and autumn population peak. The main vector in Sweden and England – T. apicalis, was caught in both countries, highlighting the networks potential as a monitoring/surveillance system for vectors. Species identification was carried out using classical taxonomy, in combination with molecular tools including non-destructive DNA extraction and DNA sequencing. Species-specific real-time PCR assays have been designed for two known, and one potential, vector to facilitate the rapid identification of species. Bulk DNA samples from suction trap catches are currently being used to test the assay sensitivity.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 23

Is methylation one of the drivers of virulence in Diuraphis noxia (Kurd.) Hemiptera: Aphididae?

Botha AM* and Breeds K

Stellenbosch University, South Africa

*Corresponding author: [email protected] Oral Presentation

Diuraphis noxia (Kurdjumov, Hemiptera: Aphididae), a specialist phloem feeder, is an economically important aphid pest afflicting wheat and barley yield in dry-land production regions of the USA, Argentina and South Africa. Populations sharing similar ecoagricultural regions and expressing different levels of virulence towards their hosts are called biotypes, and the number of D. noxia biotypes reported continues to increase, posing multiple threats to global food security. With the availability of the draft genome of D. noxia and confounding evidence of genomic plasticity, we set out to determine the extent of methylation in the genome of Diuraphis noxia in order to determine if methylation contributes to changes in virulence. To this end, the global levels of methylation as well as the methylation profiles of the different biotypes were investigated, the former done by measuring fluorescent adaptor levels when aphid DNA was restricted with isoschizomers HpaII and MspI. The latter involved the use of Methylation-Sensitive Amplification Polymorphism, and also provided insight into local regions of methylation in the genome. The global methylation results suggest an inversely proportional relationship between virulence levels and methylation, hypomethylation being associated with increased virulence. Methylation profiles of the biotypes, whilst similar, did show some clear differences indicating that differential methylation of certain genes could indeed contribute to differences in virulence. This study, being the first of its kind for Diuraphis noxia, has provided the groundwork for future research into methylation of this insect, and adds to a growing body of knowledge on the Russian Wheat Aphid.

24 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Current aphid composition of Turkish aphid fauna with new additions from Eastern part of the Turkey

Şenol Ö1*, Görür G2, Gezici G1, Parmaksız D1 and Akyıldırım Beğen H3

1Science and Art Faculty, Department of Biology, Ömer Halisdemir University, Turkey 2Science and Art Faculty, Department of Biotechnology, Turkey 3Artvin Çoruh University, Forestry Faculty, Botany Department, Turkey

*Corresponding author: [email protected] Poster 1

Aphid species are one the important group of plant suck species, they get benefit from recent climatic changes and thus invade new areas, increase their damage particularly to economically important species. Up to date nearly 532 aphid species were known from Turkey. Nearly 107 aphid species with more than 20 new records from Adıyaman, Malatya and Şanlıurfa provinces and composition of the Turkey aphid fauna increased to 550 species. These were preliminary results of the project that has been carrying out since September, 2016. Study area is one of important species entrance gate from Syria, Arabian Peninsula, desert area and some parts of Mediterranean to Anatolia. There is also one of the biggest dam namely GAP which have an impacts on climatic, floristic, crop variability, faunistic diversity of East and South Eastern part of Turkey, and recent analyses indicated that these influences are going to increase sharply in near future. Another important geographical barrier called “Anatolian diagonal” effects species entrance to the west- east part of Anatolia. When considered various specialized climatic, geographical, floristic features of Turkey, it is not going to be surprise to record more aphid species in Turkey. Findings are also going to be evaluated for possible invasive and alien species concept due to Turkey special geographical location.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 25

Evolutionary analyses of a new quantitative catalogue of candidate secreted salivary effectors in Acyrthosiphon pisum

Boulain H*, Legeai F, Guy E, Simon JC, and Sugio A

INRA, UMR Institut de Génétique, Environnement et Protection des Plantes (IGEPP), France

*Corresponding author: [email protected] Poster 2

The pea aphid, Acyrthosiphon pisum, is a sap-feeding phytophagous insect that forms a complex of 15 biotypes, each of which is specialized to one or a few species of Fabaceae. Successful establishment of aphid phloem feeding depends on the functions of salivary proteins injected into the host plant. Most of the proteins secreted with aphid saliva are thought to be produced in salivary glands (SG) of the aphid, and some are shown to suppress or trigger plant defence reactions like effectors of microbial pathogens. Transcriptomics and proteomics studies of SG or isolated saliva have created catalogues of candidate salivary proteins in the past; however, those catalogues often lack quantitative data of expression and tissue specificity. To create more informative catalogue of salivary proteins with quantitative expression data, and to analyse their evolutionary histories in A. pisum, we conducted SG and guts transcriptome by RNAseq. The data revealed around 2000 genes that were up-regulated in SG compared to gut. Among these SG induced genes, more than 600 genes are predicted to encode secreted proteins. Orthologous gene search conducted with 16 arthropod whole genomes (1 arachnid, and 15 insects including 5 aphid species) revealed that more than 50 % of our salivary gene candidates were aphid or A. pisum specific. By analysing the evolutionary rates of well-characterized salivary genes and their orthologues, we observed contrasting evolutionary patterns possibly influenced by their different roles in aphid feeding. The orthology analysis also revealed expansions of some gene families in A. pisum compare to other aphid species. Moreover, the positive selection acting on some salivary gene copies among these families suggests an important role of gene duplication in the evolution of A. pisum and may indicate the involvement of these genes in host adaptation.

26 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Single whitefly transcriptomes from Bemisia tabaci found in Brazil

De Marchi BR12*, Wainaina JM2, Kinene T2, Krause-Sakate R1, Boykin LM2

1 Faculdade de Ciencias Agronomicas de Botucatu - FCA/UNESP, Brazil 2 The University of Western Australia, UWA, Australia

*Corresponding author: [email protected] Poster 3

The whitefly, Bemisia tabaci is complex of at least 34 putative species and one of the most serious pests of vegetable, fibre and ornamental crops worldwide and has been listed as one of the world’s 100 worst invasive alien species. There is still a lack of genomic data available among the different whitefly species found in Brazil. Understanding the genetic and transcriptomic composition of these insect pests, the viruses they transmit and the microbiota is crucial to sustainable solutions for farmers to control whiteflies. Illumina sequencing was used to obtain transcriptomes of single individual of whiteflies from 6 different populations from Brazil including Middle East-AsiaMinor 1 (MEAM1), Mediterranean (MED) and New World 2 (NW2) collected in different hosts. The whiteflies mitochondrial genome, partial genome from primary and secondary endosymbionts as well as viruses present in the samples were obtained with BLASTn searches on the non-redundant nucleotide database and mapping with reference genomes for further comparison among the samples. We will discuss the utility of the single whitefly transcriptome method and resulting data.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 27

Circadian clock genes and photoperiodism in aphids

Barberà M, Collantes-Alegre JM and Martínez-Torres D*

Institut de Biologia Integrativa de Sistemes & Institut Cavanilles de Biodiversitat i Biologia Evolutiv, Universitat de València, Spain

*Corresponding author: [email protected] Poster 4

Almost all living organisms have an internal oscillator that is entrained with the external environment, the so called circadian clock, which is not only involved in the control of circadian rhythms, but also in the regulation of the photoperiodic response. In mammals, the circadian clock resides in the suprachiasmatic nucleus and regulates the synthesis of melatonin in the pineal gland via control of AANAT expression. Higher melatonin levels are generally associated to the dark phase of the day/night cycle in circadian rhythms and also to the induction of the photoperiodic response by short photoperiods. Even though the circadian clock has been characterised in several insects, its role in photoperiodism is still at debate. Aphids are typical photoperiodic insects that switch from viviparous parthenogenetic reproduction in long day seasons to oviparous sexual reproduction triggered by the shortening of photoperiod in autumn yielding overwintering diapausing eggs. We observed an increase of expression of some circadian clock genes associated to the photoperiodic response triggered by short photoperiods. Moreover, in situ localisation of per and tim transcripts allowed us to identify, for the first time, the aphid clock neurons in brain regions previously described as essential for the photoperiodic response. Additionally, we studied two hormones that may play a role as output of the circadian clock: melatonin and PTTH (prothoracicotropic hormone). We show for the first time in non-holometabolous insects the localisation of melatonin in the central nervous system. Moreover, we found an increase in melatonin levels and expression of some AANAT genes in aphids in which the photoperiodic response is activated. Finally, we identified, characterised and localised for the first time the aphid Ptth gene. Our results suggest that PTTH would not play an essential role in the photoperiodic response.

28 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Expression of candidate circadian and photoperiodic input elements in the aphid brain and eyes

Collantes-Alegre JM, Barberà M and Martínez-Torres D*

Institut de Biologia Integrativa de Sistemes & Institut Cavanilles de Biodiversitat i Biologia Evolutiv, Universitat de València, Spain

*Corresponding author: [email protected] Poster 5

Photosensitivity is fundamental in many aspects of hemipteran biology. Light is the main input signal for many vital processes such as entrainment of endogenous biological rhythms or prediction of environmental seasonal changes. Moreover, vision and color detection are key components of hemipteran ecology and behavior, including host plant discrimination in phytophagous species. Two gene families, opsins and cryptochromes, are known for their photosensitivity in many organisms. The first being responsible for chromatic vision (among other functions), and the latter taking part on the core and input pathways of the circadian clock in several insect species. Thanks to the availability of the pea aphid genome (Acyrthosiphon pisum, Hemiptera: Aphididae), we have been able to identify and validate 7 aphid opsin genes and 3 cryptochromes (one drosophila-like cryptochrome, and two mammalian-type cryptochromes). Among the anatomically relevant regions in these processes, eyes and brain have received much attention in insects, as they are known to harbor photosensitive molecules to transduce light input signals. Learning anatomical localization of the identified putative photosensitive proteins will help us understand their implication in different processes. We have designed and synthesized riboprobes to detect opsin and cryptochrome transcripts through in situ hybridization in eyes and brain on whole mount preparations. We have located 3 opsins in the aphid eye (two of them orthologous of insect short-wavelength-sensitive and one of long wavelength-sensitive opsins). The optical lobe and protocerebrum expressed another short-wavelength-sensitive opsin, a ciliary opsin (C-opsin) and one Arthropsin (an opsin only reported in dragonflies but absent from other insect species sequenced to date, despite its presence in other arthropods, such as spiders and crustaceans). Both, Drosophila and mammalian-like cryptochromes were found in regions previously related to the circadian clock and the photoperiodic response.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 29

Derivative forms of relict psyllid fauna (Hemiptera, Psylloidea) of the preglacial and postglacial biotas of the Caucasus

Gegechkori A

Ivane Javakhishvili Tbilisi State University, Georgia

*Corresponding author: [email protected] Poster 6

The Caucasus is a region with highly heterogenous psyllid fauna of recent geological time, but it encompasses relictual species of preglacial and postglacial origin. Present relict endemics, being restricted within refugial areas are the key to understand of past biotas. Homotoma ficus (L.) from tropical-subtropical genus Homotoma G-M, is largely distributed in temperate Eurasia as well. Refugia of hygro-thermophillous forests of the Arcto-Tertiary origin of South Caucasus supporting number of striking relict and endemic species of woody plants are not populated by psyllids of the same autoecology. It should be outline only Spanioneura caucasica Log. associated with Buxus colchica, typical representative of humid and shady ATG type of forests in Colchis. It South Caucasus the Mediterranean maquis communities is represented as enclaves─scattering in fragments (patches) along the coastal zone of the Black Sea. Characteristic Genisteae (Leguminosae) – eating psyllids of Arytaininae (Psyllidae) center of origin is most likely western Mediterranean region and Macaronesian archipelagos. From the Holocene time the climate in the circum-Euxinian region seem to have a filtering effect on mentioned taxa of psyllids. In extreme south-western part of Georgia penetrated only Livilla cognata (Aulm.) on Chamaecytisus.The region of the middle reaches of the Aras River in extreme southern part of Transcaucasia - an exclave of the Irano-Turanian region – is stronghold of very few deserts (Irano-Turanian) biome’s origin species: Eremopsylloides Log., Pachypsylloides Bergevin, Surreaca Burck. et Ouvr. linked with Calligonum, Egeriotrioza spp. with Populus (Turanga) euphratica, Diaphorina kopetdaghi Log. – with Zygophyllum atriplicoides. Boreal (glaciation) relict fauna is represented by Psylla sibirica Log. (feeding on Salix) splitting in its range between northern high latitudes (Siberia, Russian Far East) and the highlands of the Greater Caucasus, resulting disjunct distribution without vicariance.

30 | S1 HEMIPTERAN SYSTEMATICS &GENOMICS

Gall formed aphid species recorded from East and South Eastern parts of Turkey

Şenol Ö1, Görür G2, Gezici G1, Parmaksız D1 and Akyıldırım Beğen H3

1 Science and Arts Faculty, Department of Biology, Ömer Halisdemir University, Turkey 2 Science and Arts Faculty, Department of Biotechnology, Ömer Halisdemir University, Turkey 3 Artvin Çoruh University, Forestry Faculty, Botany Department, Turkey

*Corresponding author: [email protected] Poster 7

About 5,012 worldwide and 532 Turkey distributed aphid species were determined. Aphids, which are phloem sap feeders, infest herbaceous and woody plant. 10-20% of aphid species leads to gal formation on their host plant. About 33 galling aphid species have been recorded from Turkey. This study conducted in Adıyaman, Malatya and Şanlıurfa Provinces in Turkey from 2015 to 2016. These are preliminary findings of the planned study and nearly 121 aphid species have been determined from study area and 13 gal formed and 3 pseudo gal formed species recorded on basically Pistacia spp and Populus spp. from study area so far. Of these species, Baizongia pistaciae, Forda marginata, Geoica utricularia and Pemphigus spyrothecae were widely distributed in study area compared with others. This study aimed to find out how many aphid species distribute in East and South Parts of Turkey and give information about gall formation aphid composition of Turkish aphid fauna.

S1 HEMIPTERAN SYSTEMATICS &GENOMICS | 31

Composition, richness and abundance of Hemiptera: Cicadellidae in two areas of grassland in the Sabana de Bogotá – Colombia: preliminary analysis

Silva-Castaño A1, Brochero H2, Robin M3, Franco Lara L1

1 Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, Colombia 2 Facultad de Ciencias Agrarias, Universidad Nacional de Colombia, Colombia 3 Department of Biodiversity and Taxonomy, National Museums & Galleries of Wales, UK

*Corresponding author: [email protected] Poster 8

Leafhoppers feed on wild plants, ornamentals and crops. They are vectors of viruses and bacteria such as phytoplasmas. The information about their biology and ecology is still limited in the Sabana of Bogota, in central Colombia. A preliminary analysis on the composition, richness and abundance of Cicadellidae in the Campus of the Universidad Militar Nueva Granada (UMNG) in Cajicá, Cundinamarca (rural area) and in the campus of the Universidad Nacional (UNAL) in Bogota (urban area), was performed. In each zone, a quadrant of 25 m2 was built and the leafhoppers were collected from grass Cenchrus clandestinus (Hochst. Ex Chiov) Morrone), with leaves of between ~ 30cm and ~ 90cm of length. Samplings were done twice a month, between 11:00 h. and 13:00 h. for 1 year, from February 2016 to January 2017. The leafhoopers were captured with a sweep net and preserved in 90% ethanol. A total of 3334 individuals belong to the subfamilies Deltocephalinae, Cicadellinae, Typhlocybinae, Gyponinae, Xestocephalinae and Ideocerinae were captured. Composition similarity between the two zones was over 57% and the species accumulation curves for the two zones did not reach an asymptote, but indicated that the UNAL has less species than the UMNG. In addition, the parametric estimators Jack 1 and Boostrap suggest that the area with the highest species richness is the UMNG. However, the Chao 2 index does not indicate significant differences between sites. The most abundant species in both localities were Exitianus atratus Linnavuori with 516 individuals, Amplicephalus funzaensis Linnavuori with 1639 individuals and Borogonalia spp. with 430 individuals. The results suggest that the most abundant species in these two areas have been reported as vectors of phytoplasmas of the 16SrI and 16SrVII groups and that they complete their life cycle in C. clandestinus. (IMP- CIAS-2295).

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Xylem Feeders 2|

34 | S2 XYLEM FEEDERS

Is the role of vector choice on disease epidemiology as simple as it appears to be?

Almeida R

University of California, Berkeley, USA

*Corresponding author: [email protected] Keynote Lecture

There has been large interest on the role of vector behavior, specifically host plant choice, on plant disease dynamics. Vectors may be more or less attracted to symptomatic plants, for example, and the molecular mechanisms driving these interactions have in some cases been elucidated. In addition, vector infection status has also been shown to impact host plant preference. This elegant body of work deserves the attention it has received; it has also opened new research venues as well as novel concepts for disease control strategies. In this talk, however, I will question how these interactions may scale up to ecological scenarios. We will present data using the leafhopper-Xylella fastidiosa-grapevine disease system to illustrate how complex biological interactions lead to non-linear patterns of pathogen spread in response to disease progression and environmental variation.

S2 XYLEM FEEDERS | 35

Epidemiology of novel defensive traits against

Xylella fastidiosa in grapevines

Zeilinger A1, Beal D1, Sicard A1, Daugherty M2, Walker MA3, Almeida R1

1 Department of Environmental Science, Policy, and Management, University of California Berkeley, USA 2 Department of Entomology, University of California Riverside, USA 3 Department of Viticulture and Enology, University of California Davis, USA

*Corresponding author: [email protected] Oral Presentation

Development of resistance traits in agricultural crops is often a critical component of integrated disease management strategies. In contrast, tolerance traits—traits that reduce disease symptom severity without reducing pathogen populations in a host plant—may actually increase the risk of pathogen spread through a host population. At the same time, vector preference for hosts based on host infection status can have strong impacts on disease spread. Theoretical work from our group predicts that the precise form of defense—whether resistance or tolerance—will influence the spread of a vector-borne pathogen depending on whether vectors prefer or avoid feeding on diseased hosts. Specifically, when vectors avoid feeding on diseased hosts, resistance traits will reduce spread whereas tolerance traits will enhance spread, relative to a fully susceptible host population. The vector-borne bacterial pathogen Xylella fastidiosa causes Pierce’s Disease in grapevines and has led to reduced productivity and increased insecticide use in vineyards in California. In response, a new defensive trait from native Vitis spp. has been incorporated into commercial wine grape cultivars. While this new PdR1 trait reduces disease severity, our preliminary results indicate that it may confer a complex mix of tolerance and resistance against X. fastidiosa. Moreover, the nature of defense appears not to be constant over time but varies as the disease progresses. At the same time, the dominant sharpshooter vectors of X. fastidiosa avoid feeding on diseased grapevines, increasing the risk of enhanced pathogen spread in tolerant host populations. In this talk, I will present our theoretical work on the interactive effects of host defense and vector preference, our on-going experimental work to test these predictions, and our efforts to bridge theoretical and experimental work to aid in assessing epidemic risks prior to commercial release of novel defensive traits in agricultural crops.

36 | S2 XYLEM FEEDERS

Host-plant association and host-shifting of nymphs and adults of Philaenus spumarius L. in Italian olive orchards

Bodino N1, Plazio E2, Cavalieri V3, Dongiovanni E4, Ripamonti M2, Volani S5, Gilioli G5, Fumarola G4, Di Carolo M4, Porcelli F3,6 and Bosco D1,2*

1 Università degli Studi di Torino, DISAFA – Entomologia, Italy 2 Istituto per la Protezione Sostenibile delle Piante, CNR, Italy 3 Istituto per la Protezione Sostenibile delle Piante, CNR, Italy 4 CRSFA ‘Basile-Caramia’, Italy 5 Università degli Studi di Brescia, DMMT, Italy 6 Università degli Studi di Bari Aldo Moro, DiSSPA, Entomologia e Zoologia, Italy

*Corresponding author: [email protected] Oral Presentation

The spittlebug Philaenus spumarius is by far the most abundant xylem-sap feeder insect in the Italian olive agroecosystems and is the proven vector of Xylella fastidiosa CoDIRO strain to olive in Apulia. This highly polyphagous species has one generation per year, nymphs develop in late winter-early spring, and adults are present over an extended period from spring onwards. Four olive orchards of one ha in size were chosen in the Liguria (North-Western Italy) and in Apulia (South-Eastern Italy) regions. Host-plant association of nymphal stages on the herbaceous cover was investigated while, for the adults, the presence on herbaceous and woody hosts was recorded all through the season. Within 30 sample units of 0.25 m2 randomly distributed in each olive orchard, all nymphs were counted and their host-plants identified. Adults were sampled with sweep net on 10-30 random sample units of different vegetation compartments: grass cover, olive trees, and spontaneous shrubs. Nymphs were mainly associated to species of the families Asteraceae, Fabaceae, Umbelliferae. Host-plant shifting and a different location on the plant was recorded between early and late nymphal stages, suggesting that nymphs are rather mobile. Adult population moved from grass cover to woody plants in late spring and summer and moved back to grass cover in late summer-early autumn, thus showing a clear host-shifting during the season. This information is relevant for the development of control strategies of the vector in the X. fastidiosa infected area, based on both insecticide application and agronomic measures, as well as for risk assessment in non-infected areas.

S2 XYLEM FEEDERS | 37

Surveillance and identification of potential vectors of Xylella fastidiosa in Mallorca (Balearic Islands, Spain) after 2016 outbreak.

Miranda MA*1, Marqués A1, Paredes-Esquivel C1, Leza M1, Beidas O2, Olmo D2, Morente M3, Fereres A3 and Juan A2

1 Laboratory of Zoology. UIB-INAGEA, Spain 2 Servei d’Agricultura. Conselleria de Medi Ambient, Agricultura i Pesca, Spain 3 Instituto de Ciencias Agrarias-CSIC, Spain

* Corresponding author: [email protected] Oral Presentation

The first positive detection of the bacteria Xylella fastidiosa in Mallorca was recorded in October 2016. An extensive surveillance conducted at the beginning of 2017 showed that the pathogen was widely spread, including more than 130 positive detections and the identification of three subspecies of the bacterium: multiplex, fastidiosa and pauca distributed among the major islands of the Balearic archipelago. The bacteria is transmitted by xylem feeding insects, particularly Hemiptera: Auchenorhyncha, suborder Cicadomorpha. In March 2017, a surveillance project aiming to study the species composition of the potential insect vectors for X. fastidiosa, as well as its seasonal pattern was started in Mallorca. The project combined different methods of sampling, including sweeping net, direct sampling of immatures of vector species in herbaceous vegetation, vacuum of insects and an “ad hoc” designed passive traps. The selected sites for sampling included representative orchards of almond, olive, citrus and vineyard, which were sampled biweekly. Further, monthly sampling of tree and herbaceous vegetation was also conducted in plots were positive cases of X. fastidiosa were detected in Mallorca. Collected insects were identified according to the available literature for the European species of the families Aphrophoridae, Cercopidae and subfamily Cicadellinae. Insect specimens were also analyzed for detection of the bacteria using molecular methods. Here we present, for the first time, preliminary results on the species of Hemiptera that are associated to the outbreaks of X. fastidiosa in Mallorca.

38 | S2 XYLEM FEEDERS

Evolution of Xylella fastidiosa on plant- and insect-mimicking environments

Sicard A, Voeltz M, Almeida R

Department of Environmental Science, Policy, and Management, UC Berkeley, USA

*Corresponding author: [email protected] Poster 9

The pathogenic bacterium Xylella fastidiosa is associated with several diseases of economic importance in a wide range of plants. This bacterium must successfully colonize its host plants as well as insect vectors for effective dissemination. We are investigating the effect of restricting X. fastidiosa to only plant- or insect-mimicking environments by successively culturing the bacterium in vitro on media that include galacturonic acid or chitin as carbon sources. This study should enable us to shed light on the mechanisms leading to the loss of pathogenicity and potentially transmissibility by vectors, or gains in plant virulence, for example. In addition, we expect the work will generate a better understanding of X. fastidiosa evolution and host colonization.

S2 XYLEM FEEDERS | 39

Evaluation of olive cultivar effect on the efficiency of the acquisition and transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae)

Cavalieri V1, Dongiovanni C2, Altamura G1, Tauro D2, Ciniero A2, Morelli M1, Bosco D3, Saponari M1

1 CNR Istituto per la Protezione Sostenibile delle Piante (IPSP), Italy 2 Centro di Ricerca, S perimentazione e Formazione in Agricoltura (CRSFA) “Basile Caramia”, Italy 3 Università degli Studi di Torino, Italy

*Corresponding author: [email protected] Poster 10

The meadow spittlebug Philaenus spumarius (L.) has been identified as vector of Xylella fastidiosa subsp. pauca strain CoDiRO infecting several host plants in Apulia (Southern Italy), and causing a major bacterial disease on olive trees. Evidences of differential olive cultivar susceptibility to the bacterial infections were obtained from phenotypic characterization and molecular investigations. A higher bacterial population (up to 100 times) was consistently detected in the trees of the most susceptible cultivars (i.e. Ogliarola and Cellina di Nardò, showing severe symptoms), compared to the trees of the cultivar Leccino showing milder symptoms and erratic distribution of the bacterium within the canopy. In order to determine the epidemiological impact of olive trees harboring low bacterial population, transmission experiments were set under semi-field conditions, by caging Xf-free adults of P. spumarius on branches of field-infected trees of the abovementioned cultivars. Three independent transmission tests were carried out from June to September 2016. Insects were caged on the branches of the infected trees for 3-4 days of acquisition and then transferred (groups of five) on healthy olive plantlets for 3-4 days of inoculation access period. Quantitative PCR assays performed on the insects used in the transmissions tests showed that: (i) P. spumarius did not acquire Xf on the majority of the Leccino branches, and only on few branches 5-7% of positive insects could be detected; (ii) infected specimens were consistently detected upon feeding on the branches of Ogliarola and Cellina di Nardò, with an average of 47% of Xylella-positive insects. With regard to the transmission rate, the diagnostic assays on the recipient plants, so far, showed 20% and 35% of infected plants when exposed to the insects that fed on Cellina di Nardò and Ogliarola, respectively, while no transmission occurred on the olives exposed to the insects fed on trees of Leccino.

40 | S2 XYLEM FEEDERS

Adaptation of in vitro feeding system for acquisition and transmission of Xylella fastidiosa subsp. pauca by sharpshooters

Esteves MB*, Sales T de M, Tonini IM, Nalin JL, Lopes JRS

Department of Entomology and Acarology, Luiz de Queiroz College of Agriculture-ESALQ, University of Sao Paulo, Brazil

*Corresponding author: [email protected] Poster 11

Xylella fastidiosa is a leafhopper-borne bacterium that causes diseases in a number of economically important plants. The development of methods for in vitro acquisition of cultured bacterial cells from artificial diets by leafhoppers allowed major advances in understanding molecular mechanisms underlying transmission of X. fastidiosa subsp. fastidiosa. Such in vitro acquisition methods have not yet been tested with other subspecies of this bacterium, for which there is limited information on transmission mechanisms. Here we tested the artificial feeding system for in vitro acquisition and transmission of a citrus strain (isolate 9a5c) of X. fastidiosa subsp. pauca by sharpshooter. Isolate 9a5c was grown on two solid culture media (XFM and XFM+galacturonic acid-GA) for 15 days, and resulting colonies were resuspended in a liquid artificial diet (L-glutamine, L-aspargine and sodium citrate). The suspended cells were offered to 36 individuals of two sharpshooters vectors (Bucephalogonia xanthophis and Macugonalia leucomelas) in sachets composed of two parafilm® membranes (1 insect/sachet). Control treatment consisted of sharpshooters that fed on diets without bacterial cells. After an acquisition access period of 6 h, the sharpshooters were confined on healthy Catharanthus roseus plants for an inoculation access period (IAP) of 72 h. Insects were tested by PCR for the presence of X. fastidiosa right after the IAP, and plants were tested 120 days later. Both culture media allowed acquisition of X. fastidiosa in artificial diets and subsequent transmission to test plants. The proportions of individuals that acquired the bacterium were higher for M. leucomelas (64 and 67% for XFM+GA and XFM, respectively) than for B. xanthophis (55.5 and 44.4%, respectively). Transmission rates (3 insects per test plant) by B. xanthophis were 18 and 54% after acquisition of bacteria grown in XFM+GA and XFM, respectively; for M. leucomelas, the same media allowed transmission rates of 8 and 33%, respectively.

S2 XYLEM FEEDERS | 41

Experimental host plants for vector transmission studies of citrus strains of Xylella fastidiosa

Esteves MB and Lopes JRS*

Dept. of Entomology and Acarology, ESALQ-University of Sao Paulo, Brazil

*Corresponding author: [email protected] Poster 12

Successful transmission studies of Xylella fastidiosa depend on the availability of fast growing host plants that allow sufficient pathogen multiplication for acquisition by insect vectors and/or detection after inoculation. Although Citrus sinensis has been used as source and test (recipient) plant in transmission experiments of citrus variegated chlorosis (CVC) strains, it is a medium- to low-titer host of the pathogen and transmission rates obtained are rather low. Here we tested other experimental hosts of CVC strains of X. fastidiosa as feeding hosts of one of the main vectors of this pathogen in Brazil, the sharpshooter Bucephalogonia xanthophis (Hemiptera: Cicadellidae: Cicadellinae), as well as sources and test plants in transmission studies. Fast growing herbaceous plants that support multiplication of CVC strains, e.g. Catharanthus roseus, Medicago sativa, Nicotiana tabacum, Ocimum basilicum and Solanum americanum, were selected in mechanical inoculation assays. The highest bacterial titers were detected in C. roseus and N. tabacum, but sharpshooter feeding and survival rates were very low on the second species. Higher transmission rates were obtained when C. roseus was used as a source plant, and when C. roseus or M. sativa were used as test plants. Feeding rates by B. xanthophis were higher on O. basilicum than on the other host plants tested. Among the known hosts of CVC strains of X. fastidiosa, C. roseus appears to be the most suitable for both acquisition and inoculation assays.

42 | S2 XYLEM FEEDERS

Acquisition of Xyllela fastidiosa causes changes to the inoculation behavior (EPG X wave) of an efficient sharpshooter vector

Roddee J, Backus EA*, Cervantes FA and Hanboonsong Y

USDA Agricultural Research Service, Parlier, USA

*Corresponding author: [email protected] Poster 13

Xylella fastidiosa (Xf) is a foregut-borne bacterium that is inoculated into xylem cells of a healthy plant during feeding by sharpshooter vectors. Inoculation occurs during salivation and egestion behaviors that are likely represented by the sharpshooter X wave. The objective of this study was to test whether inoculation behaviors are altered by the presence of Xf colonies in the foregut of a vector. Age-specific, blue-green sharpshooters, Graphocephala atropunctata (Signoret), were reared on basil with weekly plant changing, to achieve minimum Xf colonization of vector foreguts. Insects were removed from basil colony and given one of four treatments prior to EPG recording: 1) old colony adults, 2) young colony adults, 3) young colony adults caged on healthy grape for 4 days that likely acquired non-Xf microbes, and 4) young colony adults caged on Xf-infected (symptomatic) grape for 4 days that likely acquired Xf. After treatment, all insects were recorded with an AC-DC electropenetrograph for 20 hours each. Waveforms for X wave components (XB1 [salivation and rinsing egestion] and XC1 [discharge egestion]) were measured and three variables (mean event duraton, mean number of events, and mean “total” waveform duration, all per insect) were calculated. Data were statistically compared among treatments via SAS. Insects from healthy and infected grape made significantly more events of both X waveforms than did insects from basil, while the mean event durations were not significantly different among treatments. “Total” durations of XB1 and XC1 per insect were highest for insects from healthy grape, lowest for basil, with intermediate durations for infected grape. These results show that colonization of vectors by Xf or other microbes increases the likelihood of performance of microbial (including Xf) inoculation behaviors. This study expands our understanding of Xf inoculation, which will be applied to improving grape resistance in the future.

S2 XYLEM FEEDERS | 43

Potential vectors of Xylella fastidiosa in the Iberian Peninsula: identification and population dynamics

Morente M*, Moreno A, Plaza M, Fereres A

Instituto de Ciencias Agrarias-CSIC, Spain.

*Corresponding author: [email protected] Poster 14

The introduction and rapid spread of Xylella fastidiosa in Europe is causing serious epidemics leading to important yield losses, especially in olive groves in Southern Italy, which entails a great risk to the European agriculture. Recent detection of the bacterium in Mallorca, Spain, has increased the need to assess the degree of threat to which Spanish agriculture is exposed. Because Xylella fastidiosa is transmitted by xylem feeders (Order Hemiptera, Suborder Cicadomorpha), understanding the biology and ecology of its potential vectors is essential for the risk assessment of pathogen spread and to develop preventive control strategies. We sampled both olive canopy and associated ground vegetation in 14 olive groves distributed in the Iberian Peninsula under different cropping practices: organic, conservation or no tillage and conventional. We found that Philaenus spumarius, the vector species in Italy, was limited to humid regions where natural vegetation is present most time of the year, mainly in the sampling sites of Seville. The first nymphs were detected in early March and its population density was low, reaching its peak between late April and mid-May on the ground vegetation, with a maximum of 0.06 insects per sweep. P. spumarius was detected in the olive tree canopy in three sampling sites, with a maximum population density of 0.06 individuals per branch in early August in Seville. Furthermore, we found other potential vectors such as Neophilaenus campestris, not yet reported as a vector, quite abundant on grasses below olive trees, mainly in Madrid. The greatest population density was 2.26 individuals per sweep in mid-May, which decreased in mid-June and increased in late October. By contrast, few individuals were found in olive canopies. Cercopis intermedia and Lepyronia coleoptrata were also present in low densities in some sampling sites. In summary, our preliminary findings suggest that xylem-feeders are present at low densities in most of the olive-producing regions sampled in the Iberian Peninsula, suggesting that they are not in their optimal habitat.

44 | S2 XYLEM FEEDERS

Phenology of the meadow spittlebug, Philaenus spumarius, in California North coast vineyards

Beal DJ, Almeida RPP and Purcell AH

Department of Environmental Science, Policy, and Management, UC Berkeley, USA

*Corresponding author: [email protected] Poster 15

The meadow spittlebug, Philaenus spumarius, is central to the current Xylella fastidiosa epidemic in Italy and potentially other outbreaks in the Mediterranean. However, its potential role in past and current outbreaks of Pierce’s disease of grapevine in California is unknown. In particular, the phenology and ecology of P. spumarius in California vineyards remains poorly described. Our current understanding of X. fastidiosa epidemiology suggests that the timing of vector immigration into vineyards will influence the vector’s contribution to disease spread. To characterize P. spumarius’ phenology within California North Coast vineyards in 2016, we collected nymphal spittlemasses from vineyard weeds and cover crops to assess seasonal patterns of nymphal development and population dynamics. Measurements of nymphal head width were used to determine instar. Upon the observation of late-instar nymphs, we also assessed adult spittlebug populations via biweekly sweep net and sticky trap collection. Spittlemasses were first observed in March and the emergence of adult spittlebugs did not occur until the first half of April. Both sweepnet and sticky trap surveys suggested that spittlebug adults did not move from the vineyard cover crops and weeds to cultivated grapevine until mid-May. Depending on the site, adult spittlebug populations within the vineyard were low throughout the late summer but returned to early-summer population levels upon the start of autumn. Adult spittlebugs were identified to sex and dissected to assess seasonal trends in sex ratio and ovarian development. Natural infectivity trials revealed that field caught spittlebugs did not successfully transmit Xylella fastidiosa to laboratory grapevines until the late summer and fall months. These preliminary findings suggest that meadow spittlebug is unlikely to contribute to typical Pierce’s disease patterns in Northern California.

S2 XYLEM FEEDERS | 45

Sensory structures of the labial tip and precibarium of Philaenus spumarius

Ruschioni S, Ranieri E, Romani R, Riolo P*, Minuz R L and Isidoro N

Dipartimento di Scienze Agrarie, Alimentari e Ambientali - Università Politecnica delle Marche, Italy

*Corresponding author: [email protected] Poster 16

The meadow spittlebug, Philaenus spumarius (Hemiptera: Aphrophoridae) is nowadays the most important vector of the xylem-limited bacterium Xylella fastidiosa, in Europe. Mouthparts associated with feeding behavior and feeding habits are important sensory and feeding structures in insects. To obtain a better understanding of feeding in P. spumarius, the ultrastructure of the sensory structure of its labial tip and precibarium were investigated by transmission and scanning electron microscopy. In P. spumarius, the tip of the labium consists of two lateral lobes separated by the stylet groove. In the dorsal part of each lobe, 8 aporous sensilla, with a single mechanosensory neuron, are present. Each lateral lobe is arranged in a pattern of 4+4+1 sensilla: seven uniporous sensilla, among which five with a set of 2 chemo- and 1 mechanosensory neurons and two with a set of 4 chemo- and 1 mechanosensory neurons; a multiporous sensillum, with a set of 3 chemo- and 1 mechanosensory neurons; an aporous sensillum, with a single mechanosensory neuron. Lastly, two aporous sensilla, with a single mechanosensory neuron, are found symmetrically located on the distal part of stylet groove. Within the meadow spittlebug precibarium, 20 putative gustatory sensilla that occur as two distinct groups are found. The distal group consists of 10 chemosensilla located on epipharynx, while the proximal group consists of 10 chemosensilla located 8 on the epipharynx and 2 on the hypopharynx. Putative functional significance of the different structures is briefly discussed.

46 | S2 XYLEM FEEDERS

Distribution patterns of Philaenus spumarius in an olive agroecosystem

Ranieri E, Minuz RL, Casavecchia S, Loreto N, Ruschioni S, Isidoro N and Riolo P*

Dipartimento di Scienze Agrarie, Alimentari e Ambientali - Università Politecnica delle Marche, Italy

*Corresponding author: [email protected] Poster 17

The meadow spittlebug Philaenus spumarius L. (Hemiptera: Aphrophoridae) is nowadays the most important vector of the xylem-limited bacterium Xylella fastidiosa, in Europe. Transmission of pathogens depends on the abundance and dispersal of their vectors, which can be affected by different cultivation practices, pedoclimatic conditions and abundance of their host plants. Dispersal of P. spumarius and its relationship with wild host plant abundances were studied over one year in an olive agroecosystem (olive orchard and surrounding areas) of the Marche region (central-eastern Italy). Adults were sampled using yellow sticky traps (n. 108) at two different heights (30 and 170 cm above the ground) positioned in a regular geo-referenced grid. Wild vegetation was surveyed around each sampling point following the Braun-Blanquet method. Moreover, spatial analysis by distance indices was performed on total insect captures and plant species abundances. A total of 744 adults were captured, with two flight peaks in May and in October, respectively. No significant differences have been observed in captures between high and low traps. In traps the numbers of males were significantly higher than females. Adults showed an aggregated distribution, with a significant spatial association with Clematis vitalba L. (Family Ranunculaceae) and Medicago sp. (Family Fabaceae). Meadow spittlebug colonization patterns throughout the sampling season have been represented in density maps of distribution. Data highlight the importance of understanding the dispersal patterns of vector species for the application of correct integrated pest-management strategies.

S2 XYLEM FEEDERS | 47

Feeding behavior of Philaenus spumarius, the main vector of Xylella fastidiosa in Europe

Cornara D*, Morente M, Garzo E, Moreno A and Fereres A

Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 18

The arthropod-borne bacterium Xylella fastidiosa is the causal agent of tremendous diseases affecting several economically important crops. The relationship between the bacterium and its vectors has been explored just for Nearctic and Neotropic insect species, mainly belonging to the sub-family Cicadellinae. On the contrary, data on X. fastidiosa relationship with Cercopoidea, the widespread candidate vectors in Europe, are limited. The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) has been reported as the main vector of X. fastidiosa within the first European hotspot of the fastidious bacterium. A deep knowledge of the feeding behavior of the vector is required in order to understand the relationship between the pathogen and its carrier. Indeed, X. fastidiosa is transmitted by its vectors without specificity, and xylem-sap feeding seems to be the only feature shared among vector species. Therefore, we carried out an electrical penetration graph (EPG) assisted study on P. spumarius feeding behavior on vetch and olive plants, the former previously used as host for the spittlebug rearing, and the latter reported as being the main host plant of X. fastidiosa in Europe. The EPG waveforms were described based on relative amplitude, frequency, and electrical origin of the observed traces. Thereafter, we correlated the observed waveforms with specific feeding behavior activities, either through histological observation of stylets sheath within the penetrated tissue, or through the analysis of the composition of the excreta, excretion rate, video-assisted EPG recording of abdominal and anal tube movements during the feeding process. Preliminary data presented here on the feeding behavior of a X. fastidiosa ascertained vector widespread in Europe, will be the base for further researches aimed at understanding the transmission mechanism of the fastidious bacterium by its spittlebug vector.

48 | S2 XYLEM FEEDERS

49 | S3 PHLOEM & MESOPHYLL FEEDERS

Phloem & Mesophyll

3 | Feeders

50 | S3 PHLOEM & MESOPHYLL FEEDERS

Plant-mediated interspecific competition between whiteflies

Shu-Sheng Liu

Institute of Insect Sciences, Zhejiang University, China

*Corresponding author: [email protected] Keynote Lecture

Whiteflies of the Bemisia tabaci complex and other members in the family Aleyrodidae include some important crop pests worldwide. Many of the species often occur sympatrically and show niche overlap, where they have complex interspecific interactions including competition. The process of interspecific competition is dynamic and should be investigated in view of the intrinsic differences between species in the context of the environments where they interact. Host plant is a major environmental factor mediating interspecific relationships that may lead to competitive displacement of some species by others, such as what would occur in biological invasions. In this presentation, first, I will show that competition trajectory and eventual outcome between competitors may vary depending on the host plant on which they live. Such host plant-mediated interspecific competition has been extensively observed under controlled conditions and further supported by circumstantial evidence from the field. I then try to explore the possible mechanisms of plant effects on whitefly behaviour, survival and reproduction. In addition, I will try to touch on the mechanisms of apparent competition mediated by various factors including natural enemies, whitefly-transmitted viruses and human-mediated disturbance such as insecticide application and vegetation management. Finally, I will try to suggest some strategies to use multidisciplinary approaches, including ecological, behavioural, genomic, metabolic, and proteomic approaches, to investigate the mechanisms underlying whitefly interspecific interactions including competition.

S3 PHLOEM & MESOPHYLL FEEDERS | 51

Newly distinguished potential drops are related to phloem cell punctures associated to the inoculation of closterovirus by aphids

Jiménez J1*, Tjallingii WF2, Moreno A 1 and Fereres A1

1 Instituto de Ciencias Agrarias-CSIC, Spain 2 EPG Systems (epgsystems.eu), The Netherlands

*Corresponding author: [email protected] Oral Presentation

Beet yellows virus (BYV) is a phloem-restricted semipersistent closterovirus that infects sugar beet (Beta vulgaris L.). Its most efficient vector is the aphid Myzus persicae Sulzer. The optimal acquisition and inoculation access periods were studied with the main aphid vectors years ago. However, it remains unclear what stylet activities are associated with BYV transmission. Feeding behaviour of M. persicae was monitored in sugar beet by using the Electrical Penetration Graph (EPG) technique. EPG waveforms reflect stylet pathway activities (waveform C) with intermittent intracellular brief punctures ('potential drop' waveforms; pd), normally followed by phloem activities; phloem salivation (waveform E1) and phloem ingestion (waveform E2). By inspecting the pd waveform features more closely, some of them, that we called type-II pds, appeared to deviate from the much more abundant and normal type-I pds. Thus, we recorded EPGs from viruliferous aphids on healthy test plants and manually ended EPG recordings after the following waveforms: 1) pathway without pds, 2) pathway with only type-I pds, 3) pathway with type-I pds and ≥1 type-II pd, 4) pathway with type-I pds + ≥1 type-II pd followed by a single E1 phase, 5) same as 4) but also including the 1st E1/E2 interphase, and 6) same as 5) but with an additional sustained E2 phase. Test plants were kept for 3 weeks in the greenhouse for expression of virus symptoms and virus detection. Successful transmission was found only in test plants with manually ended EPG recordings after treatments 3 to 6. Transmission was highest in treatment 3 with no significant differences with treatments 4, 5 or 6. Therefore, we concluded that the type-II pds are essential in BYV transmission and likely represent intracellular punctures in phloem cells, either in phloem parenchyma, companion or sieve element cells. The feature differences between the two types of intracellular punctures and the implication of type-II pds in the transmission of BYV will be discussed on the basis of our findings.

52 | S3 PHLOEM & MESOPHYLL FEEDERS

Compatible/incompatible aphid-plant interactions

Tjallingii WF

EPG Systems (epgsystems.eu), The Netherlands

*Corresponding author: [email protected] Oral Presentation

Incompatible aphid-plant interactions are very common and reflect the general situation that most plants are resistant to most pathogens and pests. Therefore, special interactions are needed in compatible interactions on 'host plants'. Aphids attempting to feed on a plant are confronted with sieve element (SE) wound responses causing phloem proteins to coagulate and occlude the SE and the food canal of the stylets. For a considerable time we thought that the SE salivation (characterized as the E1 waveforms in EPG recordings) would be responsible for avoiding or reversing these protein coagulations by binding Ca++ ions that enter sieve elements cells during stylet penetration; either by leakage through the damaged cell membrane or through Ca ion channels opening upon stylet penetration. However, it has become clear recently that protein coagulation during incompatible interactions occurs before E1 salivation starts and that coagulation is not reversed when aphids change their activity from E2 ingestion to E1 salivation. Thus in compatible interactions there must be preceding activities to avoid sieve element occlusion. A number of candidate activities before E1 salivation will be discussed.

S3 PHLOEM & MESOPHYLL FEEDERS | 53

The role of sieve element occlusion proteins in aphid resistance

Walker G* and Peng J

University of California, Riverside, USA

*Corresponding author: [email protected] Oral Presentation

Melon line TGR-1551 has long been known to be resistant to the melon aphid, Aphis gossypii (Glover). Electrical penetration graph (EPG) studies have demonstrated that when melon aphids penetrate a phloem sieve element, they usually engage in prolonged bouts of salivation (EPG waveform E1) and eventually withdraw their stylets from the sieve element without attaining ingestion (EPG waveform E2). This contrasts with their behavior on susceptible melon lines where sieve element penetration is usually followed by a brief (ca. 1 min) bout of salivation and then a prolonged bout (often hours) of ingestion. We verified these previous observations and then demonstrated that sieve element occlusion (SEO) proteins are responsible for the difficulty aphids encounter attempting to ingest phloem sap from TGR-1551. EPG monitored aphids and the leaves on which they were feeding were instantaneously frozen with liquid nitrogen several minutes after penetrating a phloem sieve element, thus fixing the stylets in place. Plant tissue was processed by freeze-substitution, stained, and examined by confocal microscopy. Penetrated sieve elements were almost always occluded by SEO proteins in TGR-1551 but were unoccluded in the susceptible melon cultivar, Iroquois. SEO protein occlusion is believed to be triggered by an influx of calcium into the sieve element, and when TGR-1551 phloem was treated with a calcium channel blocker, lanthanum chloride, SEO protein occlusion was prevented and aphids were no longer inhibited from ingesting phloem sap. They fed on lanthanum-treated phloem of TGR-1551 the same way as they fed on susceptible melons: after penetration of a sieve element, they engaged in a brief bout of salivation followed by prolonged ingestion.

54 | S3 PHLOEM & MESOPHYLL FEEDERS

Loss of resistance to Nilaparvata lugens (Hemiptera: Delphacidae) may be due to low- level expression of Bph32 in rice panicles at heading stage

Jairin J

Ubon Ratchathani Rice Research Center, Thailand

*Corresponding author: [email protected] Oral Presentation

The brown planthopper (BPH), Nilaparvata lugens Stål, is the most important insect pest of rice in Asia. Employing host plant resistance is one of the strategies to control the BPH. The resistant rice cultivar Rathu Heenati (RH) carrying Bph3 (recently, it has been renamed “Bph32”) is currently still effective despite more than 30 years of deployment. RH has been reported to be resistant against BPH at all growing stages. However, we observed that BPH could feed on panicles while it could not feed on leaf sheaths of RH. The resistance gene Bph32 was introduced into a recombinant inbred line, UBN03078. The rice line was used to observe patterns of the target gene regulation. A low-level expression of Bph32 on panicles has been hypothesized to cause susceptibility in UBN03078 at the heading stage. From gene expression analysis, the result supported the hypothesis that the regulation of the resistance gene was reduced in the upper internodes (panicle) compare to in the leaf sheaths of heading rice plants. This phenomenon may enable BPH to feed on the panicle of resistant plants and warrants further investigation.

S3 PHLOEM & MESOPHYLL FEEDERS | 55

Grapevine secondary metabolites involved in insect resistance

Webster K1*, Truong T2, Neeman T3 and Cooper P1

1 Research School of Biology, Australian National University, Australia 2 Research School of Chemistry, Australian National University, Australia 3 Statistical Consulting Unit, Australian National University, Australia

*Corresponding author: [email protected] Oral Presentation

Grapevine Scale (Parthenolecanium persicae) and Frosted Scale (Parthenolecanium pruinosum) are the most abundant Coccid species found on grapevine in Australia; mostly associated with wine grapevines. Work on the biology and ecology of Grapevine Scale and Frosted Scale on wine grapevine in Australia showed evidence of antibiosis in the grapevine cultivars tested. In general, red wine grapevine cultivars were less favourable for development of Grapevine Scale and Frosted Scale than white wine grapevine cultivars. Combined with extensive existing literature demonstrating the role of plant secondary metabolites in defence against insect feeding, grapevine secondary metabolites are a good candidate to test grapevine plant defences against scale insect feeding. We conducted surveys at four commercial vineyards in Murrumbateman, New South Wales and Canberra, Australian Capital Territory to confirm the presence of Grapevine Scale populations. Grapevine leaves were collected from the surveyed vineyards three times during the growing season at the development of fruit stage; ripening of berries stage; and after harvest. The cultivars sampled were Riesling, Shiraz, Pinot Noir, Temperanillo, Sangiovese and Grenache. Un-targeted metabolomics using gas chromatography mass spectrometry determined the differences in metabolites present among the grapevine cultivars. Additionally, liquid chromatography tandem mass spectrometry with electrospray ionisation was used to analyse salicylic acid, abscisic acid, jasmonic acid, stilbene and flavonoid content. Our results may be used to determine which cultivars to select for further metabolomics studies using greenhouse grown scale insect inoculated plants.

56 | S3 PHLOEM & MESOPHYLL FEEDERS

Settling and oviposition preferences of Russelliana solanicola Tuthill (Hemiptera: Psyllidae) among different cultivated and spontaneous plants

Kuhn TMA*, Borbón Cortés MT and Lopes JRS

Escola Superior de Agricultura “Luiz de Queiroz”, USP, Brazil

*Corresponding author: [email protected] Oral Presentation

Many psyllid species are vectors of 'Candidatus Liberibacter solanacearum', a bacterium associated with emerging diseases in vegetable crops. This pathogen has not yet been reported in South America, but there are indigenous psyllids that could act as vectors in case of introduction, especially Russeliana solanicola, which is listed as a potato pest in Andean countries. The goal of this study was to evaluate settling and oviposition preferences of R. solanicola in choice tests among vegetables (Daucus carota, Solanum tuberosum, S. lycopersicum, Capsicum annuum and C. chinese) and common weeds (Amaranthus retroflexus, Nicandra physalodes, Parthenium hysterophorus, S. americanum and Bidens pilosa). One plant of each species (vegetables and weeds were tested separately) was placed around the center of observation cages (n=20), in which 80 unsexed adults of R. solanicola were released on a flight platform located 10 cm above the top of the plants. The number of adults per plant was counted at 0.5, 1, 3, 6, 24 and 48 h after the release, and the number of eggs laid per plant was counted after 48 h. Among the vegetables, relatively higher proportions of psyllids settled on D. carota and S. lycopersicum at 48 h after the release. For weeds, relatively higher numbers were observed on P. hysterophorus, N. physalodes and S. americanum. The oviposition preferences reflected the settling behavior, with higher number of eggs observed on plants with higher number of adults, especially D. carota, S. lycopersicum and P. hysterophorous. No eggs were laid on A. retroflexus. These results indicate that R. solanicola is likely polyphagous, with potential to feed and develop on several plant species, and is not restricted to Solanaceae. Further research is needed to determine suitability of these plants for nymphal development and reproduction of R. solanicola, so that management strategies can be established.

S3 PHLOEM & MESOPHYLL FEEDERS | 57

Chemical evidence of inducible tannins as plant defense response to Lygus lineolaris feeding on cotton squares: correlation with EPG waveforms

Cervantes FA and Backus EA*

USDA Agricultural Research Service, USA

*Corresponding author: [email protected] Oral Presentation

Probing behavior of Lygus lineolaris (Palisot de Beauvois), one of the most important pests affecting cotton production in mid-southern United States, has previously been characterized with electropenetrography (EPG). Cell rupturing (CR) and Ingestion (I) EPG waveforms were identified as two of the main stylet-probing behaviors by adult L. lineolaris. However, specific plant defense responses to these two types of EPG waveforms have yet to be correlated. With the use of a 3rd generation AC-DC electropenetrograph combined with histology and microscopy, CR and I were correlated with plant responses. Events of CR and I waveforms were artificially terminated when observed during the EPG recording. Histological samples of probed pin-head cotton squares were prepared and analyzed to correlate specific waveforms with feeding damage location and plant responses to insect feeding. Both CR and I were performed in the staminal column of the cotton square. During CR events, L. lineolaris secreted saliva that elicited the production of tannins as a plant defense response. Tannin production was dependent on the number of CR events performed by L. lineolaris. Degraded plant tissue and tannins were removed from the staminal column during waveform I. These results conclude the process of defining CR and I as probing waveforms performed by L. lineolaris on pin-head cotton squares. This study increases our understanding of the interactions between L. lineolaris and cotton as a feeding host, providing new research options that could be explored for host plant resistance and pest management. 58 | S3 PHLOEM & MESOPHYLL FEEDERS

Infection of powdery mildew (Blumeria graminis) reduces the fitness of grain aphids (Sitobion avenae) through the restricted nutrition and induced defense response in wheat

Zhiwei Kang*, Fanghua Liu, Xiaoling Tan, Zhanfeng Zhang, Jingyun Zhu, Honggang Tian, Tongxian Liu

Northwest A&F University, Yangling, Shaanxi, China

*Corresponding author: [email protected] Oral Presentation

In a natural ecological system, plants are often simultaneously attacked by both insects and pathogens which can affect each other’s performance directly and indirectly. Powdery mildew (Blumeria graminis) and grain aphids (Sitobion avenae) are two common antagonists that often pose a serious threat to wheat production. Research on a single pest factor (either insects or pathogens) affecting wheat has been conducted extensively. However, the impact of one pest’s presence on the others is still unknown in wheat. Our present work investigated how the presence of powdery mildew on wheat affects the performance of grain aphids and its natural enemy – a parasitic wasp Aphidius gifuensis. Our study indicated that the infection of B. graminis reduced the feeding activity, weight, and fecundity, and prolonged developmental time of S. avenae. The proportion of essential amino acids was lower and sucrose content higher with infestation of B. graminis. Only the contents of Pro and Gln increased among all of the amino acids after B. graminis infection. Furthermore, the infection of B. graminis also increased the content of total phenolics and activities of three related antioxidant enzymes. More importantly, A. gifuensis exhibited a higher preference for wheat with B. graminis and S. avenae infected/infested than that only infested by aphids. Our findings indicate that restricted nutrition and defense response of wheat induced by B. graminis strongly affected the performance of S. avenae. Meanwhile, the simultaneous attack of plants by herbivore insects and pathogens can alter the preference of parasitoids, and consequently, this kind of effect will impact pest management.

S3 PHLOEM & MESOPHYLL FEEDERS | 59

Leaf preference of the scale insects on fern species Asplenium serratum L.

Santos MG*1, Barreto MB1, Coutinho RLM1, Ribeiro AF1, Ribeiro GM1, Portugal AS1, Tietbohl LAC2, Rocha L2 and Wolff VRS3

1 Universidade do Estado do Rio de Janeiro, Brazil 2 Universidade Federal Fluminense, Brazil 3 Fundação Estadual de Pesquisa Agropecuária do Rio Grande do Sul, Brazil

*Corresponding author: [email protected] Oral Presentation

Interactions between ferns and insects are more poorly studied than those between insects and angiosperms. The objective of this study was to evaluate the abundance of scale insects and the chemical profile of different foliar stages of Asplenium serratum L. (Aspleniaceae). The field work was conducted in the Atlantic Rain Forest of Rio de Janeiro state, Brazil. The leaves were collected in the dry (DS) and rainy (RS) seasons (years of 2013 and 2014) and divided into sterile leaves (SL), fertile leaves with sporangia in the beginning of development (FLBD), fertile leaves with mature sporangia and moderate spore release (FLMR), and fertile leaves with mature sporangia and intense spore release (FLIR). In all leaf types, the flavonoids and terpenoids profiles were analyzed, as well as, the macronutrients, micronutrients and soluble carbohydrates. All scale insects were counted and their locations on the leaves recorded. Two scale insect species of the Diaspididae and one species of the Coccidae were recorded. Hemiberlesia palmae (Cockerell, 1893) (Diaspididae) was the most frequent scale insect on the leaves. The data indicate that scale insects have a preference to occupy sori of fertile leaves with mature sporangia and intense spore release. No flavonoids were detected and no terpenoid differences were found between the foliar stages. Thus, it wasn’t possible to establish a correlation between the abundance of scale insects and the flavonoids and terpenoids profiles. The Analysis of the Principal Components demonstrated four groups: 1(FLBD13, 14 and SL14); 2(SL13, FLMR13); 3(FLIR13) and 4(FLIR14, FLMR14). The spore production requires a high nutrient investment. The sori of Asplenium serratum are borne on veins and these are a great sink region of nutrients. Probably, the scale insects are attracted by these, close to the sori, where they can find food and to be camouflaged.

60 | S3 PHLOEM & MESOPHYLL FEEDERS

Feeding behavior of the aphid Rhopalosiphum maidis on BT-maize expressing two different toxins

Paulo PD*1, Almeida MF1, Ramos GS1, Oliveira EE1, Pereira EJG1 and Fereres A2

1 Department of Entomology, Universidade Federal de Viçosa, UFV, Brazil 2 Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 19

Rapid evolution of pest resistance to transgenic crops producing a single Bacillus thuringiensis (Bt) insecticidal toxin has led to deployment of a gene pyramiding strategy, in which the Bt crop plants produce two or more Bt toxins against the same target species to delay resistance development. However, the expression of Bt transgenes in plant tissues may affect the feeding behavior of non- target species, such as hemipteran insects, which may have consequences for population fitness. Here we used the Electrical Penetration Graph (EPG) technique to assess the feeding behavior of Rhopalosiphum maidis (Hemiptera: Aphididae) on maize plants expressing two Bt crystal proteins (cry1Ab and cry1F) and on their non-Bt isoline. Our results obtained in the F1 generation of R. maidis demonstrated that these insects spent significantly more time in non-probing behavior (i.e., no stylet insertion into the plant tissue) on the Bt maize than on the non-Bt isoline. The mean duration of intercellular stylet pathway (C wave) was reduced for the aphids on Bt maize when compared to those on the non-Bt maize. The mean duration of intracellular stylet punctures (pds) was higher for aphids on non-Bt plants. On Cry1Ab+Cry1F Bt plants, aphids spent less time in phloem feeding when compared to those on the non-Bt isoline. These preliminary results indicate that R. maidis aphids showed altered feeding behavior on genetically modified maize producing toxins Cry1Ab and Cry1F. Further studies are needed to determine if the observed changes in feeding behaviour may have consequences for R. maidis population increase and fitness.

S3 PHLOEM & MESOPHYLL FEEDERS | 61

Plant-aphid interactions: role of leaf senescence on the host plant preference by Myzus persicae

López Gialdi AI1,2*, Machado-Assefh CR 1,2, Alvarez AE2, Tjallingii WF3

1 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina 2 Cátedra de Química Biológica, Facultad de Ciencias Naturales, Universidad Nacional de Salta (UNSa), Argentina 3 EPG systems (epgsystems.eu), The Netherlands

*Corresponding author: [email protected] Poster 20

Leaf senescence allows M. persicae to establish on leaves of resistant Solanum spp. genotypes, and senescence induction in Solanum tuberosum improves the aphid’s phloem ingestion and development rate. Thus, leaf senescence may influence aphid preference and feeding behavior. Our objective was to study the preference and feeding behavior of M. persicae on leaves of S. tuberosum plants under different treatments that change leaf physiology and plant nutritional status. Hence, young plants (3 – 4 weeks old) were light deprived to induce leaf senescence, and untreated plants were the control group. In addition, mature plants (7 – 8 weeks old) were treated with citokinins to inhibit leaf senescence, and untreated plants with natural leaf senescence were the control group. Leaf senescence was assessed by quantifying leaf chlorophyll content from its RGB emission in digital images. Preference was evaluated with a T-bar dual choice test and probing and feeding behavior was studied by electrical penetration graph (EPG). To avoid visual cues, all the experiments were performed in darkness. The role of volatiles on plant attraction together with the feeding behavior was assessed related to leaf senescence and host plant acceptance. Results will be discussed at the symposium.

62 | S3 PHLOEM & MESOPHYLL FEEDERS

Investigation of sex determination genes in Bemisia tabaci

Corkill RL1*, Mugford ST1, Morrison N2 and Hogenhout SA1

1 Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, UK 2 Oxitec, 71 Innovation Drive, UK

*Corresponding author: [email protected] Poster 21

The Bemisia tabaci (tobacco whitefly) species complex contains some species of whiteflies highly resistant to many insecticides and can colonize and feed on 40+ plant families. The polyphagous and insecticide-resistant nature of some species in this whitefly complex causes high economic devastation during an invasion. New control methods need to be discovered to offer better crop protection against this pest. We are investigating how sex-specific gene expression is regulated in the MED (formally known as the “Q biotype”) putative species of B. tabaci. MED is more resistant to insecticides than the MEAM1 putative species (formally known as the “B biotype”), and therefore a greater priority for new control methods. This work will provide new insights into sex determination in whiteflies and other Hemiptera, with potential utility in future genetic pest management strategies.

S3 PHLOEM & MESOPHYLL FEEDERS | 63

Transcriptional responses of selected genes involved in ascorbate biosynthesis in maize seedlings infested with bird cherry-oat aphid (Rhopalosiphum padi L.)

Sytykiewicz H, Leszczynski B*, Czerniewicz P, Lukasik I, Sprawka I, Golawska S, Sempruch C, Chrzanowski G

Department of Biochemistry and Molecular Biology; University of Natural Sciences and Humanities, Siedlce, Poland

*Corresponding author: [email protected] Poster 22

The study was aimed at evaluating influence of the bird cherry-oat aphid (Rhopalosiphum padi L.) infestation on relative expression of two genes (galdh and gldh, encoding L-galactose dehydrogenase and L-galactono-1,4-lactone dehydrogenase, respectively) in maize (Zea mays L.) plants. The analysed genes are involved in biogenetic pathway of reduced ascorbate (AsA) in maize tissues. Experiments were carried out using 14-d-old seedlings of two maize cultivars (Złota Karłowa and Ambrozja – susceptible and relatively resistant to the tested hemipterans, respectively). Plants were infested with 20, 40 or 60 apteral parthenogenetic females of R. padi per seedling. Control plants remained uninfested with the insects. Transcriptional reprogramming of two examined genes was monitored with the use of real-time qRT-PCR technique, after 1, 2, 4, 8, 24 and 48 h post-initial insect infestation. The aphids’ herbivory profoundly upregulated expression of galdh and gldh genes in a time - and insect density- dependent manner. Inter-varietal differences were also observed in expression patterns of the genes in aphid-stressed plants. Our data indicate possible involvement of galdh and gldh genes in stimulating AsA biosynthesis in maize seedlings infested with R. padi females.

64 | S3 PHLOEM & MESOPHYLL FEEDERS

EPG monitoring of the probing behavior of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae) on maize (non- preferred host) and oat (preferred host)

Brentassi ME 1*, Álvarez AE 2 and Machado-Assefh C 2,3

1 División Entomología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC), Argentina. 2 Cátedra de Química Biológica, Facultad de Ciencias Naturales, Universidad Nacional de Salta, Argentina 3 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Argentina

*Corresponding author: [email protected] Poster 23

Delphacodes kuscheli is the main vector of “Mal de Río Cuarto virus” of maize in Argentina, a disease that produces important economic losses. Although this planthopper does not breed successfully on maize (Zea mays), virus transmission occurs when adults migrate from oat (Avena sativa), its preferred winter host and a reservoir of the virus, to juvenile plants of maize following the harvest of oats. Studies of histological sections of maize and oat leaves showed that D. kuscheli is a phloem feeder with the typical salivary-sheath behavior. However, on maize the number of sheaths terminating in mesophyll was higher than in phloem and higher than the number of sheaths terminating in mesophyll in oat, indicating a different behavior on both hosts. In this work, electrical penetration graph (EPG) was used to compare the probing and feeding behavior of D. kuscheli on maize and oat. Seven waveforms were identified: np (non-probing); D1+D2+D3 (pathway phase: penetration initiation, salivation, stylet movement and extracellular activities near the phloem region); D4-a (sieve element salivation); D4-b (phloem sap ingestion) and D5 (xylem activity). EPG showed that on maize compared with oat, D. kuscheli exhibited longer periods of non-probing before the first phloem activity, and the time from first probe to first phloem activity was longer. In relation to phloem activity, the contribution of phloem salivation (D4-a) to phloem phase was higher, and the total duration of phloem ingestion (D4-b) was shorter. In addition, the percentage of probing spent in xylem was higher. These EPG results are in accordance with the previous histological studies showing that in the non preferred host, the general probing and feeding behavior of D. kuscheli is constrained. Comparison of the feeding behavior of D. kuscheli on hosts with different preference will allow identification of underlying mechanisms that could provide new targets for control.

S3 PHLOEM & MESOPHYLL FEEDERS | 65

Volatile isoprenoids as modifiers of aphid probing and feeding behaviour

Dancewicz K1, Sznajder K1, Załuski D3, Kordan B2 *, Gabryś B1

1 Department of Botany and Ecology, Faculty of Biological Sciences, University of Zielona Góra, Poland 2 Department of Phytopathology, Entomology and Molecular Diagnostics, Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Poland 3 Department of Plant Breeding and Seed Production, Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Poland

*Corresponding author: [email protected] Poster 24

Volatile isoprenoids (VIPs) are low molecular isoprene-derived compounds that are produced and emitted by plants of most groups as a protection against and in response to various biotic and abiotic stresses, and participate in direct and indirect plant defense against herbivores. Foliar VIPs may accumulate in mesophyll cells, which make them detectable by aphids in the course of different phases of probing. In this study, the peach potato aphid Myzus persicae (Sulz.) (Hemiptera, Aphididae) probing behavior was monitored using electrical penetration graph technique (EPG), following the exogenous application of camphene, α- and β- ionones, and linalool to the host plant Brassica rapa subsp. pekinensis (Lour.) Hanelt (Brassicaceae). None of the VIPs studied affected M. persicae propensity to probe: aphids spent at least 75% of experimental time on penetration in plant tissues. The application of α-ionone and linalool did not alter M. persicae behavior in relation to the control. In contrast, on camphene and β-ionone-treated plants, the proportion of non-probing to other stylet activities was very high, the success rate in reaching sieve elements and feeding was low, and the proportion of salivation in phloem phase was very high. Our experiments, using aphids as sensors, have demonstrated in an indirect way that exogenously applied VIPs may penetrate plant cuticle and epidermis, and pass into deeper tissue layers. We also determined that the transcuticular application of certain VIPs may cause serious disturbances in plant recognition and acceptance processes of aphids, which may reduce aphid infestation. The aphid reluctance to continue probing and feeding due to the exogenously applied VIPs may be considered as means for the limitation of the transmission of semi-persistent and persistent viruses.

66 | S3 PHLOEM & MESOPHYLL FEEDERS

Insight into color vision in the Asian citrus psyllid

Allan SA1*, Paris TM2 and Stansly PA2

1 United States Department of Agriculture, Agriculture Research Service, USA 2 University of Florida, Southwest Florida Research and Education Center, Immokalee, FL 34142, USA

*Corresponding author: [email protected] Poster 25

The Asian citrus psyllid, Diaphorina citri, the vector of the pathogen causing the devastating citrus disease, huanglongbing, resides primarily in citrus canopies with intermittent dispersal to new habitats. Behavior is strongly impacted by visual cues and a better understanding of these can provide insight into enhanced surveillance and management strategies. As a foundation for this research, electrophysiological studies were conducted to reveal the presence of four photopigments (one ultraviolet (UV), three in the visible range). Laboratory assays were undertaken to specifically address the impact of different wavelengths on movement-associated behaviors in the psyllids. Ultraviolet and green light differentially affected klinotaxis with strong orientation towards ultraviolet light and little movement under green light. Additionally, age affected walking responses with older psyllids less mobile under green light. The presence of UV in orientation to plant visual cues was also examined. These results will be discussed in relation to movement of psyllids within tree canopies and between trees.

S3 PHLOEM & MESOPHYLL FEEDERS | 67

Effect of two essential oils of Mentha pulegium L. and Pistacia lentiscus L. on the feeding and orientation behavior of the green peach aphid Myzus persicae Sulzer

Behi F1,2*; Giordanengo P3; Bachrouch O2 and Bouhachem-Boukhris S2

1 Faculté des Sciences de Bizerte 7021 Jarzouna, Tunisie. 2 Laboratoire de Protection des Végétaux, INRAT, Tunisie. 3 Biologie des Entomophages (UPRES EA 3900), Université de Picardie Jules Verne, France.

*Corresponding author: [email protected] Poster 26

The green peach aphid Myzus persicae is a sap sucking feeder implicated in the transmission of more than 100 plant viruses causing loss of crop yield and quality. The control of this aphid is based on intensive and repeated use of chemicals which causes aphid resistance to almost all the classes of pesticides. Essential oils provide new control methods developed to avoid the resistance problems. The present study aims to investigate the effect of essential oils from Mentha pulegium and Pistacia lentiscus on the orientation and feeding behavior of M. persicae. The orientation test was conducted in a static olfactometer (dual choice test). The results detected no significant differences between both essential oil treatments and the control. In fact, the use of the essential oils doesn’t change the attractiveness of the plant to aphids. Furthermore, electrical penetration graph (EPG) was used to estimate the durations of phloem and xylem sap consumption by aphids feeding on treated leaves. The results of EPG show that the number of aphids accessing the phloem and xylem were reduced by 50 % and 30 %, respectively. We also demonstrated that the time spent by aphids feeding on phloem sap with treated leaves is shorter than in the control. In addition, the consumption duration of the xylem sap was doubled on treated leaves compared to the control. This seems to be associated with a need to recover from dehydration due to the essential oil treatments. Our results show that essential oil treatments have no repulsive effect on aphids but they may disturb their feeding.

68 | S3 PHLOEM & MESOPHYLL FEEDERS

Insight into feeding behavior and salivary proteome of two pentatomids

Serteyn L*, Mabola JCF and Francis F

Functional and Evolutionary Entomology – Gembloux Agro-Bio Tech – University of Liege – Gembloux, Belgium

*Corresponding author: [email protected] Poster 27

The invasive Halyomorpha halys Stål, the brown marmorated stink bug, has widely spread throughout North America and Europe. Its ability to adapt to many host families is probably a boost for world colonization and may be closely related to its feeding activity. Therefore we investigated the feeding behavior on faba bean (Vicia faba L.) of two Hemipteran pests with different levels of polyphagy: brown marmorated stink bug and Nezara viridula (L.), the southern green stink bug. Despite the lack of complete electropenetrography (EPG) waveform libraries published for these pests, we could deduce some functional parameters associated with non- probing, test probes and phloem feeding events. In comparison with N. viridula, H. halys presents a longer penetration phase into phloem sieve elements, which is accompanied by liquid salivation and sap tasting. Complementary proteomics on stink bugs salivary glands was performed by liquid chromatography coupled with tandem mass spectrometry. The role of the identified proteins in plant-insect interactions is discussed. By pairing EPG and analysis of salivary compounds, we suggest an interesting way of investigation on pest adaptation to numerous host plants.

S3 PHLOEM & MESOPHYLL FEEDERS | 69

Elevated carbon dioxide concentration reduces alarm signaling in aphids

Boullis A, Serteyn L*, Fassotte B, Sarles L, Lognay G, Heuskin S, Vanderplanck M, Bartram S, Haubruge E, Francis F and Verheggen FJ

Functional and Evolutionary Entomology – Gembloux Agro-Bio Tech – University of Liege – Gembloux, Belgium

*Corresponding author: [email protected] Poster 28

Temperature and atmospheric greenhouse gas concentrations are rising and are not likely to stabilize in the foreseeable future. Assessment of these changes on species interactions is one ecological challenge. Like many insect species, aphids communicate with each other using semiochemicals, and because of their economical importance in agriculture, the understanding of their adaptation to a changing climate remains a major issue. Here, we test the hypothesis that changes in atmospheric carbon dioxide (CO2) concentration affect alarm signaling in aphids, and specifically (E)-β-farnesene (Eβf - the main compound of alarm pheromone) (1) production, (2) emission, (3) perception and (4) induced behavioral response. Indeed, it is important to consider the effect of elevated CO2 concentration on all the steps that a semiochemical has to pass through, from its biosynthesis and release by the emitting individual, until its perception and behavioral response expressed by the receiver. Using pea aphids (Acyrthosiphon pisum) reared under ambient or elevated CO2 concentrations (respectively a[CO2] and e[CO2]), we first quantified the whole body content of Eβf and the amount of Eβf released by pea aphids under predation. Pea aphids reared under e[CO2] were found to contain and release lower amount of Eβf. There were no significant differences in terms of neuronal responses to Eβf when using electroantennography. Finally, the escape behavior expressed by aphid colonies when exposed to natural doses of Eβf was strongly lessened when reared under e[CO2]. We conclude that the chemical communication of aphids reared under CO2 concentrations that mimic those predicted for the coming century will be affected. The potential impact of [CO2] and other abiotic parameters associated with climate change should be evaluated on each trophic level.

70 | S3 PHLOEM & MESOPHYLL FEEDERS

Climate change: impact on plant-aphid-ant interactions

Blanchard S¹², Detrain C², Lognay G³, Serteyn L¹* and Verheggen FJ¹

¹ Entomologie fonctionnelle et évolutive, Gembloux Agro-bio Tech, Université de Liège, Belgium ² Ecologie Sociale, C.P. 231, Université Libre de Bruxelles, Belgium ³ Chimie analytique, Gembloux Agro-bio Tech, Université de Liège, Passage des déportés, Belgium

*Corresponding author: [email protected] Poster 29

While the impact of climate change on plant or insect communities has been receiving increasing attention during the last decade, studies on plant-insect interactions under a changing environment are yet limited. These interactions are of particular importance in regard to the economic dimension of some crop plants and to the pest status of some insect species. In this project, we raise the hypothesis that the predicted changes in climate for the end of the 21st century – i.e. elevation of carbon dioxide concentration beyond 700 ppm, with an increase of 2 to 4°C (IPCC 2007) – will impact multitrophic relationships between herbivorous pests, their host plants and mutualist partners. These relationships are mediated by plant secondary metabolites, including volatile organic compounds (VOCs). Some modifications in atmospheric temperature or greenhouse gases concentration could alter the semiochemical interactions and behaviours in such multitrophic systems. Studying relations between plants, aphids and ants is an emblematic case of multitrophic relations involving an insect pest and mutualism. In this case, we will evaluate the impact of elevated atmospheric carbon dioxide concentration and elevated temperature, alone or in combination, on the secondary metabolites previously identified as important mediators of a multitrophic interaction model including a plant (Vicia faba), an aphid pest (Aphis fabae) and an aphid-tending ant (Lasius niger). We pay special attention to the plant semiochemicals, aphid biology and pheromones, and ant biology and semiochemicals.

S3 PHLOEM & MESOPHYLL FEEDERS | 71

Characterization of the electrical penetration graphs of the psyllid Bactericera trigonica on carrots

Antolinez CA*, Moreno A, Appezzato-da-Gloria B and Fereres A

Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 30

The psyllid Bactericera trigonica Hodkinson (Hemiptera, Triozidae) is a carrot and celery pest recently described as a vector of the plant pathogenic bacterium Candidatus Liberibacter solanacearum (Lso) on Apiaceae. Detailed information on vector stylet penetration activities is essential in the study of Lso transmission. In this study we used the electrical penetration graph (EPG) technique, characterized waveforms produced during the different stylet penetration activities in carrot leaves, and correlated them with stylet tracks and salivary sheath termini in plant tissues as well as with Lso inoculation. In addition, the effect of Lso in B. trigonica on the stylet penetration activities was tested. The EPG waveforms identified were: waveforms C1 and C2 detected in the mesophyll, waveforms D, E1, E2 near or in phloem sieve elements, and waveform G in xylem vessels. A waveform pattern not previously reported for psyllids was the "pseudo- potential drop" (pseudo-pd); characterized by sudden voltage dips similar to potential drops. However, the lowered voltage appeared to be inverted when the plant voltage is negative, indicating that it is caused by an increased resistance period and not due to a cell puncture. In addition, a direct correlation is shown between the waveform E1 and salivation into phloem sieve elements by B. trigonica as the inoculation of Lso occurred in a period as short as 30 seconds of E1; transmission percentage 48% (17/35 plants). On the other hand, stylet activities during waveforms C or D had no consequences on the inoculation of Lso. Interestingly, Lso infection directly affects the probing behaviour of B. trigonica by increasing the total duration of C and D waveforms, but not on variables related to phloem salivation or ingestion. The new information will be helpful to identify the psyllid vector behavioural traits associated with transmission of Lso to Apiaceae.

72 | S3 PHLOEM & MESOPHYLL FEEDERS

Population dynamics and damage of the invasive phloem-feeder psyllid Acizzia uncatoides (Hemiptera : Psyllidae) on the endemic tree Acacia heterophylla on la Réunion Island

Marquereau L1, Dervin S3, Ah Peng C1, Salamolard M2, Wagner PA1, Rouget M3 and Reynaud B1*

1 UMR PVBMT, University of La Réunion, France. 2 Parc National de la Réunion, France. 3 UMR PVBMT, CIRAD, La Réunion, France.

*Corresponding author: [email protected] Poster 31

Phloem feeders, such as psyllids are known as pests in agriculture and in natural ecosystems. Acizzia uncatoides originates from Australia and is an invasive psyllid pest on Acacia koa in Hawaii. This psyllid species has been recently found on Acacia heterophylla Willd, an endemic tree on la Réunion Island (Ouvrard et al, 2016). This tree is the dominant canopy tree at mid elevations in endemic forests, and is distributed throughout the National Park of the island. In the present study, population dynamics and the effects of the psyllid on A. heterophylla were studied, as well as the diversity of its potential natural enemies present on the tree. A survey was conducted by monitoring three altitudinal transects set up within the National Park (three to five points between 1350 to 2350 m). Damage on A. heterophylla was recorded and A. uncatoides larvae and adults were counted. Severe damage was observed such as leaf chlorosis, defoliation and dieback. The higher the elevation, the more numerous and damaging the psyllid appeared to be. Acizzia uncatoides was found in all transects in the 13 sites. During the sampling process, some predators like net-winged insects (especially Hemerobidae and Chrysopidae larvae) and predatory mites occurred in abundance, and to a lesser extent Coccinellidae predators. The risks to endemic forests of Reunion Island and the possibilities of biological control of A. uncatoides are discussed.

S3 PHLOEM & MESOPHYLL FEEDERS | 73

Scaphoideus titanus performs a dual-meaning, sharpshooter-style EPG X wave in xylem and phloem: Could S. titanus be a potential vector of Xylella fastidiosa in European vineyards?

Chuche J, Sauvion N*, Backus E and Thiéry D

INRA, UMR BGPI 0385 (INRA-CIRAD- SupAgro Montpellier), France

*Corresponding author: [email protected] Poster 32

In phytophagous hemipterans, salivary sheath-feeding species are described as xylem or phloem sap-ingesting specialists. Because these two food sources have very different characteristics, two different feeding tactics are associated with this supposed specialization. Study of feeding behavior is crucial to elucidate transmission abilities of vectors of plant pathogens. In a recently published DC-EPG study, results showed that the leafhopper Scaphoideus titanus (subfamily Deltocephalinae) ingests significantly from both phloem and xylem. S. titanus is an invasive species originally from North America that is now common in French vineyards. Until now, this insect has been considered a phloem-feeder; however, this attribute was solely based on its role as a vector of the Flavescence dorée phytoplasma. More importantly from a vector standpoint, S. titanus recordings revealed the first dual-meaning X wave, containing two different-appearing ingestion waveforms that were correlated with phloem and xylem ingestion, respectively. In addition, the S. titanus X wave strongly resembled that of sharpshooter leafhoppers (subfamily Cicadellinae), vectors of Xylella fastidiosa ssp. fastidiosa, the xylem-inhabiting bacterium that causes lethal Pierce’s disease of grape. Recent research has shown that the sharpshooter X wave represents the essential feeding behaviors responsible for inoculation of X. fastidiosa into grape xylem cells. Although many hemipterans ingest from xylem, probably only those few species that perform a sharpshooter-style X wave can be X. fastidiosa vectors. Therefore, is it possible that S. titanus could also become a vector of Xylella fastidiosa? Until now, no deltocephaline leafhopper has been formally proven to be a vector of X. fastidiosa. Also, North American X. fastidiosa ssp. fastidiosa has not yet been introduced to Europe. However, if this subspecies were introduced into European vineyards, could S. titanus (already present in the vineyards) be a potential vector?

74 | S3 PHLOEM & MESOPHYLL FEEDERS

Exploring non-nutritional, nutritional and biophysical parameters regulating antibiotic and antixenotic interactions between cotton- germplasms and two phloem-feeders, Bemisia tabaci and Amrasca devastans

Asrar M1, *,Wakil W2, Gogi MD2 and Zia K2

1 Department of Zoology, GC University Faisalabad, Pakistan 2 Department of Entomology, University of Agriculture Faisalabad, Pakistan

*Corresponding author: [email protected] Poster 33

Study was carried to explore non-nutritional, nutritional, biophysical plant-parameters regulating antobiotic and antoxenotic interactions between cotton-germplasms including one conventional non-bt (FH-942) and ten transgenic cotton-germplasms (FH-113, Bt-121, FH-114, VH-282, IR-901, BT-886, N-121, CRSM-2007, Al- samei and VH-144) and two phloem-feeders, Bemisia tabaci (whitefly) and Amrasca devastans (jassid). The results of both studies revealed that Bt-121 and FH-113 demonstrated comparatively stronger antibiotic and antixenotic interactions in form of resistance. Irri-901, FH-114, CRSM-2007 and N-121 exhibited intermediate; whereas, Al-saemi, Bt-886, VH-282, VH-144 and FH-942 exhibited least level of antibiotic and antixenotic interactions against whitefly and jassid. Among biophysical plant-parameters, area of leaf-lamina and density of gossypol-glands were dominating parameters attributing 71-73% variation in settling, eggs- density and progeny-development of both phloem-feeders on tested cotton-germplasms. The germplasms exhibiting stronger antibiotic and antixenotic interactions had less leaf-area and more gossypol-glands than those exhibiting intermediate and least level of antibiotic and antixenotic interactions. Area of leaf-lamina exhibited a significant positive while gossypol-glands demonstrated strong negative correlation to settling, eggs-density and progeny-development of both phloem-feeders. Bt-121 and FH-113 had less moisture (< 80%), protein (< 10%), amino-acid (< 40%), carbohydrate (<10%) and ash contents (<10%) while higher potassium (> 0.4 ppm), phosphorous (> 2%) and phenol contents (> 2%) in leaves than those demonstrating intermediate and least level of antibiotic and antixenotic interactions. Moisture, nitrogen, protein, amino- acid, carbohydrate and ash contents had strong positive whereas, phosphorus, potassium and phenol contents exhibited strong negative correlations to settling, eggs-density and progeny-development of whitefly and jassid. All above-mentioned leaf-contents explained 83%, 95%, 84.2% and 94.75% variation in settling, eggs-density and progeny-development of whitefly and jassid. Phenol, ash (non-nutritional), carbohydrates, amino-acids, proteins (nutritional), area of leaf-lamina and density of gossypol-gland (biophysical) were determined as the prominent plant-parameters regulating antixenotic and antibiotic interaction between cotton-germplasms against two phloem-feeders.

Hemiptera-Symbiont

4 | Interactions

76 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Mutualism turns into aggressive mimicry in an aphid

Salazar A, Pérez-Hidalgo N, Moreno-González V and Martínez-Torres D*

Institut de Biologia Integrativa de Sistemes & Institut Cavanilles de Biodiversitat i Biologia Evolutiva. Universitat de València, Spain

*Corresponding author: [email protected] Oral Presentation

Myrmecophily is the term applied to the association between ants and a variety of other organisms like plants, arthropods and fungi. Numerous insect species have been cataloged as mymecophilous. Within Hemiptera, especially in the suborders Sternorrhyncha (aphids, mealybugs and whiteflies) and Cicadomorpha (cicadas and membracids) many species exhibit some degree of myrmecophily. Many of these mutualisms are facultative and unspecialized, but all have a common denominator: the interaction is mainly based on the production by these insects of sugary excretions that are consumed by the ants which, in return, provide protection against predators and cleaning services. This type of association is known as trophobiosis and about 40% of aphid species participate obligately or facultatively in this type of association with different ant species. Although this description constitutes the paradigm of the interactions between aphids and ants, the ants do not only exhibit mutualistic relations with aphids. It is possible to find ants that combine care and predation (they harvest both the honeydew and the "meat" of the aphid) and others that are only predatory on aphids. Recently, we described an unprecedented relationship between the aphid species Paracletus cimiciformis and ants of genus Tetramorium. In the root dweling phase of P. cimiciformis, two alternative clonal morphs have evolved which maintain very different relationships with the ants. While the so-called green morph maintains a typical trophobiotic relationship with ants of the genus Tetramorium, the white morph possesses a cuticle whose chemical composition (and therefore its odor) mimics that of the ants’ larvae. This causes ant workers to mistakenly treat white aphids as if they were their on larvae transporting them to their breeding chambers along with their true ant larvae. Once there, we showed how white aphids suck on ant larvae hemolymph, thus constituting the first case of aggressive mimicry reported in aphids.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 77

Horizontal transmission of insect symbiont Serratia symbiotica via phloem and new insight into the acquisition of the symbiont

Pons Guillouard I*, Renoz F, Noël C and Hance T

ELIB – Université catholique de Louvain, Louvain-la-neuve, Belgium

*Corresponding author: [email protected] Oral Presentation

Symbiosis is quite a common phenomenon in insects where many species have been shown to harbor symbiotic bacteria within their gut, tissues, and/or cells. Thanks to their well-studied associations with an obligate nutrient-provisioning symbiont and a wide range of facultative symbionts, aphids represent a valuable model to study symbiotic relationships. Among this symbiotic diversity, S. symbiotica is one of the most common facultative partners found in various aphid species. Recently, several S. symbiotica strains were isolated from A. fabae and cultivated, constituting the first symbiotic bacterium of aphids able to grow outside its host. These special strains are located in gut level of aphids. The great diversity of S. symbiotica supposes a means of horizontal transmission, but few mechanisms for this transmission has been described. Here, we used the black bean aphid Aphis fabae to examine the horizontal transmission of free-living S. symbiotica strain via phloem of broad bean Vicia faba and the acquisition of the symbiont by plants and aphids. Using laboratory experiments, molecular analyses and hydroponic cultivations, we found that free-living S. symbiotica were transferred from A. fabae host to a plant, moved inside the phloem, and could be acquired by other aphids. We also showed the possibility of uptake of free-living S. symbiotica by roots of hydroponically grown broad bean plants. Our study highlights the importance for considering plants as a reservoir for horizontal transmission of symbionts and can promote the emergence of novel symbiotic associations.

78 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Microflora analyses of the Australian eggplant psyllid, Acizzia solanicola

Morris J1,2,3*, Shiller J1,3, Mann R1,3, Smith G1,4, Yen A1,2,3 and Rodoni B1,2,3

1 Plant Biosecurity Cooperative Research Centre, , Australia 2 La Trobe University, Australia 3 Department of Economic Development, Jobs, Transport and Resources, AgriBio, Australia 4 Plant & Food Research Lincoln, New Zealand

*Corresponding author: [email protected] Oral Presentation

The causative agent of zebra chip disease, ‘Candidatus Liberibacter solanacearum’ (CLso), and the causative agents of citrus huanglongbing (HLB) and citrus greening disease, ‘Ca. Liberibacter asiaticus’ (CLas), ‘Ca. Liberibacter americanus’ (CLam) and ‘Ca. Liberibacter africanus’ (CLaf), are all quarantinable pathogens in Australia. If introduced, they would pose a severe threat to the Australian potato and citrus industries respectively. The psyllid species that vector these pathogens are not present in Australia, however, Australia is a centre for psyllid diversity and little is known about whether there is liberibacter-like organisms in native psyllids. Additionally, the potential role of Australian psyllid species in transmitting non-pathogenic and pathogenic Ca. Liberibacter species is not known. Acizzia solanicola, commonly known as the eggplant psyllid, is a native Australian psyllid that has broadened its host range to have plant-host crossovers with Bactericera cockerelli, the tomato potato psyllid. Without disease symptoms present or reported for A. solanicola hosts, liberibacter generic primers were designed and a multilocus sequence analysis approach using next generation sequencing performed to target liberibacter-like species. These methods were able to detect a novel species of liberibacter, ‘Candidatus Liberibacter brunswickensis’ (CLbr) and can aid the detection of liberibacters globally. The biology of CLbr in A. solanicola feeding on eggplants and genomics of liberibacter genus is explored.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 79

Characterization of bacteria associated with the leafhopper Matsumuratettix hiroglyphicus (Matsumura) vector of sugarcane white leaf disease

Wangkeeree J*and Hanboonsong Y

Thammasat University, Thailand

*Corresponding author: [email protected] Poster 34

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) is the key vector of phytoplasma which causes the sugarcane white leaf disease. It is the most destructive disease in Southeast Asia and vector management has been considered as one of the important control strategies of the disease. We studied both uncultured and cultured bacteria for possible use as tools to control the disease. For uncultured bacteria identification, the 16S rRNA genes were amplified and analyzed by cloning and sequencing. Two types of dominant uncultured bacteria, Candidatus Sulcia muelleri and an unknown type belonging to Betaproteobacteria class which we named BAMH (Bacterium Associated with M. hiroglyphicus) were identified. They were suggested as primary endosymbionts because they were found to co-localize in the leafhopper’s bacteriome and were revealed throughout the insect vector life cycle. For cultured bacteria identification, the bacterial colonies were isolated and identified using molecular methods. The predominant bacterial genera were Bacillus, Microbacterium and Arthrobacter. Among of these, six bacterial isolates including M. testaceum, B. licheniformis, B. subtilis, B. megaterium, A. woluwensis and A. nicotianae were selected to be tested for their pathogenicity and their effect on the leafhopper fecundity. The insect mortality reached 6-23% after 7 days feeding with a bacteria suspension of (p≤0.05 compared with feeding without bacteria) and the average number of eggs laid decreased to about 20-70% after 72 hrs feeding with bacteria suspension before mating (p≤0.05). This study of bacteria and impact on insect vector could be beneficial to use as a candidate control agent for vector management.

80 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Scorpion antimicrobial peptides affect fitness of pea aphid by impairing its bacterial symbionts

Skaljac M1*, Luna-Ramírez K1, Grotmann J2, Kirfel P2 and Vilcinskas A2

1 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Bioresources Project Group, Germany 2 Institute for Insect Biotechnology, Justus Liebig University of Giessen, Germany

*Corresponding author: [email protected] Poster 35

Aphids are notorious agricultural insect pests with remarkable reproductive potential. They cause damage to numerous crops by feeding on the phloem sap and vectoring hundreds of plant viruses. For millions of years aphids have lived in symbiosis with several bacteria that enabled their ecological and evolutionary expansion. On the other side, scorpions developed over 450 million years of evolutionary history potent and specific anti-insect compounds and antimicrobial peptides (AMPs). We hypothesized that scorpion AMPs might be highly effective in disrupting aphid‘s relationship with their symbiotic bacteria. By feeding pea aphid Acyrthosiphon pisum (Harris) with natural and enhanced scorpion AMPs, we aimed to disrupt and eliminate bacterial symbionts and therefore reduce aphid fitness. Results showed that scorpion AMPs drastically reduced aphid survival, delayed their reproduction and reduced density of bacterial symbionts in aphids. Chemical insecticides are still important control strategy against aphids, but due to resistance concerns, environmental contamination and risk for humans and beneficial organisms, there is a constant need for alternative control strategies. This communication shows that scorpion AMPs hold potential to be developed as biodegradable insecticides and could be deployed through bioengineering of host plants or symbiotic bacteria.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 81

Role of endosymbionts in the pear psylla, Cacopsylla pyri

Le Goff G*, Fakhour S, Lebbe O and Hance T

Université catholique de Louvain, Earth and Life Institute, Louvain-la-Neuve, Belgium

*Corresponding author: [email protected] Poster 36

Many sap feeders are living in strong association with obligate endosymbionts that produced the essential amino-acids, like Buchnera aphidicola in aphids. In the same way, psyllid insects host an obligate endosymbiont, Carsonella ruddii. However, in this case, about half of the biosynthesis pathways for essential amino-acids are missing partially or entirely in this species, questioning the role of this bacterium as essential an amino-acid provider. In that context, we were interested to determine the whole bacteria community living in C. pyri. The presence of endosymbionts was determined by Illumina sequencing in psyllids of different origins (lab and field populations) and stages (eggs, larvae, adults), in the phloem sap, and in the honeydew. At the same time, we compared the phloem sap to what is excreted in the honeydew in order to determine which amino-acids are ingested or transformed. The bacteriome of C. pyri is dominated by the secondary endosymbiont of C. pyricola (84%) followed by the primary endosymbiont of psyllids, C. ruddii (11%). These two symbionts were found in all tested individuals, suggesting a nutritional role or at least obligate functions that they have in the psyllid. Our study also revealed that two amino-acids were found in higher proportion in the honeydew than in the phloem sap: asparagine and glutamine suggesting that the psyllids received an oversupply of these two amino-acids from another source than the phloem sap. Furthermore, two essential amino acids were not found in phloem sap samples: methionine and tryptophan suggesting that these two aminoacids are also synthesized by endosymbiont(s). The results obtained in this study allow better understanding the role of endosymbionts in the nutrition of C. pyri. Discovery of the metabolic pathways involved in the synthesis of the amino-acids will be interesting continuation of this study.

82 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Are gut bacteria involved in whiteflies' plant adaptation?

Santos-García D1*, Mestre-Rincon N1, Zchori-Fein E2, Morin S1

1 The Hebrew University of Jerusalem, Robert H. Smith Faculty, Department of Entomology. Rehovot, Israel 2 Department of Entomology, Newe-Ya’ar Research Center, ARO, Ramat-Yishai, Israel

*Corresponding author: [email protected] Poster 37

Polyphagous insects require an ability to overcome different sets of host plant defense compounds. In general, during host-plant switching, there is a quick response of the insect, which shows a reduced performance that only rebounds few generations later. Natural selection (a slow process) seems not be the main force driving this relatively short-term adaptation. Indeed, phenotypic plasticity and host-gut bacteria interactions are fast processes that could be involved in the host switching process. Gut-associated bacteria can help the host by processing the food and/or degrading the plant’s defense compounds. In addition, bacteria can acquire new genetic material to deal with new environmental conditions. The whitefly Bemisia tabaci, a tiny phloem- feeder, is considered an important polyphagous pest species. Reported microbiomes of B. tabaci are dominated by bacterial endosymbionts. However, because insects’ gut microbiota can play a major role in host-plant adaptation and are mainly acquired from the diet/environment, we hypothesized that B. tabaci might acquire environmental bacteria that can provide different benefits/costs related to host adaptation. Accordingly, our goal was to explore the putative role of gut bacteria in the plant-adaptation process of B. tabaci under field conditions. We conducted a field-like assay by following the adaptation process of a B. tabaci population switching from watermelon (suitable host) to pepper (unsuitable) along four generations. Each generation, we collected samples for gut dissections (cDNA 16S rRNA amplicons) and performance assays (offspring survival). Amplicons were generated using a blocking strategy to decrease the endosymbionts’ load and analyzed with USEARH and phyloseq. Our results shown that B. tabaci adaptation to pepper took 2-3 generations but also some microbiome differences between watermelon/pepper populations. Genera significantly associated with watermelon or pepper diets and with generational time were identified. These data suggest that gut bacteria might help B. tabaci to adapt to new host plants.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 83

Influence of symbionts in the biology of Heteropteran insects de Souza Prado S

Embrapa Environment, SP, Brazil

*Corresponding author: [email protected] Poster 38

Although bacterial associations occur within animals and plants, insects are considered to be the largest group that has substantial advantages of these associations. This close relationship between both, microorganisms and insects usually provide innumerous advantages for both. Symbionts can provide some essential nutrients and/or help in the insect digestion, which is really important since insects feed in a poor diet such as many important agricultural pests and disease vectors within Heteroptera. In this suborder, symbionts can be found extracellularly in their midgut. Transmission mechanism of symbionts by the heteropteran insects occurs in different manners (vertical or horizontal routes) within the families, however several families need to be investigated. As many heteropteran symbionts are not cultured outside their hosts, it is being used the phylogenetic placement of the gene 16S RNA of the symbionts to show that some symbionts have coevolved with stink bugs to the point that host fitness is impacted with the elimination or alteration of their symbiont, and others symbionts are more diverse indicating multiple evolutionary origins of the symbiotic associations and/or frequent horizontal transmission of the symbiotic bacteria. The notable collection of the genomic information of the symbionts allied to the recent advanced sequencing methods will facilitate the analysis of the host and symbiont genome which will allow a better understanding of the heteropteran/symbiont associations, and their ecological and physiological features. Additionally, the more we know about the evolutionary and biological aspects of these associations the development of alternative techniques for effective heteropteran pest control will became just a matter of time.

84 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Aphids RNAi efficiency and RNA viruses

Jinzhi Niu, Chao Ye*, Guy Smagghe, Jinjun Wang

Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, China

*Corresponding author: [email protected] Poster 39

Aphids are important agricultural pests with about 250 species known to cause damage not only by feeding on plant phloem but also transmitting plant viruses. RNA interference (RNAi) and RNA viruses are better potential approaches to manage these deleterious aphids due to the resistance of insecticides resistance and chemical residue. To obtain insights of the possible effect of RNA viruses in influencing RNAi efficiency of aphids, we analyzed the RNA viruses (or related sequences) based on viral metagenomics by sequencing small RNAs and the transcriptome coupled with bioinformatics analysis. Next, RNAi efficiency of the same strains of aphids (Acyrthosiphon pisum, Myzus persicae, and Aphis citricidus) were evaluated by both micro- injection and leaf-mediated feeding of dsRNAs targeting various genes including C002, Acetylcholinesterase gene, hunchback, and insulin receptor genes. In addition, the correlation of RNAi efficiency and infections of sequenced viruses were also analyzed by quantitative real-time PCR. Based on the available genomes of A. pisum and M. persicae, and the coming genome of A. citricidus sequenced by the third generation sequencing technology PacBio in together with Bionano, the core genes of RNAi machinery based on the homologies of Drosophila were compared. The expressions of these genes for three aphids were also detected upon the injection and ingestion of dsRNA. In total, the possible interactions of RNAi and infected RNA viruses in three aphids are analyzed and their implications would be discussed.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 85

Population settlement of Bemisia tabaci Mediterranean species (BIOTYPE Q) in Brazil

Moraes LA*, Marubayashi JM, Bello VH, Yuki VA, De Marchi BR, Krause-Sakate R, Pavan MA

Faculdade de Ciências Agronômicas de Botucatu- FCA/UNESP, Brazil

*Corresponding author: [email protected] Poster 40

Bemisia tabaci (Hemiptera: Aleyrodidae), is a complex of cryptic species and one of the most important agricultural pests worldwide, since it is a highly polyphagous insect and considered a virus supervector. More than two decades after the species B. tabaci Middle East Asia Minor 1 (MEAM1) invaded and settled in Brazil through ornamental plants, the presence of B. tabaci Mediterranean species (MED) was first reported in Rio Grande do Sul in 2014, and recently in São Paulo and Paraná States. In 2015, a first survey in São Paulo State revealed that MED species was present only in commercial ornamentals greenhouses and not in open field neither in vegetable cultures, where MEAM1 prevailed ever since. In 2016, however, a second and more extensive survey performed in São Paulo and Paraná showed that MED has spread through several important vegetable cultures, like tomato, cucumber and sweet pepper, either in greenhouses and open fields located near where MED was detected in ornamental plants. In addition, the secondary endosymbionts Arsenophonus, Hamiltonella and Rickettsia were detected by PCR and confirmed by sequencing and FISH analysis for MED from São Paulo and Paraná in 2015, differing from MED detected in Rio Grande do Sul that harbored Hamiltonella and Cardinium, and from the MED collected in 2016, whose sets were composed by Arsenophonus, Hamiltonella, Rickettsia and Wolbachia. Through phylogenetic analysis of gene mtCOI using the whitefly global database, specimens has shown to represent different haplotypes divided into two groups within the species MED. The settlement of MED represents a new concern to Brazilian agriculture.

86 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Comparing the genomes of Buchnera aphidicola from 10 aphid species

Burger, NFV and Botha A-M*

Genetics Department, JC Smuts Building, Stellenbosch University, South Africa

*Corresponding author: [email protected] Poster 41

All members of the order Hemiptera harbour Buchnera aphidicola as an endosymbiont tasked with enhancing their nutritionally poor phloem diet. This endosymbiotic association is believed to have established itself prior to the radiation of aphids 200 – 150 million years ago. As aphids radiated, it is believed that isolated B. aphidicola either adapted to their local environments, or suffered uncontrollable genome deterioration, which resulted in substantial gene losses and genome shrinkage. The genome of the largest known B. aphidicola endosymbiont (Myzus persicae, 643,517bp) is 35.29% larger than that of the smallest known B. aphidicola endosymbiont (Cinara cedri, 416,380bp) and contains 205 more genes. To determine whether this genomic discrepancy between B. aphidicola endosymbionts is as a result of a loss of dependence on B. aphidicola by its host, or an adaptation by B. aphidicola to better suit the needs of its host, we compared the genomes of B. aphidicola from 10 different aphid species to ascertain the level of conservation. Whole genome alignments of 10 B. aphidicola genomes were performed using MAFFT v7.222. The resulting consensus sequence of 715,488 bases showed sequence conservation of only 19.2% across all genomes (or 113, 779 bases per genome) with an overall average pairwise identity of 59.2% (a high of 84.2% and low of 40.8%). Gene content and identity of the different B. aphidicola strains were then compared to their counterpart genes in Escherichia coli, the closest free-living relative of B. aphidicola. Protein sequence similarity of different B. aphidicola to that of E. coli ranged from 100% up until below 10% and was positively correlated with an increase in GC content and gene length. This tentative shaping of B. aphidicola’s genome thus appears to be a more controlled reduction than that of genomic drift and mutational accumulation.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 87

Symbionts of the tomato potato psyllid in New Zealand and Honduras

Frampton RA, Sullivan KL, Barnes AM, Agnew NM, Smith GR, Scott IAW, Vereijssen J*

The New Zealand Institute for Plant & Food Research Limited, New Zealand

*Corresponding author: [email protected] Poster 42

The tomato potato psyllid (Bactericera cockerelli Šulc; TPP) is a significant pest in New Zealand on Solanaceous crops. It transmits ‘Candidatus Liberibacter solanacearum’, the putative causal agent of zebra chip disease of potatoes. TPP also has a range of bacterial symbionts associated with it. Little is known about the interactions between these associated bacteria and their interactions with TPP. In this study TPP were collected from different host plants in potato growing regions in New Zealand and Honduras during 2013 and 2014. Complete DNA was extracted from 192 individual insects from six New Zealand and two Honduras locations and sequenced. The obligate endosymbiont, Carsonella, was present in all samples. The sequences revealed a range of other bacteria also associated with TPP, including Wolbachia and Hamiltonella. TPP from the two different countries and locations harboured different combinations of bacteria with lower titres of Wolbachia in insects from New Zealand compared to those from Honduras. Additionally, using a network analysis, we observed differences in single nucleotide polymorphisms (SNPs) within bacterial species that separate samples based on the country of origin. The major differences in the composition of bacteria associated with TPP were related to the country of origin, with no association in respect to host plant or season. TPP populations are separated into four biotypes based on differences in the mitochondrial cytochrome oxidase I gene sequence. TPP in New Zealand are all of the Western biotype, while the Honduras samples were all of the Central biotype. Further samples from other locations are needed to investigate if these differences are due to the insect biotype or the geographic location. This information provides a baseline for understanding the complex interactions that occur between bacteria and their insect hosts.

88 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

Scale insects (Hemiptera: Coccomorpha) as trophobionts of ants (Vespoidea: Formicidae)

Masten Milek T*, Markotić V, Šimala M, Pintar M

CCAFRA Institute for plant protection, Croatia

*Corresponding author: [email protected] Poster 43

Scale insects feed on sap of their host plants and majority of them subsequently excrete abundant amounts of honeydew that attracts ants. In this mutualistic relationship scale insects act as trophobionts providing food for ants. In return, ants protect them from their natural enemies. Additionally, ants can stimulate scale insects into producing honeydew by tapping on their bodies and help them spread into new areas and to new hosts by partaking in their transmission (Maschwitz, 1990; Johnson et al. 2001). During twelve years of faunistic research on scale insects in Croatia (2006-2016), the following six ant species from Formicidae family have been found in association with scale insects: Crematogaster schmidti (Mayr), Crematogaster scutellaris (Olliver), Formica gagates Latreille, Lasius niger L., Lasius paralienus Seifert and Temnothorax affinis (Mayr). Scale insect species found in association with ants, as well as their host plants and distribution, will be shown.

S4 HEMIPTERA- SYMBIONT INTERACTIONS | 89

Portiera as a phylogenetic tool on whiteflies

Santos-García D1*, Mestre-Rincon N1, Netanel N2, Ouvrard D3, Zchori-Fein E2, Morin S1

1 The Hebrew University of Jerusalem, Robert H. Smith Faculty, Department of Entomology, Israel 2 Department of Entomology, Newe-Ya’ar Research Center, ARO, Israel 3 Department of Life Sciences, Natural History Museum, UK

*Corresponding author: [email protected] Poster 44

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are small phloem feeding insects that include several agricultural pests. In contrast to other insects, whiteflies taxonomy is based on nymphal stages morphology. However, these stages show phenotypic plasticity, which produces several inconsistencies at the genus level. All whiteflies have specialized cells (bacteriocytes) that host a bacterial endosymbiont named Candidatus Portiera aleyrodidarum. Portiera is an obligatory mutualistic endosymbiont that complements its host’s unbalanced diet, being essential for the whitefly. Portiera has been vertically transmitted since the divergence of whiteflies from their Psyllinea ancestor (circa 160 Mya), and therefore can reflect its host’s phylogeny. Also, Portiera from Bemisia tabaci lineage has lost the polymerase proofreading subunit (dnaQ) and presents an uncommon genome instability. Our main goal was to establish a phylogenetic framework based on several Portiera’s genes. This framework will be used to improve whiteflies classification and to trace the dnaQ loss and the rise of genomic instability. For these reasons, a PCR screening with specific primers targeting five Portiera genes (16S and 23S rRNA, groEL, dnaK, rpoD and dnaQ), plus some Portiera rearrangements, was performed on 22 whiteflies species (including exsiccata museum specimens). Phylogenetic methods were used for tree inference, divergence dating, species delimitation and ancestral node reconstruction of dnaQ/rearrangements (RaxML, BEAST2, bPTP and ape). We noticed that teh use of Portiera sequences outperform the universal mitochondrial cytochrome oxidase subunit I (mtCOI) region used so far. In summary, they show low signal saturation, avoid common misclassification of similar insects and can be used on parasitized samples. Therefore, it solves some problems/inconsistencies in current whiteflies phylogeny. The loss of dnaQ and the associated rearrangements are unique to the Bemisia genus.

90 | S4 HEMIPTERA- SYMBIONT INTERACTIONS

91 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Hemiptera-Plant

5 |Pathogen Interactions

92 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Differential transmission of criniviruses and begomoviruses by whiteflies of the Bemisia tabaci complex

Navas-Castillo J

Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" (IHSM-UMA-CSIC), Spain

*Corresponding author: [email protected] Keynote Lecture

Emerging virus diseases limit the production of important vegetable crops in tropical, subtropical and temperate regions worldwide, most of the causal viruses being transmitted by whiteflies (Hemiptera: Aleyrodidae). The majority of the whitefly-transmitted viruses belong to the genus Begomovirus (Geminiviridae), although whiteflies are also vectors of other virus groups including the genus Crinivirus (Closteroviridae). Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops worldwide. The symptoms of infected plants consist of yellowing, reduced size and upward curling of leaves, and drastic reduction in plant height and yield. TYLCD is caused by a complex of viral species in the genus Begomovirus, including Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), all of them transmitted by species of the Bemisia tabaci complex. Another emerging virus affecting tomato crops in many regions of the world is the crinivirus Tomato chlorosis virus (ToCV). ToCV causes a yellow leaf disorder with symptoms of interveinal yellowing and thickening of leaves, reducing production due to decreased fruit size and number. ToCV is transmitted by B. tabaci, Trialeurodes vaporariorum and T. abutiloneus. In Spain, populations of two species belonging to the B. tabaci complex have been recently identified in addition to the widespread Mediterranean (MED) species: South-Saharan Africa 2 (SSA2) and Italy 3. In this talk, I will present experimental evidence for reproductive isolation between MED, SSA2 and Italy 3 species as well as the results of experiments which demostrate differential transmission of TYLCVs and ToCV by these whitefly species.

S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS | 93

Clathrin-mediated endocytosis is involved in Tomato yellow leaf curl virus transport across the midgut barrier of its whitefly vector

Li-Long Pan*, Qun-Fang Chen, Juan-Juan Zhao, Tao Guo, Xiao-Wei Wang, Aliza Hariton- Shalev, Henryk Czosnek, Shu-Sheng Liu

Institute of Insect Sciences, Zhejiang University, China

*Corresponding author: [email protected] Oral Presentation

Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci (Gennadius). The organs involved in the circulative translocation of the virus in the insect and the time course of this process are known in their broad line. However, the transit of TYLCV from the digestive tract into the haemolymph is poorly understood. Here we studied the involvement of clathrin in this process by disrupting the clathrin-mediated endocytosis and the endosome network using inhibitor feeding, antibody blocking and dsRNA silencing. We monitored the quantities of TYLCV in the whitefly whole body, midgut and haemolymph as well as virus transmission efficiency to test plants. Following endocytosis and endosome network disruption, the quantity of virus was higher in the midgut relative to that of the whole insect body, and the quantity of virus in the haemolymph was reduced. The transmission efficiency of TYLCV by the treated insects was also reduced. These findings indicate that clathrin-mediated endocytosis and endosomes play an important role in the transport of TYLCV across the whitefly midgut. They suggest that new strategies for managing the whitefly-transmitted TYLCV can be developed by targeting clathrin-mediated endocytosis.

94 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Host-vector-pathogen interactions in the context of mixed infections by hemipteran- transmitted viruses

Domingo-Calap ML1, Moreno AB1*, Díaz-Pendón JA2 and López-Moya JJ1

1 Centre for Research in Agricultural Genomics CRAG, CSIC-IRTA-UAB-UB, Spain 2 Institute for Mediterranean and Subtropical Horticulture "La Mayora" (IHSM-UMA-CSIC), Spain

*Corresponding author: [email protected] Oral Presentation

Plant-pathogenic viruses transmitted by hemipterans vectors are responsible of relevant economic losses in agriculture worldwide. While numerous viral diseases are known to be caused by a single infection, field surveys and metagenomics studies are revealing the frequent occurrence of mixed viral infections in plants. Except in a few cases where severe synergistic interactions have been reported, many others remain unnoticed due to the lack of specific symptoms. Furthermore, the possible impact of mixed infections on the transmission of viruses by insects is largely unknown. Our project aims to address the mixed infection caused by the combination of certain relevant viruses affecting melon and tomato. Our interest is to evaluate the outcome of these mixed infections, first considering how they affect the severity of the disease on the host plant, but also the effects produced on virus-vector relationships. To initiate the experimental work, melon plants were co-infected under controlled conditions with isolates of the potyvirus Watermelon mosaic virus (WMV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV), using their natural vectors, aphids and whiteflies respectively. Viral loads were measured by qRT-PCR for both viruses at different time points. Transmission experiments using single and mixed infected plants as virus source are being performed to determine the influence of the disease in virus dispersion by their natural vectors. Also, free-choice experiments have been designed to explore the influence of the mixed infection on vectors behaviour. Similar approaches are being applied to a tomato pathosystem with co-infections of the begomovirus Tomato yellow leaf curl virus (TYLCV) and the crinivirus Tomato chlorosis virus (ToCV). The purpose of this project is to generate knowledge on the dynamics of mixed infections in melon and tomato plants, aiming to derive recommendations for disease control that could help to maintain the productivity of these high-value horticultural crops.

S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS | 95

The N-terminus of the Cauliflower mosaic virus aphid transmission protein P2 is involved in both transmission and microtubule interaction

Then C1*, Malouvet E1, Bak A2, Macía JL1, Blanc S1 and Drucker M1

1 INRA, UMR BGPI, TA A-54/K, Montpellier, France 2 Department of Plant Pathology, University of California, Davis, USA

*Corresponding author: [email protected] Oral Presentation

To move between hosts, many plant viruses including Cauliflower mosaic virus (CaMV) use aphids as vectors. When the insects feed on infected leaves, virus particles already present in the leaf attach to their mouth parts (stylets). This enables the viruses to be transferred to a new host plant when the aphids change plants. As a prerequisite for CaMV transmission, the viral helper protein P2 acts as a molecular linker and mediates binding of the virus particles to the aphid stylets. P2 is available in infected plant cells in a specific structure that is formed beforehand during CaMV infection. This structure is specialized for transmission and named the transmission body (TB). When inserting its stylets into an infected leaf, the aphid triggers ultra-rapid massive influx of tubulin into the TB, followed by its disruption. As a consequence, P2 is redistributed onto cortical microtubules, together with viral particles (that are simultaneously set free from intracellular storage sites) and forms the so-called mixed networks (MNs). The MNs are the predominant structure from which virus is acquired by aphids; inhibiting their formation reduces drastically transmission rates. To find out which are the P2 motifs and domains involved in MN formation, we generated a set of P2 mutants by Alanine scanning. These P2 mutants were analyzed in vitro and in planta (in and without viral context) for their capacity to interact with microtubules and to transmit the virus. One knock-out mutant with N-terminal alanine substitutions lost the capacity to form MNs and to support transmission. Other mutants had altered kinetics of MN formation and transmission capacity, P2 protein stability and phenotypes of TB and viral factories. The results allow to correlate P2's capacity for network formation and microtubule-binding with transmission, confirming the importance of MNs for transmission.

96 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Drought stress facilitates conditional mutualism in a plant-virus-vector interaction

Bosque-Pérez NA1*, Eigenbrode SD1 and Davis TS2

1 University of Idaho, USA 2 Colorado State University, USA

*Corresponding author: [email protected] Oral Presentation

Both biotic and abiotic conditions influence crop responses to stress, but studies on interactions between environmental conditions and insect-borne plant pathogens remain limited. Using the Barley yellow dwarf virus (BYDV) - wheat- Rhopalosiphum padi (bird cherry-oat aphid) pathosystem we tested the hypothesis that drought stress drives conditional outcomes in host- pathogen and host-vector relationships. Our results demonstrated that when water was abundant, BYDV infection reduced plant performance parameters such as aboveground growth, seed set, seed yield and germination. However, when water was chronically limiting, wheat infected with BYDV did not exhibit significantly more injury than plants exposed to nonviruliferous aphids, or control plants not exposed to aphids. When plants were subjected to acute drought stress, BYDV- infected plants surpassed both control plants and plants exposed to nonviruliferous aphids in all measured performance traits. Rhopalosiphum padi feeding experiments and subsequent life table analysis revealed that aphid fecundity improved 47% when feeding on BYDV-infected plants compared to noninfected plants when water inputs were chronically low. However, when plants received ample water, aphid fecundity was enhanced by only 23% after feeding on BYDV-infected compared to noninfected plants. Our data showed that varying water stress shifts a host-virus interaction from antagonistic to commensal to mutualistic. Similarly, aphid vectors of virus benefitted from feeding on infected hosts, especially when water stress was high. Further research demonstrated that effects are in part mediated by changes in plant hormone concentrations, with water availability and BYDV infection having primarily additive, rather than interactive effects on hormone profiles in wheat. Hormonal responses to virus infection may have effects on subsequent plant responses to environmental stress. Our findings are consistent with the hypothesis that mutualistic interactions are facilitated under elevated abiotic stress, suggesting that environmentally-driven ecological changes potentially favor coexistence of hosts, vectors, and pathogens in stressful environments.

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Is viral transmission by aphids enhanced upon manipulation of host Brassicaceae by Turnip yellows virus?

Halter D1, Chesnais Q2, Claudel P1, Baltenweck R1, Boissinot S1, Meyer S1, Hugueney P1, Krieger C3, Ziegler-Graff V3, Ameline A2, Brault V1*

1 UMR 1131 SVQV INRA-UDS, France 2 FRE CNRS 3498 EDYSAN, France 3 IBMP-CNRS-UDS, France

*Corresponding author: [email protected] Oral Presentation

Vector-borne plant pathogens can alter plant phenotype and vector behaviour in such a way that virus transmission may be affected (Mauck et al., Curr. Op. Plant Biol. 2016). We investigated the host plant manipulations by a circulative aphid-transmitted virus and the potential impact on its transmission by the aphid vector. The pathosystem is composed of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae) which is efficiently transmitted by the polyphagous aphid Myzus persicae, and two Brassicaceae host plants, Camelina sativa and Arabidopsis thaliana. Behavioural and physiological approaches were conducted to assess aphid fitness, colonization and feeding behaviour on infected and non-infected plants. In addition, volatile emissions and metabolite composition of leaves and phloem exudate were analysed on the same host plants. We observed that aphid preference for infected or non-infected plants varied depending on the plant species. Moreover, the feeding behaviour on infected plants was differentially affected on camelina or arabidopsis. Overall, the aphid behaviour on infected camelina was more conducive to virus transmission than on infected arabidopsis. Metabolites accumulation clearly distinguished infected from non-infected plants in both camelina and arabidopsis. Identification of compounds involved in camelina palatability is in progress. Volatile emission analysis highlighted a higher abundance of headspace volatiles in infected camelina plants compared to infected arabidopsis. These exploratory and comprehensive data suggest that the same virus is able to trigger different metabolomic changes in two plant species within the Brassicaceae family. Such plant modifications induced highly contrasting changes in the aphid behaviour, which issue may have important impact on virus transmission by aphids and on disease outbreak.

98 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Effects of Barley yellow dwarf virus infection on the interaction of virus susceptible and tolerant barley genotypes with different clones of the aphid species Rhopalosiphum padi

Will T*, Meiners T, Schliephake E, Habekuss A and Ordon F

Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Germany

*Corresponding author: [email protected] Oral Presentation

Plants are confronted with different biotic stresses at the same time and the response to one of those may change the interaction with another. In this context, it was demonstrated that virus infection of plants affects aphid behaviour in a way that virus free aphids appear to prefer virus infected plants due to a change of plant volatile composition. However, observations mainly focussed on one aphid clone feeding on infected and non-infected virus susceptible plants. In contrast to this, the aim of our study was to get information whether different virus free clones of R. padi behave in a similar way when confronted with virus infected susceptible plants or virus infected tolerant genotypes. Therefore, we initially conducted a comparative analysis of the effect of a Barley yellow dwarf virus (BYDV) infection on the volatile patterns of susceptible and tolerant (Ryd2 and Ryd3) barley genotypes by using gas chromatography-mass spectrometry. Virus infected and non-infected plants were offered to different clones of the aphid species Rhopalosiphum padi and their preference was examined in choice tests. To examine a potential correlation of host preference with BYDV acquisition and transmission efficiency aphid clones were previously selected for their efficiency in acquiring and transmitting BYDV. Behaviour of the selected R. padi clones was analysed by using electrical penetration graph (EPG) technique. First results of choice tests demonstrate that aphid clones with low BYDV acquisition and transmission efficiency prefer infected plants of the susceptible barley genotype and that uninfected plants of the tolerant barley genotypes are preferred by the aphid clone with high virus acquisition/transmission efficiency. Results will be discussed with regard to the importance of susceptible and tolerant barley genotypes as virus pools in the field.

S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS | 99

The effect of ant-aphid mutualisms and plant diversity on the spread of aphid-vectored plant viruses

Eubanks MD, Coppler L and Murphy JF

Texas A&M University and Auburn University, USA

*Corresponding author: [email protected] Oral Presentation

We tested the hypothesis that food-for-protection mutualisms between an abundant ant and aphids affected the spread of an aphid-vectored plant virus. We studied the invasive red imported fire ant (Solenopsis invicta), several common aphids that form facultative mutualisms with these ants, and a ubiquitous, aphid-vectored plant virus (Cucumber mosaic virus (CMV)) in a three-year field experiment conducted on tomato farms. We found that the abundance of fire ants in weedy areas of tomato farms was positively correlated with the density of alate aphids in nearby tomato fields and the density of alate aphids was positively correlated with CMV incidence in tomato. We also found that suppressing fire ants in weedy areas near tomato fields reduced the number of aphids in tomato. Most importantly, we found that the abundance of fire ants was associated with increased CMV incidence and that suppression of fire ants reduced CMV incidence in some fields. The effects of fire ants on the spread of CMV, however, depended on the diversity of the herbaceous plant community surrounding tomato fields. Fire ants were more likely to increase the spread of CMV from herbaceous plants to tomato when the herbaceous plant community was relatively diverse. At our field sites, the two main non-crop hosts of CMV (greenbriar (Smilax spp.) and ivy-leaf morningglory (Ipomoea hederacea)) and the two main non-crop hosts of aphids that vectored CMV (ivy-leaf morningglory and blackberry (Rubus spp.)) were more likely to co-occur in relatively diverse plant communities than in species poor plant communities. This is one of the few studies to document the effect of a mutualism on the spread of a pathogen and perhaps the first study to show that diversity can mediate the role of mutualisms in disease ecology.

100 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Elevated CO2 impacts aphid and plant physiology, virus incidence and interactions between them

Trębicki P1*, Bosque-Pérez N2, Rodoni B1 and Fitzgerald G3

1 Department of Economic Development, Biosciences Research, Australia 2 Department of Plant, Soil and Entomological Sciences, University of Idaho, USA 3 Agriculture Research Division, Australia

*Corresponding author: [email protected] Oral Presentation

Atmospheric carbon dioxide (CO2) concentration has been increasing rapidly, currently exceeding

400 µmol mol-1. Cumulative CO2 emissions, together with human population growth, will put further pressure on the future CO2 level, which is projected to double by the end of this century.

As a result, elevated CO2 will impact plants, including food crops directly and indirectly through increase in temperature, changes in weather patterns and severity. Consequently, this can affect epidemiology of vector transmitted pathogens as well as performance, population size and efficiency of pathogen spread by many insect vectors. In this study, we examined the effects of ambient CO2 (aCO2; 400 µmol mol-1) and elevated CO2 (eCO2; 650 µmol mol-1) on the interactions between the bird cherry-oat aphid (Rhopalosiphum padi), Barley yellow dwarf virus and wheat plant growth in plant growth chambers. Due to a reduction of plant nitrogen content in virus free wheat, which was mediated by eCO2, prolonged aphid feeding and lower offspring production was shown. Although no changes to aphid fecundity and feeding rates were observed on BYDV- infected plants, irrespective of CO2 treatment. We also report virus epidemiology from the free air carbon dioxide enrichment (FACE) facility, its effects on the wheat and aphid vectors. Results from both FACE and growth chamber experiments, influencing interactions between aphid, virus and plant will be presented and the potential ecological and epidemiological consequences discussed.

S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS | 101

Understanding the transmission mechanism of Rice stripe virus by its insect vector Laodelphax striatellus

Xifeng Wang* and Wenwen Liu

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China

*Corresponding author: [email protected] Oral Presentation

Yeast two-hybrid system was used to study proteomic interactions of Rice stripe virus, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication and transovarial transmission1. Then, several techniques, including pull-down, immunofluorescence, immunoelectron microscopy and RNA interference, were used to verify the RSV ingeniously utilizing the vitellogenin (Vg) of L. striatellus to invade nurse cells in the germarium of ovary, then spread to the oocyte through the nutritive cord for completing transovarial transmission2. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidences that viruses can use existing vector proteins to aid their survival and transmission in vector insect.

102 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Inoculation of Wheat dwarf virus strains to wheat and barley plants by Psammotettix alienus

Abt I12, Souquet M1, Derlink M3, Angot G1, Gauthier K12, Mabon R1,2, de Keyzer A2, Letroublon M2, Thébaud G1, Virant-Doberlet M3 and Jacquot E1*

1 UMR BGPI, INRA, Montpellier SupAgro, Campus international de Baillarguet, Montpellier, France 2 Bayer S.A.S./Bayer CropScience, France 3 National Institute of Biology (NIB), Department of Organisms and Ecosystems Research, Slovenia

*Corresponding author: [email protected] Oral Presentation

Wheat dwarf virus (WDV) infects plants of the Poaceae family. WDV species includes the WDV-w and the WDV-b strains. Contradictory data present in the literature led to a conclusion that WDV- w and WDV-b preferentially infect wheat and barley, respectively. WDV is transmitted by the leafhopper Psammotettix alienus. However, species identification in the genus Psammotettix, based on morphometric characterization of the aedeagus, is a challenging procedure. Indeed, due to high aedeagus variability, species identification can lead to the incorrect assignment of males. Moreover, it makes impossible the identification of larvae and females. Thus, in some published works on WDV, it is possible that leafhoppers were not accurately characterized before use in transmission experiments. To improve knowledge on WDV host range, our aim was to describe Psammotettix leafhoppers at the species level using multiple criteria and then to use P. alienus to inoculate WDV to wheat and barley. Leafhopper characterization included recordings of vibrational signals used during mating and morphometric analyses. In addition, sequences of the COI gene were obtained and compared to sequences available in databases. Field-collected P. alienus were characterized, reared and then used in WDV transmission experiments. Larvae, males and females were tested for their ability to transmit WDV strains. Moreover, the abilities of WDV strains to infect wheat and barley plants was tested using host-alternation procedures based on single and dual viral acquisitions/inoculations. Inoculation of WDV-w and WDV-b to barley and wheat, respectively, failed to produce infected plants while WDV-w/WDV-b dual inoculation made it possible to detect WDV-w in barley and WDV-b in wheat. These results suggest a helper effect between WDV strains during host infection. Such information is crucial for future work on the epidemiology of wheat dwarf disease.

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Molecular characterization of phytoplasmas to trace the epidemiological routes of Flavescence dorée between Vitis spp. and the leafhopper vector

Marzachì C1*, Pegoraro M1,2, Rossi M1, Ripamonti M1, Beal D2, Giraudo A12 and Bosco D 12

1 Istituto per la protezione Sostenibile delle Piante, CNR, Italy 2 Università degli Studi di Torino, DISAFA-Entomologia, Italy

*Corresponding author: [email protected] Oral Presentation

Grapevine Flavescence dorée (FD) is one of the major problems of viticulture in several European countries. FD is associated with phytoplasmas of the 16Sr-V ribosomal group, and is transmitted in a persistent propagative manner by the leafhopper Scaphoideus titanus. FD is epidemic in the Piemonte region of Italy since 1998. In spite of the application of the compulsory control methods (insecticides against the vector, rogueing of infected plants, use of healthy propagation material) the disease is still spreading and represents a serious threat to viticulture. The vector transmits FD phytoplasma (FDp) from vine to vine but can also acquire the pathogen from gone-wild rootstocks and fly to the vineyard, thus spreading the disease. A detailed molecular characterization of the pathogen aimed at tracing its movement between the wild compartment and the vineyard was undertaken in order to provide grounds for designing more effective control strategies. Adults of S. titanus were collected with yellow sticky traps inside the vineyard and in the surrounding wild areas at seven sites of the Piemonte region. At the same time, leaf samples of cultivated grapevines, gone-wild Vitis spp. and other potential reservoirs of FDp, were sampled. Insect and plant samples were analyzed by PCR for the presence of FDp and representative samples were genetically typed. After a thorough analysis of the suitability of several candidate genes, dnaK, malG and vmpA were selected. Vector population level and infectivity were determined inside the vineyards and in the wild compartments. The genetic characterization, still in progress, showed high variability of the pathogen at six sites, often in mixed infection in both vectors and plants. In the remaining site, FDp was very homogeneous in both plant and vectors, inside and outside the vineyard. FDp genotypes identified in S. titanus from the wild compartment and cultivated grapevines largely overlapped, supporting the hypothesis of frequent primary infections from outside the vineyard. Interestingly, FDp genotypes found in the alternative host Clematis vitalba were always specific and different from those found in grapevine and S. titanus.

104 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Do phytoplasma effectors target specific processes in insect vectors?

Kliot A* and Hogenhout SA

Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, UK

*Corresponding author: [email protected] Oral Presentation

Phytoplasmas are causative agents of over 700 plant diseases worldwide. They are transmitted by insect vectors and are one of few microbes that invade and colonize cells in organisms from both the plant and animal Kingdoms. Insects transmit phytoplasmas in a persistent, propagative, circulative manner and in some cases are reported to show changes in behavior, fecundity and longevity, but it is not fully understood how these bacteria navigate through the various insect organs and modulate insect behavior and fitness. Aster Yellows strain Witches’ Broom (AY-WB) phytoplasma is transmitted by the polyphagous leafhopper Macrosteles quadrilineatus and induces witches’ broom symptoms in many commercial crops. Phytoplasmas produce an arsenal of virulence proteins, termed effectors, which are deposited inside the cytoplasm of plant/insect cells where they modulate processes to enable successful phytoplasma invasion. Sequencing of AY-WB genome revealed 56 such effectors. Previously, about 20 were found to be up-regulated during infection of the insect vector, M. quadrilineatus, indicating a possible role in modulating insect processes. To investigate if one or more of the 20 effectors plays a role in AY-WB-insect interactions, we use yeast two-hybrid and protein co-immunoprecipitation followed by mass spectrometry to identify insect targets. Further analyses of the AY-WB effector targets will shed more light on the processes taking place within the insect vector during phytoplasma transmission and may reveal new approaches to control phytoplasma outbreaks.

S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS | 105

Invasive mutualisms between a plant pathogen and insect vectors in the Middle East and Brazil

Elliot SL1*, Queiroz RB1, Donkersly P1, Silva FN1, Carvalho CM1, Al-Mahmooli IH2 and Al- Sadi AM2

1 Universidade Federal de Viçosa, Brazil 2 Sultan Qaboos University, Oman

*Corresponding author: [email protected] Oral Presentation

Complex multi-trophic interactions in vector-borne diseases limit our understanding and ability to predict outbreaks. Arthropod-vectored pathogens are especially problematic, with the potential for novel interspecific interactions during invasions. Variations and novelties in plant-arthropod- pathogen triumvirates present significant threats to global food security. We examined aspects of a phytoplasma pathogen of citrus across two continents. ‘Candidatus Phytoplasma aurantifolia’ causes Witches’ Broom Disease of Lime (WBDL) and has devastated citrus production in the Middle East. A variant of this phytoplasma currently displays asymptomatic or ‘silent’ infections in Brazil. We first studied the capacity and fitness impacts of the pathogen on its vectors. The potential for co-occurring weed species to act as pathogen reservoirs were analysed and key transmission periods in the year were also studied. We demonstrate that two invasive hemipteran insects – Diaphorina citri and Hishimonus phycitis – can vector the phytoplasma. The phytoplasma greatly increases reproduction of its invasive vector D. citri both in Oman and Brazil; suggesting that increased fitness of invasive insect vectors thereby further increases the pathogen’s capacity to spread. Based on our findings, this is a robust system for studying the effects of invasions on vector-borne diseases and we highlight concerns about its spread to warmer, drier regions of Brazil. Financial support: FAPEMIG, CNPq, CAPES.

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Genetic and metabolic analyses of Candidatus Liberibacter solanacearum infecting carrot

Katsir L1, Piasetzky A1, Sela N1, Zhepu R2, Freilich S2 and Bahar O1*

1 Department of Plant Pathology and Weed Research, ARO - Volcani Center, Rishon LeZiyyon, Israel 2 Department of Entomology, Newe Ya'ar Research Center, ARO, Israel

*Corresponding author: [email protected] Oral Presentation

Insect-vectored plant bacterial pathogens are gaining attention in recent years due to crop threatening outbreaks around the world. Candidatus Liberibacter spp. are infecting crops of different botanical families: Solanaceae, Rutaceae, and Apiaceae and are vectored by psyllids. Five genetic haplotypes (A-E) have been described thus far for the species Ca. Liberibacter solanacearum (Lso). Haplotypes A and B infecting solanaceous plants, haplotypes C-E infecting Apiaceae crops. To better understand the genetic basis that governs host specificity of Lso haplotypes, we sequenced the genome of haplotype D (LsoD). The LsoD genome size is 1.23 Mbp, with a GC content of 34.8% and 1167 predicted genes. Enzyme Commission (EC) numbers were assigned using the JGI software tool and 358 ECs were identified. ECs were mapped to metabolic pathways and compared with other sequenced Liberibacters. Phylogenetic analysis based on ECs and assigned metabolic pathways shows that LsoD groups together with Lso haplotypes (A and B) and is clearly different than Liberibacter species infecting citrus. Differences between LsoD and LsoA/B haplotypes were also found, hinting on host specific enzymes. The LsoD genome was also scanned to identify putatively secreted proteins using the SignalP tool. Thirty-one putative genes were identified, most of them with unknown function. While some genes have homologous in other Lso haplotypes, some were unique to LsoD. By quantitative-PCR we examined the expression of the putatively secreted proteins in the different hosts; the psyllid vector Bactericera trigonica, and carrot. Several genes with significantly higher expression levels in carrot compared with psyllid and vice versa were identified. These genes may have host specific functions. Overall, our analyses reveal genetic and metabolic elements differentiating the carrot-infecting Lso from Lso haplotypes infecting potato/tomato. Research is underway to identify the function of these elements.

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New insights into psyllid-liberibacter interactions

Ghanim M*1, Jassar O1, Kontsedalov S1, Gosh S1, Achor D2, Levy A2

1 Department of Entomology, Volcani Center, Israel 2 University of Florida, USA

*Corresponding author: [email protected] Oral Presentation

The citrus greening disease known also as Huanglongbing, caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) has resulted in tremendous losses and the death of millions of citrus trees worldwide. CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The closely-related bacterium Candidatus Liberibacter solanacearum (CLso), which is associated with vegetative disorders in carrots and the zebra chips disease in potatoes, is transmitted by other psyllid species including Bactericera trigonica in carrots and B. cockerelli in potatoes. Chemical sprays are currently the prevailing method for managing these diseases for limiting psyllid populations; however, they are limited in their effectiveness. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is very limited. We have recently reported that CLas induced changes in the nuclear architecture in the midgut of ACP, and activated programmed cell death (apoptosis) in this organ. Strikingly, CLso displayed an opposite effect in the gut of B. trigonica, showing limited apoptosis, but widespread necrosis. Electron and fluorescent microscopy further showed that CLas induced the formation of Endoplasmic reticulum (ER) inclusion- and replication-like bodies, in which it increases and multiplies. ER involvement in bacterial replication is hypothesized to be the first stage of an immune response leading to the apoptotic and necrotic responses. ER exploitation and the subsequent events that lead to these cellular and stress responses might activate a cascade of molecular responses ending up with apoptosis and necrosis. Understanding the molecular interactions that undelay the necrotic/apoptotic responses to the bacteria will increase our knowledge of ACP-CLas, and BT-CLso interactions, and will set the foundation for developing novel, and efficient strategies to disturb these interactions and inhibit the transmission.

108 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Psyllid-Ca.Liberibacter interactions at molecular and cellular interfaces

Brown JK*, Rast TJ and Fisher TW

University of Arizona, USA

*Corresponding author: [email protected] Oral Presentation

Ca. Liberibacter asiaticus (CLas) is the obligate, fastidious bacterial pathogen that causes citrus greening disease, or Huanglongbing. CLas is transmitted in a circulative, propagative manner by Asian citrus psyllid (ACP) Diaphorina citri Kuwayama adults. Identification and functional characterization of effectors involved in invasion of the psyllid gut and its presumed entry into the salivary glands was investigated using transcriptomic, proteomic, yeast-2 hybrid (Y2H) and co- immunoprecipitation (Co-IP) analyses. In silico annotation and differential expression analysis of contigs from ACP nymphs and adults, and adult midgut and salivary gland tissues identified transcripts and proteins with altered expression in response to CLas infection. Several differentially expressed transcripts were selected and used as bait for Y2H detection of protein- protein interactions. Those positive by Y2H were subjected to verification by bait to prey co- transformation and Co-IP. Proteins positive in one or both assays were tested in ‘knock down’ experiments using dsRNA to induced RNA-interference (RNAi) and quantified by qPCR. Candidates of the greatest interest have been those with a predicted role in virulence and invasion, that if disrupted by RNAi could abate CLas accumulation in and exit from the gut, circulation in hemolymph and systemic infection, and acquisition e.g. in salivary glands. Collectively, the results suggest a model for invasion in which CLas- and prophage-encoded effectors transform the endocytic host pathway into a ‘pathogen-mediated phagocytic scenario’ utilizing membrane ruffling in CLas interactions with the gut, leading to bacterial exit into the hemocoel and systemic invasion of other psyllid host tissues and organs.

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Looking for elicitors and receptors of Cauliflower mosaic virus (CaMV) transmission activation

Then C*, Macía JL, Thebaud G and Drucker M

INRA, UMR BGPI, France

*Corresponding author: [email protected] Poster 45

Cauliflower mosaic virus (CaMV) is, like many other plant viruses, transmitted by aphids and using the non-circulative transmission mode: when the insects feed on infected leaves, virus particles from infected cells attach rapidly to their stylets and are transmitted to a new host when the aphids change plants. Mandatory for CaMV transmission, the viral helper protein P2 mediates as a molecular linker binding of the virus particles to the aphid stylets. P2 is available in infected plant cells in a specific structure that is formed beforehand during CaMV infection. This structure is specialized for transmission and named the transmission body (TB). When puncturing an infected leaf cell, the aphid triggers an ultra-rapid viral response, necessary for virus acquisition and called transmission activation: tubulin flows massively into the TB, followed by its disruption. As a consequence, P2 is redistributed onto cortical microtubules, together with virus particles (that are simultaneously set free from intracellular storage sites) and forms the so-called mixed networks (MNs). The MNs are the predominant structure from which virus is acquired by aphids; inhibiting their formation reduces drastically transmission rates. We want to identify elicitors that trigger the TB response. For this, we screen different compounds for their capacity to induce MNs in CaMV- infected protoplasts and correlate it with aphid transmission rates obtained using these protoplasts. Preliminary results confer a role to chitin, which is an important compound of the aphids’ exoskeleton and to Myzus persicae saliva effector proteins MP1 and MP2. In parallel, an Arabidopsis thaliana mutant screen has been initiated to dissect the genetic and molecular basis of signal perception and signal transduction pathways involved in the TB response. The latest results will be presented.

110 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

The leafhopper effect: how plant-leafhopper- phytoplasma interactions drive large-scale patterns of infection

Tomkins M*, Maree S, Hogenhout S

Computational and Systems Biology, John Innes Centre, Norwich

*Corresponding author: [email protected] Poster 46

Phytoplasmas are specialised bacteria that colonise a wide range of plants. They are transmitted by vectors, normally sap-feeding insects such as leafhoppers, which carry the bacteria from plant to plant. Phytoplasma increases the plants' attractiveness for the insect vector while damaging or killing the plant itself, resulting in massive crop damage. We build computational models to untangle some of this complexity, while developing new hypotheses regarding the dynamics of the spread of the pathogen that can be tested in the field. Phytoplasma infections occur at a number of scales, both spatially and temporally. Although focus tends to be on single infection events during one growing season, leafhoppers can migrate over thousands of kilometres, and therefore the infection in one single field forms part of a much larger-scale pattern. Understanding the dynamics of these large patterns in both space and time allows researchers to make predictions about the spread of infected leafhoppers over large areas and multiple years. However, this necessitates the use of models that can take into account spatial heterogeneity, including patchiness of plants type, quality, and availability. Here, we present individual-based models (IBMs) designed to investigate the temporal and spatial scales of infection, along with a novel method to directly compare their predictions to that of equivalent ordinary differential equation (ODE) models. We derived a technique which allows us to prove the impact of the non-uniform distribution of leafhoppers and infection on the overall population dynamics. We predict coexistence of infected and uninfected leafhoppers, together with large spatial variation in phytoplasma-induced plant damage. We finally discuss how modellig can help determining what are the important parameters for the infection dynamics. This can then drive novel data collection, improving the predictive value of future model simulations.

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Influence of BYV virus in sugar beet on the aphid parasitoid Lysiphlebus fabarum foraging behaviour and development

Albittar L1*, Ismail M2, Lohaus G3; Bragard C1 and Hance T1

1 Université catholique de Louvain, Belgium 3 Bergische Universität Wuppertal, Germany 2 Wuhan Botanical Garden/Institute, Chinese Academy of Science, China

*Corresponding author: [email protected] Poster 47

Aphis fabae is a major pest on sugar beet. It causes yield loss by transmitting Beet yellow virus (BYV). The interaction among plant viruses, their insect vectors and parasitoids has attracted increasing attention. Our aim was to test the foraging strategy of the parasitoid Lysiphlebus fabarum in the presence of the plant virus, and to verify whether immature parasitoid will successfully develop inside the aphids that developed on inoculated plant. We investigated whether L. fabarum would able to discriminate between aphids that were reared on either healthy or inoculated BYV plants. 25 females of L. fabarum aged ≤ 24h were individually exposed for 30 minutes to a group of 10 aphids (L3). Three series were tested: 10 individuals of A. fabae reared on healthy plant, 10 individuals of A. fabae reared on inoculated plant, Mix: 5 individuals of A. fabae reared on healthy plant and 5 individuals of A. fabae reared on inoculated plant. We observed: number of antennal contacts, number of probing with the ovipositor, emergence rate and patch residence time. We also measured the tibia size of A. fabae and the contents of sucrose and amino acids in the leaves. Our results showed that the number of antennal contact increased significantly in virus patches comparing with healthy patches. L. fabarum was not able to distinguish between aphids reared on healthy or inoculated virus plants. No influence of the virus on the development as well as the emergence rate of parasitoids. Aphids reared on inoculated plant had bigger size than aphids reared on healthy plants. We found that inoculated plants had higher concentrations of sugars and amino acids comparing with healthy plants. Even if physiological changes appeared on virus infected plants and thus on the aphids, these changes seem not to influence the parasitism rate and the development of parasitoid larvae.

112 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Transcriptome changes in the whitefly, Bemisia tabaci MEAM1 in response to feeding on melon infected with the Crinivirus, Cucurbit yellow stunting disorder virus.

Kaur N1*, Chen W2, Fei Z2 and Wintermantel WM1

1 US Department of Agriculture-Agricultural Research Service, Crop Improvement and Protection Research, USA 2 Boyce Thompson Institute, Cornell University, USA

*Corresponding author: [email protected] Poster 48

CYSDV, a crinivirus transmitted by the whitefly, B. tabaci, causes widespread losses in melon in many parts of the world. The virus emerged in the southwestern United States (Arizona and California) and Sonora, Mexico in 2006 and rapidly became established in local crops and weeds, from which it is transmitted to cucurbits by the resident population of B. tabaci MEAM1 each year. CYSDV has a semipersistent mode of transmission and can be retained for seven to nine days in the whitefly vector. In order to understand the specific interactions between B. tabaci and CYSDV, RNA-Seq was performed on whiteflies following acquisition feeding on CYSDV-infected melon leaves at three different time points, 24 h, 72 h, and 7 days. Of the 15,664 genes present in the whitefly, B. tabaci MEAM1, 275 genes were differentially expressed in whiteflies in response to feeding on CYSDV-infected melon host plants over the three time points. Transcriptome analysis of the whiteflies identified differences in gene expression among the three time points, with only 3 down-regulated genes at 24 h, followed by higher numbers of genes differentially expressed at 72 h (221 DEGs: 82 up-regulated and 139 down-regulated), and 7 days (51 DEGs: 49 up-regulated and 2 down-regulated). Several distinct gene categories were represented among the DEGs in the whiteflies. As was found in previous studies involving Tomato chlorosis virus (ToCV), a large percentage of the DEGs were orphan genes that are unique to the whitefly and do not show any homology to known genes in other species. Further, we found 59 DEGs common between whiteflies fed on CYSDV-infected melon and ToCV-infected tomato plants suggesting a common response by whiteflies to feeding on crinivirus infected host plants.

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Effect of Tomato chlorosis virus infection in Capsicum sp. genotypes

Fecury-Moura M*, Vallado NA, Ruschel RG, Ribeiro-Junior MR, Pavan MA and Krause- Sakate R

Universidade Estadual Paulista “Júlio de Mesquita Filho” – Faculdade de Ciências Agronômicas, UNESP-FCA, Brazil

*Corresponding author: [email protected] Poster 49

Tomato chlorosis virus (ToCV) is an emergent pathogen in tomato but also infect pepper plants. There are few studies performed on infection of ToCV in Capsicum and regarding this lack of information, 10 Capsicum genotypes were inoculated with ToCV. The test was performed with “No choice assay” using 50 whiteflies (Bemisia tabaci, MEAM1 cryptic species) per plant and the acquisition and transmission periods were 24h and 48h, respectively. To confirm ToCV infection, a retro-inoculation was performed on susceptible tomato plants cv. Mariana, since detection of ToCV directly from Capsicum is difficult. All Capsicum genotypes tested were susceptible to ToCV infection and the symptoms observed were leaf and vein yellowing to assymptomatic plants. Also two commercial hybrids ‘Magali R’ and ‘Rubia R’ were inoculated with ToCV, since they are commonly grown in Brazil and number and weight of the fruits were analyzed and the statistical analysis performed based on Scott-Knott test (P=0.05 of significance limit) using the ASSISTAT software. Significant losses in number of fruits, 16.48% and 39.28% for Magali R and Rubia R, respectively, were observed. The total weight of the fruits was also negatively affected showing a significant loss of 12.29% and 15.30% for Magali R and Rubia R, respectively, similar results observed for the single infection of theses hybrids with the Tomato severe rugose virus, the predominant species of begomovirus in Brazil. The mixed infection of Magali R and Rubia R with ToSRV and ToCV resulted also in loss of productivity. We can conclude with this work that ToCV in single infection, or in mixed infection with the begomovirus ToSRV, affects production of important pepper hybrids cultivated in Brazil.

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Effect of temperatures to leafhopper vector Matsumuratettix hiroglyphicus and amount of sugarcane white leaf phytoplasma

Kaewmanee C1* and Hanboonsong Y2

1 Division of Agricultural Science, School of Interdisciplinary Studies, Mahidol University, Kanchanaburi Campus, Thailand 2 Division of Entomology, Department of Plant Science and Agricultural Resources, Khon Kaen University, Thailand

*Corresponding author: [email protected] Poster 50

Sugarcane white leaf (SCWL) disease is an important disease of sugarcane in Asia region, and is caused by phytoplasma. The characteristic disease symptoms are presence of leaf with total chlorosis, proliferating tillers, pronounced stunting, side shoots on the infected stalks and finally death in infected plants. The disease is spread primarily by cuttings obtained from the infected sugarcane stalks and secondly by transmission through insect vectors Matsumuratettix hiroglyphicus and Yamatotettix flavovittatus (Hemiptera: Cicadellidae). The objectives of this research were to evaluate the impact of temperatures on the life-history of the leafhopper M. hiroglyphicus and transmission efficiency of SCWL phytoplasma. The leafhopper was reared at natural temperature (26±2°C) and under controlled conditions; 20±1, 25±1, 30±1 and 35±1°C. The result of rearing the leafhopper indicated that the life span of M. hiroglyphicus at higher temperature was shorter in all developmental stages. In addition, the developmental stage of male adults was shorter than female adults at all tested temperatures. The highest percentage survivorship of the leafhopper from egg to adult stage was obtained at natural temperature (26±2°C). For transmission, the result of nested PCR showed that the female insects transmitted the SCWL phytoplasma more efficiently than the male insects. Moreover, the real-time PCR result showed that the highest amount of SCWL phytoplasma was within the infected insects reared at optimal temperature (26±2°C) both in infected female and male insects, and the amount of phytoplasma found in infected female insects was higher than in infected male insects. Therefore, the natural temperature (26±2°C) is the optimal temperature for development of the leafhopper M. hiroglyphicus and transmission of SCWL phytoplasma.

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Gene expression of Diaphorina citri effectors on different life stages de Souza Pacheco I*, Manzano Galdeano D, Machado MA

Centro de Citricultura Sylvio Moreira. SP, Brazil

*Corresponding author: [email protected] Poster 51

Effectors are molecules secreted/released by an organism which alters the physiology of another organism. Secreted mainly by insect salivary glands, these molecules act modulating plant physiology and/or favoring establishment and transmission of phytopathogens. Differences in morphology, physiology and gene expression are observed among insect nymphs instars and adults. Those changes are a critical point for colonization and dispersal of bacteria Candidatus Liberibacter asiaticus (Ca. Las), pathogen of Huanglongbing disease (HLB), through its insect vector Diaphorina citri. Significant differences on acquisition and transmission of Ca. Las by D. citri were observed on different life stages. Based on this information, we evaluate the gene expression of D. citri candidate effectors of 4th, 5th nimph instar and adults. Bioinformatic pipeline allowed the identification of 18 D. citri putative effectors. To confirm the potential of the effectors been secreted through salivary glands, differential gene expression of putative effectors were realized using psyllid body and head, separately. Unaltered gene expression for most of candidate effectors among life stages analyzed were observed however, a few genes were overexpressed on D. citri immature stages, showing fold change more than 25 higher in nymphs compared to adults. Differences about expression of candidate effectors were also observed between infected/Ca las- free psyllids. Once nymphal stages shows high rates of acquisition and transmission of Ca. Las, these results indicate that D. citri effectors could play a key role on HLB pathosystem.

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Effects of vector density and time of infection on zebra chip disease development both at harvest and during storage

Wenninger EJ1*, Rashed A2, Olsen N1, Wharton P2 and Karasev A3

1 University of Idaho, Kimberly Research & Extension Center, USA 2 University of Idaho, Aberdeen Research & Extension Center, USA 3 University of Idaho, USA

*Corresponding author: [email protected] Poster 52

Zebra chip (ZC) disease is associated with a bacterium, Candidatus Liberibacter solanacearum, and vectored by the potato psyllid, Bactericera cockerelli. ZC is now a well-known threat to potato production in the Pacific Northwestern (PNW) region of the US and since 2011 has resulted in increased costs through additional insecticide applications. Monitoring results show that potato psyllids in the PNW often exhibit a late-season peak in abundance despite occurring at relatively low levels during much of the growing season. However, effects of late-season infections on ZC disease development under PNW conditions need to be clarified to inform management recommendations. We conducted field trials over two seasons in which individually caged ‘Russet Burbank’ potato plants were inoculated with liberibacter-positive potato psyllids at different densities (two or five psyllids) and times (2 days, 1 week, 3 weeks, or 7 weeks before vine kill). ZC symptoms (which were confirmed by PCR) were rarely observed for plants inoculated within 1 week of vine kill, for both at-harvest and post-storage (88 days) ratings. At harvest, incidence of ZC symptoms generally was higher for plants inoculated at 7 weeks versus 3 weeks before vine kill, and ZC incidence generally was positively related to vector density. After storage, plants inoculated at 3 weeks before vine kill showed some increased incidence of ZC symptoms, though incidence still tended to be lower than that observed for the 7 week treatments. These data suggest that under PNW growing conditions, Russet Burbank plants that are infected very close to vine kill (within 1 week) are at lower risk of ZC disease development; however, for plants infected earlier, ZC can develop over time during storage.

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Interaction between plant responses and transmission of TuMV by Myzus persicae

Berthelot E1,2*, Macia JL1, Khelifa M2 and Drucker M1

1 INRA, UMR BGPI, France 2 SIPRE, France

*Corresponding author: [email protected] Poster 53

Many viruses are transmitted by aphids in a non-circulative manner. This indicates that when aphids feed on an infected plant, the transmitted virus particles attach within seconds to the stylets, and are transported in them to a new host plant. This is the case of the Caulimovirus Cauliflower mosaic virus (CaMV), which follows the molecular strategy called “transmission helper component” for transmission. Its viral helper component, protein P2, intervenes by creating the molecular link between virus particles and stylets. In presence of aphid vectors, CaMV forms transmissible P2-virus complexes in the cell. This phenomenon, called "Transmission Activation (TA)", boosts CaMV transmission and depends on CaMV interfering with aphid-plant responses. We are interested in the transmission of the Potyvirus Turnip mosaic virus (TuMV), another non- circulative virus using the transmission helper component strategy with its viral protein Helper Component Protease (HC-Pro). We wanted to determine whether this virus also uses TA. Aphid transmission tests with Myzus persicae were carried out using infected protoplasts as virus source that were incubated with different reagents, mimicking plant defense responses, before the transmission tests. The results showed that a blocker of calcium signaling, LaCl3, reduced transmission while the reactive oxygen species (ROS), H2O2, activated it. This suggests that different plant defense responses modify the cellular environment in which HC-Pro is located, induce TA of TuMV and therefore its acquisition by aphids. Moreover, western blotting showed that LaCl3 inhibited and H2O2 induced formation of intermolecular cysteine bonds linking HC-Pro oligomers. Taken together, our results demonstrate that TuMV transmission can be activated, that calcium signaling and ROS are involved in it and that it correlates with oxidation of HC-Pro. Thus, TuMV is a second example for the TA phenomenon, and a model of Potyvirus transmission.

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Zebra chip symptom and Lso development in potato with respect to vector density and the time of infestation: A greenhouse evaluation

Rashidi M, Wenninger EJ, Liang X, Olsen N, Karasev A, Wharton P and Rashed A*

University of Idaho, Dept. of Plant, Soil and Entomological Sciences, USA

*Corresponding author: [email protected] Poster 54

Zebra chip (ZC) disease, a serious threat to potato production, is associated with the potato psyllid (Bactericera cockerelli)-borne bacterium ‘Candidatus Liberibacter solanacearum’ (Lso). A greenhouse study was conducted to evaluate Lso transmission success, Lso quantity, and ZC severity (0-3, scale) in relation to three vector densities (1, 2, and 5 psyllids) and the three infestation times of 84, 21 and 2 days before vine-kill (DBVK). Symptom severity and Lso titer were evaluated in 2-4 tubers/plant, 21 days after vine-kill. Lso titer within the same tubers was re- quantified following 4 months of storage at 55 °F to determine pathogen development. Plant photosynthetic rates were recorded throughout the experiment. Overall, the rate of successful Lso transmission ranged between 36.8 and 55.5% across the three infestation times. Transmission success in 1-psyllid treatments was lower than for 2- and 5-psyllid treatments in 84- and 2- DBVK infestations. This pattern however, was not observed among the three density treatments in the 21-DBVK infestation. Lso titer in 2-DBVK tubers was significantly lower that those in 84- and 21- DBVK; the majority of 2- DBVK tubers remained asymptomatic, a few exhibited minimal visual discoloration (mean ZC score ± SE: 0.22 ±0.04). ZC severity score was significantly higher in 84- DBVK (1.30 ±0.25) and 21-DBVK (1.67 ±0.20) infestations. Inoculated plants exhibited lower photosynthetic rates compared to non-inoculated plants. During storage, Lso titer increased in 2- and 21-DBVK tubers, but declined in 84-DBVK tubers (based on the first-year results). 21- and 2- DBVK plants did not develop foliar symptoms. Psyllid monitoring and management is recommended until vine-kill. Future studies are needed to evaluate germination success of tubers infested as late as 2 days before vine-kill.

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The variable membrane protein VmpA of Flavescence dorée phytoplasma interacts with cells of the insect vector Euscelidius variegatus

Arricau-Bouvery N1*, Duret S1, Dubrana MP1, Batailler B1, Desqué D1, Béven L1, Danet JL1, Monticone M2, Bosco D2, Malembic-Maher S1 and Foissac X1

1 UMR Biologie du Fruit et Pathologie 1332, INRA, Université de Bordeaux, France 2 Università di Torino, DISAFA – Entomologia, Largo Paolo Braccini, Italy

*Corresponding author: [email protected] Poster 55

Phytoplasmas are uncultivated plant pathogens, cell wall-less bacteria and transmitted from plant to plant by Hemipteran. Their circulative propagative cycle in insects implies the crossing of eukaryotic cells of midgut and salivary glands at least. Adhesion of bacteria to cells is an initial step to the invasion process. The Flavescence Dorée phytoplasma possess a set of variable membrane proteins (Vmps) exposed to its surface and supposed to interact with insect cells. However no mutant is available to study these candidates putatively implicated in phytoplasma adhesion to insect cells. We thus used S. citri devoid of the adhesins ScARPs, S. citri G/6 (Béven et al., 2012) and expressing VmpA proteins at their surface, and VmpA-coated fluorescent latex beads for studying interaction between VmpA and insect cells of the experimental vector Euscelidius variegatus in ex vivo adhesion assays and in vivo ingestion assays experiments. The results show that VmpA interacts specifically with insect cells in culture and allows VmpA-coated beads to be retained in the midgut of E. variegatus. In this latest case VmpA-coated fluorescent microspheres were localized embedded in the perimicrovillar membrane of the insect midgut. These results show that VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasma.

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Psammotettix alienus-mediated inoculations of barley and wheat strains of Wheat dwarf virus to wild gramineous species

Abt I1,2*, Souquet M1, Derlink M3, Temple C1, Gauthier K1,2, de Keyzer A2, Letroublon M2, Dallot S1, Thébaud G1, Virant-Doberlet M3 and Jacquot E1

1 INRA, Montpellier SupAgro, CIRAD, UMR BGPI, France 2 Bayer S.A.S., 16 rue Jean-Marie Leclair, France 3 National Institute of Biology, Slovenia

*Corresponding author: [email protected] Poster 56

Wheat dwarf virus (WDV, family Geminiviridae, genus Mastrevirus) is the causal agent of dwarfing, mottling and yellowing symptoms in wheat and barley. WDV is exclusively transmitted by leafhoppers from the genus Psammotettix in a persistent manner and infects plants of the Poaceae family. Phylogenetic analyses carried out with WDV genomic sequences showed that this viral species comprises several strains including the originally described wheat (WDV-w) and barley (WDV-b) strains. Psammotettix leafhoppers collected in different French cereal-growing areas were characterized using recordings of vibrational signals used during mating, morphometric analyses and sequences of the COI gene. Characterized P. alienus individuals were used as WDV vectors to assess the host ranges of isolates from the WDV-w and WDV-b strains. Thirty-seven wild gramineous species were identified in a 5-km² cereal-growing area located in Annoix (Cher, France) where WDV and Psammotettix leafhoppers are usually highly prevalent (Gauthier et al., 2017). Whenever seeds were available, susceptibility of wild gramineous species to WDV was tested using P. alienus-mediated WDV inoculations. The characterization of field-collected Psammotettix showed that P. alienus is the prevalent Psammotettix species and sometimes it is found syntopically with P. confinis. Leafhopper mediated transmission experiments highlighted that (i) the two WDV strains have different but slightly overlapping host ranges, (ii) three gramineous species not yet reported to be WDV hosts (i.e. Alopecurus myosuroides, Cynosurus cristatus and Poa annua) could be infected by WDV, and (iii) Setaria viridis, described in the literature as non-host for WDV, was successfully infected by the WDV-w isolate.

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Epidemiology of barley yellow dwarf and wheat dwarf diseases

Gauthier K1,2*, Abt I1,2, Deshoux M1,2, Marchat M1,2, Temple C1, de Keyzer A2, Letroublon M2, Dallot S1, Thébaud G1 and Jacquot E1

1 UMR BGPI, INRA, Cirad, France 2 Bayer S.A.S./Bayer CropScienceFrance

*Corresponding author: [email protected] Poster 57

The most important cereal viruses are Barley/Cereal yellow dwarf virus (B/CYDV, genera Luteovirus/Polerovirus, family Luteoviridae) and Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae). B/CYDV is a complex of at least nine viral species and WDV consists of two main strains. B/CYDV and WDV are exclusively transmitted by aphids and leafhoppers, respectively. In spite of a wealth of data on both B/CYDV and WDV pathosystems, little information is available processes related to (i) the emergence, spread and maintenance of these viruses in a landscape including cultivated and wild compartments and (ii) the interactions between these viral species and between their vectors. An intensive survey was conducted to collect plants and insects in a 5 km² landscape (Annoix, Cher, France) where B/CYDV and WDV, and their vectors (i.e. cereal aphids and Psammotettix alienus), are usually highly prevalent. Previously characterized and putative host species of the Poaceae family present in the visited areas were sampled. Simultaneously, aphids and leafhoppers were caught using yellow water traps and sticky traps. The presence of BYDV-PAV, BYDV-MAV, CYDV-RPV and WDV in collected plants and insects was assessed using serological and molecular tools. A total of 37 wild gramineous species were identified. Serological analyses revealed that B/CYDV was present in 9 wild gramineous species. Surprisingly, none of the thousands collected leaves were infected by WDV. To complement the description of the gramineous species present in the studied landscape and their possible role as viral reservoirs (Abt et al., 2017), interactions between viruses and between vectors with overlapping host ranges will be analyzed in order to determine (i) how these pathogens (and their vectors) compete for host resources and (ii) the consequences of such interactions on the epidemiology of barley yellow dwarf and wheat dwarf diseases.

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Tissue-specific transcriptional responses related to the horizontal and vertical transmission of a bacterial pathogen by its hemipteran insect vector

Badillo-Vargas IE*, Bedre R, Esparza-Díaz G, Avila C and Mandadi KK

Texas A&M AgriLife Research, USA

*Corresponding author: [email protected] Poster 58

The potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae), is the insect vector of the fastidious bacterium “Candidatus Liberibacter solanacearum” (Lso). This bacterial pathogen causes diseases in several solanaceous crops, including zebra chip, an economically important disease of potato in United States, Mexico, Central America, and New Zealand. Lso is transmitted in a persistent propagative manner by B. cockerelli where it infects and multiplies in the digestive track, reproductive organs, and salivary glands of its insect vector. Lso infection of the reproductive organs of B. cockerelli is hypothesized to be a pre-requisite for transovarial transmission of the pathogen to the insect offspring. It has been previously shown that Lso has a detrimental effect on the fecundity and nymphal survival rate of B. cockerelli. To better understand the molecular bases of these biological consequences in the hemipteran insect vector due to the bacterial infection, we have conducted a tissue-specific transcriptome analysis of B. cockerelli organs involved in the horizontal and vertical transmission of Lso. Total RNA was extracted from pools of dissected salivary glands and ovaries from non-infected (Lso-) and infected (Lso+) insects using three biological replicates. Preparation of libraries using poly-A enriched RNA and the TruSeq Library kit followed by mRNA sequencing using Illumina Hi-Seq technology was performed by the Texas A&M AgriLife Genomics and Bioinformatics Services Facility in Texas A&M University. Bioinformatics analyses are being conducted to identify the transcriptional changes in these insect tissues in response to the bacterial infection. Identification of responsive candidate genes from B. cockerelli is expected to increase our understanding of a vector-bacteria interaction that results in some detrimental effects to the animal host and might ultimately aid in the development of novel control strategies to mitigate losses caused by this economically important pathosystem.

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Studying transmission activation of aphid- vectored Cauliflower mosaic virus

Dáder B*, Macia JL and Drucker M

INRA, UMR BGPI, France

*Corresponding author: [email protected] Poster 59

It has been assumed for a long time that virus acquisition was simple contamination of the stylets occurring when aphids feed on infected plants. However, non-circulative Cauliflower mosaic virus (CaMV) forms during aphid punctures specific transmission morphs in infected cells. More precisely, the CaMV transmission helper protein P2 is dispatched from cytoplasmic transmission body inclusions and virus particles from virus factory inclusions to form together and transiently transmission morphs on microtubules that are acquired and transmitted by the vector. This 'Transmission Activation' (TA), discovered first for CaMV and recently described for the Potyvirus Turnip mosaic virus, requires that the virus recognizes the presence of aphids via the plant perception systems, and then induces TA. We want 1) to characterize TA reaction in living tissue after biotic (aphid punctures) and abiotic stresses, and 2) to capture and identify plant partners involved in TA. For this, we chose GFP, which serves as a fluorescent in vivo reporter, and can be used to immunocapture interaction factors by the GFP trap technique. Since the CaMV genome cannot accommodate the entire GFP sequence, we implemented the split GFP system, based on spontaneous auto-assembling of the non-fluorescent small GFP11 (16 amino acids) and big GFP1- 10 (rest of the molecule) fragments to fluorescent GFP. A recombinant CaMV with GFP11 fused to P2 was constructed and used to infect transgenic Arabidopsis expressing GFP1-10. The virus was infectious and infected cells displayed P2-GFP fluorescence in transmission body-like structures. When validated, the system might help to identify TA elicitors by tracking GFP11-P2 behavior after application of candidate molecules. The same approach will be used for viral protein P6, with the goal to potentially detect early infection events at the site of aphid-inoculated cells. P6 is particularly suited for this because it is the first viral protein to appear during infection.

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A fluorescent infectious clone of Turnip yellows virus to study the effect of aphid infestation on virus distribution in planta

Boissinot S1*, Pichon E2, Sorin C3, Scheidecker D4, Ziegler-Graff V4 and Brault V1

1 UMR INRA-UDS Virus-Vection group, France 2 CIRAD UMR-BGPI TA A-54/K Campus International de Baillarguet, France 3 Université Paris-Diderot, Institute of Plant Sciences Paris Saclay, France 4 CNRS-IBMP, France

*Corresponding author: [email protected] Poster 60

Turnip yellows virus (TuYV) is a polerovirus (family Luteoviridae) restricted to phloem tissue and obligatorily transmitted by aphids in a circulative and non-propagative mode. This virus infects several plant species of economic importance. The icosahedral viral particles contain a single positive strand RNA genome. In order to track virus movement in plants and to measure the impact of aphids presence on virus distribution in plants, a GFP-tagged virus was engineered (TuYV-GFP). In this construct, the GFP sequence was inserted in a non-structural protein sequence involved, to some extent, in virus movement. The fluorescent signal is expected to label virus- replicating cells. The TuYV-GFP sequence was introduced into a binary vector to inoculate several plant species by agroinfiltration. TuYV-GFP was able to replicate and to move in A. thaliana, N. benthamiana and M. perfoliata. Accumulation of TuYV-GFP was however reduced when compared to the wild type virus. The impaired ability of the recombinant virus to reach efficiently non- inoculated leaves is likely due to the insertion of the GFP sequence in the viral genome. The TuYV- GFP genome was stable in N. benthamiana while it was subjected to partial deletions in A. thaliana and in M. perfoliata. Nevertheless, we observed fluorescent phloem cells in non-inoculated leaves of the three plant species. Importantly, fluorescent phloem cells were also observed after virus acquisition and inoculation by aphids, both in inoculated and non-inoculated leaves. This major breakthrough in the study of poleroviruses persistently transmitted by aphids will now be used to address whether the virus localisation in planta is affected following aphid infestation.

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Infection and colonization of Nicotiana benthamiana by GLRaV-3

Prator CA*, Kashiwagi C and Almeida RPP

UC Berkeley, USA

*Corresponding author: [email protected] Poster 61

Grapevine leafroll disease (GLD), caused by Grapevine leafroll-associated virus-3 (GLRaV-3), is an increasing problem in California and all grape-growing regions of the world. GLRaV-3 has never been shown to infect species outside of the genus Vitis, resulting in a challenging and often labor- intensive host-pathogen system. It is evident that GLRaV-3 research would benefit from infection in a plant model organism that could help potentially overcome these issues. To determine if GLRaV-3 could infect a non-grapevine host, virus transmission to several species of widely used plants for research was tested using the vine mealybug, Pl. ficus, as an insect vector. We show that GLRaV-3 is able to infect the model organism Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and Transmission Electron Microscopy (TEM) observations, respectively. Immunogold labeling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected source V. vinifera vs. N. benthamiana revealed that sequences were identical. These results open new venues and opportunities for GLRaV-3 research.

126 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Genetic characterization of ‘Candidatus Liberibacter solanacearum’ strains infecting carrot psyllids in southwestern France

Bergey B, Eveillard S* and Foissac X

UMR1332 Fruit Biology and Pathology, INRA and University of Bordeaux, France

*Corresponding author: [email protected] Poster 62

The phloem-limited bacterium, Candidatus Liberibacter solanacearum (CaLsol), is responsible of several diseases on Solanaceae (USA, New-Zealand) and Apiaceae such as carrots in several European countries (Spain, France Scandinavia, Germany). This bacterium is vectored by psyllids, Bactericera cokerelli to potatoes, or B. trigonica and Trioza apicalis to carrots. In France, since the 1970s, proliferation of carrot was shown to be associated with a psyllid-transmitted, phloem limited bacterium. In order to reassess disease impact and better characterize the bacterium involved, five organic or non-organic carrot fields were surveyed in 2016 for CaLsol and psyllids in three production areas of Southwestern France. Psyllids were present at spring and summer only in an organic experimental field, but could be captured from September in all fields. The highest level of psyllid populations was reached in October when up to 350 specimens were collected upon 100 sweeps. Psyllids collected on carrots were most probably Bactericera trigonica. CaLsol could be detected by Taqman real-time PCR (Teresani et al., 2014) in psyllids as well as in carrots exhibiting stem proliferation and stunting. CaLsol detection ranged from 0 to 18% in individual psyllids and from 0 to 9% in carrots. In Europe, the two ribosomal CaLsol haplotypes D and E have been characterized based on the 16S rDNA and the 16S-23S spacer sequences. In the present survey, haplotype E was identified in both carrot and psyllids, as well as haplotype D2, a new haplotype differing from the haplotype D by two SNPs. Genotyping using the five most variable gene markers described by Glynn and collaborators (2012) showed that the two haplotypes differed for all five markers dnaG, metG, recA, mutS and adk, with mutS being the most discriminant with 8 SNPs between haplotype E and D2.

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Transcriptional response of the leafhopper, Exitianus exitiosus (Uhler) to Spiroplasma kunkelii infection

Andreason S1, Cougar B2 and Wayadande A1*

1 Department of Entomology and Plant Pathology, Oklahoma State University, USA 2 High Performance Computing Center, Oklahoma State University, USA

*Corresponding author: [email protected] Poster 63

The intricate molecular interactions between phytopathogens and their insect vectors are yet elusive. From a behavioral perspective, plant pathogens such as spiroplasmas have been demonstrated to have positive, neutral, and negative effects on the fitness of their leafhopper vectors. This variation in vector host effects raises questions about what occurs transcriptionally in the leafhopper when it acquires phytopathogenic spiroplasmas. To begin investigating this transcriptional response, Spiroplasma kunkelii-infected versus -naïve gray lawn leafhopper, Exitianus exitiosus (Uhl.), transcriptomes were compared. Total RNA was extracted from three replicate pools of male and female leafhoppers 25 days (latent period) after a 24-hour acquisition access period from S. kunkelii-inoculated feeding sachets. Three similar replicates were simultaneously completed for the naïve leafhopper control groups after exposure to S. kunkelii- negative feeding sachets. RNA was poly-A selected and sequenced using the Illumina NextSeq500 platform. De novo assembly was performed using the Trinity pipeline. Datasets were normalized, and transcript abundances were estimated. Differentially expressed transcripts were identified and annotated. Over 300 transcripts were highly supported (FDR < 0.05) as differentially expressed in E. exitiosus infected with S. kunkelii. Of particular interest was the evidence of differential expression of immune response related genes, considering that, until recently, spiroplasmas, including phytopathogenic species, were not thought to induce a defense response in their insect hosts. This work provides a first look at the potentially persistent differential gene expression in a leafhopper vector infected with a phytopathogenic spiroplasma.

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The Ephrin receptor: a potential actor in polerovirus transmission by aphids

Mulot M1, Boissinot S1, Monsion B1†, Rastegar M2, Bochet N1 and Brault V1*

1 UMR1131 INRA-UDS SVQV, France 2 Shiraz University, Plant Protection Department, Iran † Present address: INRA, UMR BGPI INRA-CIRAD-SupAgro, France

*Corresponding author: [email protected] Poster 64

Poleroviruses are phloem-limited RNA viruses strictly transmitted by aphids in a circulative and non-propagative mode. Virus particles, acquired by aphids on infected plants, successively cross intestinal and accessory salivary gland cells by transcytosis before being released, together with saliva, into a plant during aphid feeding. Virus transport through the epithelia relies on the presence of specific virus receptors. Identification of these receptors could eventually result in the development of innovative strategies to block virus acquisition by aphids. The Ephrin receptor (Ephr) from Myzus persicae has been previously identified by yeast two hybrid as a potential partner of the structural proteins of Turnip yellows virus (TuYV, genus Polerovirus, family Luteoviridae). Ephr is a membrane protein involved in cell communication and endocytosis in mammalian cells. In order to address its function in TuYV transmission by M. persicae, we developed several strategies (1), all based on the RNA interference mechanism, to inhibit expression of Ephr in M. persicae. The oral acquisition of in vitro synthetized dsRNA targeting Ephr and the acquisition of dsRNA, or siRNA, from transgenic plants expressing an RNA hairpin, resulted in an inhibition of the accumulation of Ephr-mRNA in M. persicae. When fed on a viral source, these Ephr-silenced aphids showed a lower internalization of viral genomes into their body. Although the feeding behaviour and the fecundity of the Ephr-silenced aphids were not affected by the reduction of Ephr expression, TuYV transmission was reproducibly reduced. Preliminary experiments showed that transmission inhibition of another related polerovirus, the Beet mild yellowing virus, was also obtained when using Ephr-silenced aphids. Taken together, these experiments strongly suggest implication of Ephr in polerovirus transmission. Whether Ephr encodes a true virus receptor or a co-receptor still needs to be elucidated.

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Landscape influence on the occurrence of Laodelphax striatellus and its vectored maize virus

Clemente-Orta G*, Albajes R, Madeira F and Achón MA

University of Lleida, Agrotecnio Center, Spain.

*Corresponding author: [email protected] Poster 65

Planthoppers are pest of maize and more importantly vectored a large number of viruses. Laodelphax striatellus Fallen (Delphacidae, Fulgoroidea) breeds on winter cereals, rice, maize and weeds. Its importance as a primary pest of crops is negligible but it transmits Maize rough dwarf virus (MRDV) in a propagative and transovarial manner. MRDV is the main constraint to maize production in Spain. It is widely accepted that the epidemiology of MRD disease is strictly linked to the abundance and distribution of its vector. Previous studies in Northeast of Spain over 2006- 2009 period showed that adults of L. striatellus occurred in maize fields from mid-May to the beginning of October and that the incidence of MRDV is correlated with accumulated numbers of adults at early developed stage of maize plants. Besides, these studies suggested that agroecosystem management could affect the population of the planthopper. In this study we monitored the population dynamics of L. striatellus on 28 sites with three different agriculture landscapes: 6 sites in a landscapes mosaic mainly dominated by arable crops (MA), 4 sites in landscapes mainly dominated by orchards (MO), and 18 sites in a mosaic landscape including arable crops and orchards (MAO). Insects were captured using yellow sticky traps from Mach to September. Virus incidence was determined by symptoms and selective isolation of viral genome. We observed that the large populations of the insect were in MA landscape followed by MAO and MO. The incidence of MRDV was 8% lower in MAO landscape than in MA one. This could be a result of different weed covers observed among landscapes and orchards management. However, the high abundance of winter cereal crops on MA landscapes could not be discarded since act as reservoirs of these insects. Our results suggest that crop diversity could contribute to reduce the incidence of specialist virus.

130 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Transmission of important Brazilian viruses by Bemisia tabaci Mediterranean species

Bello VH 1*, Watanabe LFM1, Marubayashi JM1, Yuki VA2, Moraes LA1, Pavan MA1, Krause-Sakate R1

1 Faculdade de Ciências Agronômicas de Botucatu – FCA/UNESP, Brazil 2 Instituto Agronômico (IAC), Brazil

*Corresponding author: [email protected] Poster 66

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important agriculture pests and an excellent virus vector. The Middle East-Asia Minor 1 (MEAM1) cryptic species, formely known as biotype B, is prevalent in Brazil since its first detection at the 90’s and caused the emergence of begomoviruses in tomatoes. The Mediterranean species (MED), also called as biotype Q, was detected in 2014 in the South of Brazil and more recently in São Paulo and Parana States. Here we evaluated the ability of MED to transmit important viruses in Brazil: the carlavirus Cowpea mild mottle virus (CpMMV) and the begomoviruses Tomato severe rugose virus (ToSRV) and Bean golden mosaic virus (BGMV). Two different MED populations, one harboring 14 % of Hamiltonella and 29 % of Rickettsia secondary endosymbionts (“MED 1”) and the second with 97% of Hamiltonella, 33% of Rickettsia and 12% of Arsenophonus (“MED 2”) were tested. The acquisition access period (AAP) and the inoculation access period (IAP) performed was 24 hours at 30°C, using 10 insects and 30 plants. MED 2 transmitted BGMV and CpMMV to beans with 100% efficiency (30 plants tested), while MED 1 with 56.6 % and 53.3 % efficiency, respectively. Comparatively, the MEAM1 species harboring 98 % of Hamiltonella and 91 % of Rickettsia transmitted the BGMV and the CpMMV with 90% efficiency. Regarding to the ToSRV, both MED populations tested transmitted this begomovirus with 83.3 % efficiency, while the MEAM1 species with 80 % efficiency. The crinivirus ToCV was transmitted by MED1 and MED2 with 93.3 % and 83.3 % efficiency, respectively, and with 80% efficiency by MEAM1. We conclude that the invasive Mediterranean species detected recently is an excellent vector of important beans and tomato viruses in Brazil.

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Phytoplasma effectors modulate plant development and plant resistance to insect vectors

Wouters RHM*, Maclean AM and Hogenhout SA

Department of Cell and Developmental Biology, John Innes Centre, UK

*Corresponding author: [email protected] Poster 67

Phytoplasmas are bacterial plant pathogens that are transmitted by plant-sucking insect species of the order Hemiptera. Phytoplasmas occur worldwide and have caused yield losses in a number of economically important crops. Phytoplasma-infected plants show dramatic symptoms, such as witch’s broom (proliferation of shoots), phyllody (formation of leaf-like flowers) and virescence (green pigmentation of flower structures that are normally not green), that suggest interference with plant development. Moreover, it was previously found that some phytoplasma-infected plants become hosts for leafhoppers that normally do not like a plant species, suggesting that phytoplasmas interfere with non-host resistance to the hemipterans. Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) produces the virulence proteins (effectors) SAP11 and SAP54 that induce shoot proliferation and leaf-like flowers, respectively, in Arabidopsis thaliana. However, 54 additional candidate effector genes were identified in the genome of this phytoplasma. To study if any of these effectors modulate A. thaliana non-host resistance to the corn leafhopper Dalbulus maidis, AY-WB-infected and transgenic plants producing AY-WB effectors were exposed to 8 D. maidis females and two males. Survival rates of these insects were assessed over a period of 4 days and leaves were searched for D. maidis eggs and nymphs 14 days later. D. maidis showed higher survival rates and laid eggs on AY-WB-infected plants confirming published results. D. maidis survival increased on transgenic plants for one effector. Interestingly, this leafhopper laid eggs and produced nymphs on transgenic plants for two AY-WB effectors. Thus, three different AY-WB effectors appear to promote D. maidis survival/fecundity suggesting their involvement in modulating non-host resistance during phytoplasma infection. Other screens using transgenic plants revealed that two effectors induce stunting of A. thaliana, and two effectors increase phytoplasma titers in the plants. In summary, AY-WB effectors modulate a range of plant processes that contribute to phenotypes seen in phytoplasma-infected plants.

132 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

A Tomato necrotic dwarf virus isolate from Datura with poor transmissibility by the whitefly, Bemisia tabaci

Wintermantel WM1*, Hladky LL1, Cortez AA 1 and Natwick ET2

1 United States Department of Agriculture-Agricultural Research Service, USA 2 University of California Cooperative Extension, USA.

*Corresponding author: [email protected] Poster 68

Tomato necrotic dwarf virus (ToNDV); genus Torradovirus, is a whitefly-transmitted virus that caused significant losses for tomato production in the Imperial Valley of California during the 1980s. The virus causes severe stunting, dwarfing of leaves, foliar and fruit necrosis, and greatly reduced fruit and seed production. ToNDV has a limited host range with an affinity for members of the Solanaceae, and infection of Imperial Valley tomato contributed to the loss of the tomato seed industry from the region. After the loss of tomato production the virus disappeared and was not identified in the region again until 2015 when it was found infecting wild Datura species. Like many other tomato-infecting members of the genus, Torradovirus, ToNDV produces icosahedral virions approximately 30 nm in diameter, and can be transmitted in what appears to be a semipersistent manner by at least three whitefly species; Bemisia tabaci MEAM1 and NW1, Trialeurodes abutilonea, and T. vaporariorum, as well as mechanically and by grafting. The ToNDV genome is composed of two RNA molecules of 7.2 and 4.9 Kb, with both RNAs expressing large polyproteins. Three tomato isolates of ToNDV characterized to date share approximately 96 percent or greater sequence identity and are transmitted efficiently by T. abutilonea and B. tabaci MEAM1 in laboratory studies, whereas transmission by T. vaporariorum appears to be less efficient. A new isolate from Datura shares only 80 and 87% nucleotide identity for RNA1 and 2 of the original ToNDV-R, respectively, whereas the polyproteins encoded by these RNAs share 93 and 96% identity. The Datura isolate has been found infecting new Datura plants from an isolated desert weed plot for two years, suggesting the possibility of either seed transmission in Datura, or more efficient transmission to solanaceous weeds than to tomato.

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Species of plant source of Tomato chlorosis virus influences the transmission rate of the virus by Bemisia tabaci MEAM1

Esquivel AF* and Rezende JAM

University of São Paulo, USP, Brazil

*Corresponding author: [email protected] Poster 69

Tomato chlorosis virus (ToCV), a crinivirus transmitted by Bemisia tabaci MEAM1, in a semi- persistent manner, has become an important problem for tomato production in Brazil. The Brazilian isolate of ToCV infects a range of alternative hosts in Solanaceae and Amaranthaceae families, which show different levels of susceptibility to infection with the virus and preference for oviposition of B. tabaci MEAM1. In order to better understand the complexity of virus-host-vector interaction, which might help on the establishment of some other alternative for disease management, the aim of this study was to evaluate the transmission rate of the virus by B. tabaci MEAM1 from different hosts of ToCV, which showed different levels of preference for vector oviposition. Transmission tests were performed using ToCV-infected eggplant (Solanum melongena), tomato (S. lycopersicum), and Chenopodium quinoa as sources of inoculum. Previous studies showed that the averages oviposition of B. tabaci on these species were 10.6; 2.7, and 0.0 eggs/cm2, respectively. When ToCV-infected eggplant, tomato, and C. quinoa were separately used as sources of inoculum for virus transmission to tomato plants by B. tabaci, average infected plants from two independent assays were 5%, 75% and 0%, respectively. Although eggplant was the most preferred host for oviposition of B. tabaci, tomato was the most efficient source of inoculum for ToCV. Further works are under way to evaluate the susceptibility of those species to ToCV infection, as well as their efficiency as sources of inoculum, under field conditions.

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Bemisia tabaci MEAM1 can acquire Tomato severe rugose virus (ToSRV) during a probe lasting only one minute

Mituti T1*, Toloy RS1, Tanaka FAO1, Maluta NKP1, Lopes JRS1, Fereres A2, Rezende JAM1

1 ESALQ/USP, Piracicaba, SP, Brazil 2 Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 70

ToSRV has been the prevalent begomovirus affecting tomato crops in Brazil. The virus is transmitted by Bemisia tabaci MEAM1 (Middle East-Asia Minor 1), in a persistent circulative manner. This study aimed to evaluate ToSRV transmission by B. tabaci MEAM1 after very short acquisition access period (AAP) and analyze by fluorescence in situ hybridization (FISH) the localization of ToSRV in tomato leaf tissue cells. Transmission tests conducted under different AAP showed that B. tabaci MEAM1 was able to acquire the virus after very short probing time. Average results from five independent experiments showed that for AAP of 1, 3, and 5 min, transmission efficiencies with 30 insects/tomato test-plant were 75%, 73%, and 67%, respectively. For 24-h AAP transmission efficiency was 90%. A total of 77 insects were analyzed by RCA-PCR after one minute probing time on tomato leaves infected with ToSRV. For 33 individual insects analyzed, one tested positive for the virus. For two groups of two insects, only one was positive, while for four groups of 10 insects, two were positive. As positive control, 14 individual insects were analyzed after 24 hours AAP, and in 9 insects the virus was detected. Insects fed for 1 min on tomato leaves systemically infected with the crinivirus Tomato chlorosis virus – ToCV (phloem restricted) were also used as control. ToCV was not detected by RT-PCR in seven groups of 10 insects. Healthy and ToSRV-infected tomato leaves were sectioned in the region of the midrib and analyzed by FISH. Fluorescence was observed in phloem parenchyma cells as well as in the epidermal cells. These results show that B.tabaci MEAM1 can acquire ToSRV during very short AAP.

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Biological and behavioral responses of Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae) to tomato plants infected with Tomato chlorosis virus (ToCV)

Maluta NKP 1*, Fereres A2, Lopes JRS1

1 ESALQ, Universidade de São Paulo, Brazil 2 Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 71

Several phytoviruses are capable of influencing the behavior and fitness of their insect vectors in order to promote their spread among host plants. Tomato chlorosis virus (ToCV, Crinivirus) is an emergent pathogen in solanaceous crops whose effects on behavior and biology of its main vector, Bemisia tabaci (Genn.) are unknown. We investigated the alighting preference and feeding behavior of B. tabaci MEAM1 (Middle East Asia Minor 1) on non-infected (mock-inoculated) vs. ToCV-infected tomato plants, and the indirect effect of ToCV on the biological performance of this species. Additionally we correlated stylet activities of B. tabaci MED (Mediterranean) with the inoculation of ToCV, which is phloem-limited and semi-persistently transmitted. The results showed that tomato infection by ToCV influenced alighting preference and fitness of B. tabaci MEAN1, with negative effect on nymphal viability (only 32% on ToCV-infected plants, contrasting with 77% in non-infected plants). In free-choice assays, ToCV-viruliferous insects respond indistinctly to non-infected or ToCV-infected plants, whereas non-viruliferous insects prefer non- infected ones - a behavior response that does not seem to favor ToCV spread. Electrical Penetration Graph (EPG) studies showed no significant effect of tomato infection on stylet activities of B. tabaci MEAM1 in the phloem sieve elements, which are associated with ToCV inoculation (a significant increase in transmission rate (52%) occurs in waveform E1). Overall, the data show that ToCV does not manipulate whitefly behavior or fitness in a way that would enhance its own spread in tomato crops. Preventing stylet activities in the phloem should be focus of ToCV control strategies targeting this important vector.

136 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Effect of Maize Bushy Stunt Phytoplasma in biological parameters of its vector, Dalbulus maidis and of a non-vector species, Agallia albidula

Ramos A* and Lopes JRS

Luiz de Queiroz College of Agriculture, University of Sao Paulo (ESALQ/USP). SP, Brazil

*Corresponding author: [email protected] Poster 72

Several studies have shown that plant pathogens can manipulate their vector behavior and biological fitness by promoting physiological changes in their host plants, which may become attractive or more suitable to the vector, improving the pathogen spread in nature. However, there is little information about effects of plant infection on performance of non-vector herbivores in the same feeding guild. In the present study, we compared the effects of Maize bushy stunt phytoplasma (MBSP) on oviposition and development of its leafhopper vector, Dalbulus maidis, which is maize specialist and of a polyphagous non-vector leafhopper, Agallia albidula, which was collected on Solanum americanum, a common weed in maize fields. Three couples of each species were submitted, in no-choice assays, for a 2-day oviposition access period on maize plants (n=6) infected with MBSP (still asymptomatic) and non-infected (mock-inoculated); plants were at the 4- leaf stage and had been inoculated 15 days before. Both leafhopper species laid more eggs on asymptomatic MBSP-infected plants than on non-infected ones. The number of eggs laid by the two species was not statistically different, but the numbers of first-instar nymphs and emerged adults were much higher for D. maidis on both infected and non-infected plants, indicating a low survival of A. albidula on maize. The larger number of leafhopper eggs observed on infected plants suggests that MBSP may suppress plant defenses against oviposition. However, the low survival of nymphs and adults of A. albidula on both infected and non-infected plants indicates that MBSP does not make maize a good host for development or may be pathogenic to this non-vector species.

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Involvement of leafhoppers of the Typhlocybinae subfamily in the transmission of Potato Y virus

Duparrois A1,2, Leconte V3, Lebecque P3, Pinchon V3, Gobert V1 and Khelifa M1,2*

1 Semences, Innovation, Protection, Recherche et Environnement, France 2 Centre de ressources régionales en biologie moléculaire. Université de Picardie Jules Verne, France 3 Fédération Régionale de Défense contre les Organismes Nuisibles de Picardie, France

*Corresponding author: [email protected] Poster 73

Potato virus Y (PVY) affects mostly plants of the Solanaceae family. It is the most important viral pathogen affecting both quantity and quality of potato production worldwide (Singh et al., 2008) and the NTN strain inducing the potato tuber necrotic ringspot disease takes over in Europe in recent years (Rolland et al., 2008). It is well known that the PVY is only transmitted by potato colonizing aphids (Radcliffe and Ragsdale, 2002), and non-colonizing species which contribute significantly to its spread (Pelletier et al., 2008; Verbeek et al., 2010). Traps carried out in 2014 on several potato fields distributed in the north of France reveal the prevalence of sap-sucking insects belonging to a taxonomic order different from the aphids, the leafhoppers of the Typhlocibinae subfamily. The analysis of the trapped specimens indicates that a number of them were carriers of PVY and the transmission tests have revealed that the leafhoppers are competent to transmit the virus. These results show for the first time that the leafhoppers of the Typhlocybinae subfamily are vectors of a non-circulative virus and highlight a potential phytosanitary risk for potato crop.

138 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Hypervirulence in Cowpea mild mottle virus in soybean and its effects on fitness of whitefly vectors and the virus itself

Carvalho CM*, Zanardo LG, Trindade TA, Alves MS and Elliot SL

Universidade Federal de Viçosa, Brazil

*Corresponding author: [email protected] Poster 74

High virulence of vectorborne pathogens can lead to reduced fitness if vector performance is affected negatively. Cowpea mild mottle virus (CPMMV; Betaflexviridae: Carlavirus) has emerged as a major threat to soybean production to the high virulence of some of its strains. At its most extreme, CPMMV causes systemic necrosis and plant death, yet some isolates cause far milder mosaic symptoms. In a serial passage experiment with mechanical inoculation, we showed that a highly virulent isolate of CPMMV loses its capacity to cause systemic necrosis consistently after six passages. Besides showing the role of recombination in this change, we show that the titre of the virus within the plant increases with reduced virulence. Critically, we show that the fitness of the whitefly vector (Bemisia tabaci MEAM1, Hemiptera: Aleyrodidae) on plants infected with the necrogenic isolate is zero – nymphs do not survive long enough to reach adulthood – while its fitness on plants infected with the same isolate that is no longer necrogenic is equivalent to that on uninfected control plants. We conclude that the virus has the capacity to switch its virulence rapidly, through recombination, depending on the host it is infecting, but the existence of highly necrotic isolates appears paradoxical as our results show that necrosis precludes development of the insect vector. We propose that the non-persistent mode of transmission of this carlavirus in whiteflies is a key aspect that allows this situation to persist, and suggest some scenarios in which transmission might occur even for necrogenic isolates. Financial support: FAPEMIG, CNPq, CAPES.

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Multi’omics reveal adult female and male Diaphorina citri respond similarly to ‘Candidatus Liberibacter asiaticus’ exposure

Coates L1*, Mahoney J2,3, Warwick ER4, Johnson R5, MacCoss M5, Ramsey J2,3, Moulton K4, Shatters R4, Hall D4, Cilia M2,3,6 and Slupsky C1

1 Department of Food Science and Technology, University of California, USA 2 Boyce Thompson Institute for Plant Research, USA 3 Robert W. Holley Center for Agriculture and Health, Emerging Pests and Pathogens Research Unit, USDA Agricultural Research Service, USA 4 U.S. Horticultural Research Laboratory, Unit of Subtropical Insects and Horticulture, USDA Agricultural Research Service, USA 5 Department of Genome Sciences, University of Washington, USA 6 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, USA

*Corresponding author: [email protected] Poster 75

Citrus greening disease is a major threat to citrus industries worldwide. It is associated with the phloem- restricted bacterium, ‘Candidatus Liberibacter asiaticus’ (CLas), and transmitted by the phloem-feeding hemipteran, Diaphorina citri. D. citri can acquire CLas as nymphs and adults, and transmit CLas in a persistent-circulative mode. However, not all individuals within a population transmit CLas, particularly among individuals that acquire CLas as adults. This study confirms previously published research which suggests that D. citri may defend against CLas infection and that the protective response is more pronounced in adults. A systems biology approach was taken to understand the response of D. citri to CLas infection, and if the response varies by sex. Female and male adult D. citri were analyzed separately after feeding on healthy or CLas-infected citrus. From each unique sample of female or male D. citri, metabolites, RNA, DNA and protein were sequentially isolated. Upon multivariate analysis of D. citri metabolite abundance, we found that whether adult D. citri were fed on CLas-infected or uninfected citrus explained greater differences in metabolism between D. citri compared to the metabolic differences associated with sex. In particular, diaphorin -- a cytotoxic polyketide produced by the bacteriome endosymbiont ‘Candidatus Profftella armatura’ -- is more abundant in D. citri exposed to CLas-infected citrus compared to D. citri reared on healthy citrus, perhaps serving a protective role against CLas infection. Analysis of transcript abundance in D. citri revealed that the predicted biosynthetic pathway for (S)-methylmalonyl-CoA – a probable extender unit of diaphorin – is expressed to a greater degree in CLas-exposed D. citri than unexposed D. citri. Ultimately, we found that there are a core set of D. citri pathways differentially regulated in response to CLas, regardless of sex. These pathways provide candidate targets to manipulate and assay for involvement in CLas infection of D. citri. 140 | S5 H EMIPTERA- PLANT PATHOGEN INTERACTIONS

Candidatus Liberibacter asiaticus localizes inside ER-associated bodies in Diaphorina citri gut cells

Levy A1*, Achor D1 and Ghanim M2

1 University of Florida, CREC, USA 2 Department of Entomology, Volcani Center, Israel

*Corresponding author: [email protected] Poster 76

Citrus greening is caused by the phloem-restricted bacteria Candidatus Liberibacter asiaticus (CLas), and is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. Current management options for greening rely on the application of chemical insecticides but for the most part those strategies are still not effective enough. As an alternative, it may be possible to block the bacteria propagation and/or movement inside the psyllid, but very little is currently known about the bacteria-insect interactions. We recently identified a novel immune response that takes place during this interaction, showing that the acquisition of CLas by ACP induced significant apoptosis in the insect gut (Ghanim et al., 2016). Here we focused on the role that the ER is playing in this process. Confocal microscopy of ER labeled gut cells of infected ACP adults showed that in the presence of CLas the ER undergo a dramatic re-organization, forming intracellular bodies. Immunolocalization identified CLas accumulation inside these bodies. Electron microscopy studies verified these observations. Our results suggest that after entry into the gut cells, CLas modifies the host ER to create an isolated and safe environment to support its own propagation inside the psyllid. This mechanism may also enable to bacteria to escape apoptosis. Ghanim, M., Fattah- Hosseini, S., Levy, A., and Cilia, M. (2016). Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus.

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Implementation of DNA barcodes for Amplicephalus funzaensis Linnavuori and Exitianus atratus Linnavuori (Hemiptera:Cicadellidae), insect vectors of phytoplasmas of the Sabana de Bogota, Colombia

Alfaro JP1, Silva AF1, Montaño DC1, Brochero E2, Michael R3, Franco-Lara L1*

1 Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, , Colombia 2 Facultad de Ciencias Agrarias, Universidad Nacional de Colombia, Colombia 3 Department of Biodiversity and Taxonomy, National Museums & Galleries of Wales, UK

*Corresponding author: [email protected] Poster 77

The presence of phytoplamas have been associated with urban trees and crops in the Sabana de Bogotá, Cundinamarca, Colombia showing severe decay symptoms and deviation from normal morphology. These pathogenic bacteria of the class Mollicutes are transmitted by insect vectors, including some species of leafhoppers. DNA Barcode is molecular tool that allows species identification by amplification and sequencing of a short conserved sequence among a taxon. In the case of arthropods, the gene COI (Citrochrome oxidase I) has been widely used. The objective of this work was to provide DNA barcodes for five leafhoppers species that inhabit the grasslands of the Sabana de Bogota. Samples of leafhoppers from two grassland areas were taken at Universidad Militar Nueva Granada (UMNG), in Cajicá-Cundinamarca (rural area) and Universidad Nacional de Colombia (UNAL) in Bogotá (urban area). Representative individuals of five leafhopper morphotypes, including the phytoplasma vectors Amplicephalus funzaensis and Exitianus atratus, were identified by classic taxonomic procedures. Polymerase chain reaction (PCR) amplifications were performed using primers LCO1490/HCO2198 that amplify COI; amplicons of 720 pb were sequenced and sequences compared with NCBI and BOLD databases by BLAST-n. None of sequences obtained in this work corresponded to previously reported species. However, in the dendograms A. funzaensis and E. atratus clustered with species of their genuses, morphotype 3 with Deltocephalinae, morphotype 6 with genus Xestocephalus and morphotype 2 was inconclusive. Sequences are being uploaded in the BOLD database. Phytoplasmas of groups 16SrI and/or 16SrVII were detected by PCR with universal primers in A. funzaensis and E. atratus, morphotype 2, morphotype 3 and morphotype 6, in at least one sample of each species. In the future, DNA barcodes will help to confirm the identification of males and females of the same species and of the different juvenile stages, helping the biological and ecological characterization of these species.

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Insect-Plant Dialog 6|

144 | S6 INSECT- PLANT DIALOG

Role of MIF immune regulators in plant-aphid interactions

Coustau C*, Naessens E, Michelet C and Keller H

Institut Sophia Agrobiotech, France

*Corresponding author: [email protected] Keynote Lecture

Little is known about the molecular processes that allow aphids to exploit their host plants. We have identified a family of cytokine MIF (Macrophage Migration Inhibitory Factor) in the pea aphid Acyrthosiphon pisum and the green peach aphid, Myzus persicae. Interestingly one family member is expressed in the salivary glands of aphids, and secreted during feeding. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We showed that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. The ectopic expression of aphid MIF1 in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. The existence of three MIF orthologs in the plant Arabidopsis thaliana (AtMDL) suggests that aphid MIF could possibly use the AtMDL molecular and cellular machinery for modulating plant defense to the aphid’s advantage. Our first results suggest that plant AtMDL genes participate to plant immune responses and interactions with aphids. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.

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Chemical and behavioral analysis of the whitefly Bemisia tabaci attraction to rosemary (Rosmarinus officinalis)

Sadeh D1*, Ghanim M2 and Dudai N1

1 The Unit of Aromatic and Medicinal plants, Newe Ya’ar Research Center, Agricultural Research Organization (ARO), Israel 2 Department of Entomology, Volcani Center, ARO, Israel

*Corresponding author: [email protected] Oral presentation

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a polyphagus insect pest that considered a complex of cryptic species, some of which are known for their ability to develop high levels of resistance to pesticides. Therefore, the development of additional control methods is urgently needed. Field observations revealed the presence of rosemary chemo-types, which differ in their attractiveness to whiteflies. Rosemary (Rosmarinus officinalis, Lamiaceae), is native to the Mediterranean region as an aromatic evergreen plant. Rosemary is grown commercially in Israel as a fresh herb and for preservative extractions as well. The general aim of this research is to characterize the resistance mechanisms of rosemary against B. tabaci based on its volatile compositions. A series of choice and non-choice laboratory experiments using two chambers connected with a glass tunnel and rosemary seedlings, was conducted. A Solid Phase Micro- Extraction (SPME) and GC-MS analysis were used to characterize volatiles that might influence the insect’s behavior. In order to test the response of whiteflies to various pure volatile components, a series of non-choice experiments was done. In the preference tests of B. tabaci adults with rosemary seedlings we found diversity among chemo-types with different oil contents. Variety "2" was significantly more attractive than variety "11", and the two varieties also differed in their oil content (0.67% and 1.43%, respectively). Significant differences in B. tabaci preferences were recorded and exhibited a bell curve behavior for the volatiles β-caryophyllene and D-limonene in doses that ranged between 0.002–0.04 and 0.0002-0.008 ppm, respectively. The differences in essential oil content, its composition and the percentage of the volatiles, which may influence B. tabaci preferences, suggest complex plant-insect interactions mediated by these volatiles. Breeding for volatile compositions that influence B. tabaci interactions with plants is a new strategy that could be adopted for combating this important pest.

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Modulation of legume defense signaling pathways by native and non-native pea aphid clones

Sanchez-Arcos C, Gershenzon J and Kunert G*

Max Planck Institute of Chemical Ecology, Germany

*Corresponding author: [email protected] Oral presentation

The pea aphid (Acyrthosiphon pisum), a phloem-sucking insect, is a complex of at least 15 genetically different host races that are native to specific legume plants, but can all develop on the universal host plant Vicia faba. It is still not clear why pea aphid host races are able to colonize their native hosts while other host races are not. When aphids test plant suitability they penetrate the plant and salivate into plant. Thus plants might react differently to the various pea aphid host races and might vary in their defense responses to different pea aphid host races. In order to test this, we measured the amounts of salicylic acid (SA), the jasmonic acid-isoleucine conjugate (JA- Ile), other jasmonate precursors and derivatives, and abscisic acid in four different species (Medicago sativa, Trifolium pratense, Pisum sativum, V. faba) after infestation by native and non- native pea aphid clones of various host races. We also assessed the performance of the clones on the four plant species. On M. sativa and T. pratense non-native clones were barely able to survive or reproduce, and triggered a strong SA and JA-Ile response. The infestation with native clones however, led to lower levels of both phytohormones. On P. sativum, non-native clones survived or reproduced to a certain extent, and induced fluctuating SA and JA-Ile levels, whereas the native clone triggered only a weak SA and JA-Ile response. On the universal host V. faba all aphid clones triggered only low SA levels initially, but induced clone-specific patterns of SA and JA-Ile later on. Our results suggest that A. pisum clones manipulate plant-defense signaling to their own advantage, and perform better on their native hosts due to their ability to modulate the SA- and JA-defense signaling pathways.

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Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar- induced aphid resistance in maize

Tzin V13*, Lindsay PL1, Christensen SA2, Meihls LM1, Blue B1 and Jander G1

1 Boyce Thompson Institute for Plant Research, USA 2 USDA-ARS Chemistry Unit, Center for Medical, Agricultural, and Veterinary Entomology, USA 3 Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Israel

*Corresponding author: [email protected] Oral presentation

Plants in nature have inducible defenses that sometimes lead to targeted resistance against particular herbivores, but susceptibility to others. The metabolic and genetic diversity available for maize make this a suitable system for a mechanistic study of within-species variation in such plant- mediated interactions between herbivores. Beet armyworms and corn-leaf aphids are naturally occurring maize herbivores with different feeding habits. Whereas chewing herbivore-induced methylation of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) to form 2- hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) promotes caterpillar resistance, lower DIMBOA-Glc levels favor aphid reproduction. Thus, caterpillar-induced DIMBOA- Glc methyltransferase activity is predicted to promote aphid reproduction. To test this tradeoff, the impact of caterpillar feeding on aphid progeny production was assessed using genetically diverse maize inbred lines. Whereas aphid progeny production was increased by prior caterpillar feeding on lines B73, Ki11, Ki3, and Tx303, it decreased on lines Ky21, CML103, Mo18W, and W22. Genetic mapping of this trait in a population of B73 x Ky21 recombinant inbred lines identified significant quantitative trait loci on maize chromosomes 1, 7 and 10. There is transgressive segregation for aphid resistance, with the Ky21 alleles on chromosomes 1 and 7 and the B73 allele on chromosome 10 increasing aphid progeny production. The chromosome 1 QTL coincides with a cluster of three maize genes encoding benzoxazinoid O-methyltransferases that convert DIMBOA- Glc to HDMBOA-Glc. Gene expression and benzoxazinoid measurements indicate that caterpillar induced responses in this pathway differentially affect aphid resistance.

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Identification and expression analysis of candidate odorant-receptors (ORs) by antennal transcriptome of Sitobion avenae

Fan Jia*, Zhang Qian, Xue Wenxin, Jiang Xin, Chen Julian

Institute of Plant Protection, Chinese Academy of Agricultural Sciences. China

*Corresponding author: [email protected] Oral presentation

More than 4000 species with their various host plants make aphids the perfect model to study evolution and co-evolution between plants and herbivorous insects. Moreover, developing novel strategy on population control of aphid, major pest in worldwide agriculture, drives aphid-plant chemical communication one of the most active research area. Insect ORs are insect specific and critical for info-chemical perception. To date, both OR family of pea aphid Acyrthosiphon pisum (ApOR) and cotton aphid Aphis gossypii (AgoOR) have been annotated based on the available genomic data. Here we report the identification of 31 ORs in Sitobion avenae, the most serious stenophagic pest in cereal fields. We sequenced the transcriptome for the antenna as well as head from winged and wingless individuals of Sitobion avenae. Among 133,331 unigenes obtained, 31 ORs putative transcripts (SaveORs) were identified according to ApORs and AgoORs. Except Orco as a co-expressed protein has been reported by us before, 30 ORs include an OR5 orthologues, the alarm pheromone E-β-farnesene receptor, are first discoveries from S. avenae. By phylogenetic analysis of the proteins encoded by the 31 AgoORs along with the 73 ApORs, we identified 12 SaveOR/ApOR orthologous subgroups, which contain 18 SaveOR in total. Other than Orco, orthologous subgroups with identities higher that 80% include the SaveOR1 (Orco), 2, 3, 4, 5, 9,18,20,22, 25, 33, 42, 43, 47, 54. All SaveORs have identities with either ApORs or AgoORs higher than 50%. The expression levels of the SaveORs varied depending on different tissues. 18 ORs are antenna-specific and 5 ORs are significantly high expressed in antenna. Compare to other ORs, Orco, OR3, OR22 and OR68 showed significantly higher expression. Of which, OR22 is antenna- specific in wing aphid. Further molecular and functional studies of these olfactory system genes will explore their potential as novel targets for controlling S. avenae.

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Linking plant-soil feedbacks to several trophic levels in sweet pepper-aphid system

Vaello T1,2*, Kos M2, Bezemer M2, Marcos-García MA1 and Pineda A2

1 CIBIO, University of Alicante, Spain 2 NIOO-KNAW, Wageningen, The Netherlands

*Corresponding author: [email protected] Oral presentation

Changes in soil properties that are caused by plants, which influence the performance of plants that grow later in the soil are termed ‘plant–soil feedbacks’ (PSF). Plant-soil feedbacks can be driven by changes in soil microorganisms, such as bacteria, mycorrhizal or pathogenic fungi. Soil microbial communities can influence primary and secondary plant metabolite concentration and consequently affect the performance of above-ground herbivores, such as aphids. While most of this knowledge comes from studies with single microbes, not much is known how different soil microbial communities affect the performance of plants and insects. The aim of this study was to test PSF effects on four trophic levels: sweet pepper plants (Capsicum annuum), the aphid Myzus persicae, its natural enemy Aphidius colemani, and the hyperparasitoid Dendrocerus aphidum. We also assessed molecular mechanisms usually involved in plant defences by measuring the response of salicylic and jasmonic acid-responsive genes. Seeds of sweet pepper were sown in different types of soil and sterile soil as control. Aphid development was then monitored during two weeks for each type of soil, and performance parameters as survival and weight of each individual, were measured for parasitoids and hyperparasitoids. Our results show that plant-soil feedbacks do not only affect plants but also higher trophic levels.

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An aphid effector protein localizes to the cytoplasm of mesophyll cells and suppresses the first layer of the plant defence response

Mugford ST1*, Drurey C1, Chen Y1, Corkill B1, Mathers T2 and Hogenhout SA1

1 Cell and Developmental Biology, John Innes Centre, UK 2 The Earlham Institute, UK

*Corresponding author: [email protected] Oral presentation

Aphids and other hemipteran herbivores use specialised mouthparts (stylets) to navigate to the phloem and feed from the vasculature. To successfully establish a feeding site, the aphid must overcome plant defences. Plants respond to the presence of insects through activation of the pathogen-associated molecular pattern (PAMP)- triggered immunity (PTI) signalling pathway, leading to the activation of downstream defence factors detrimental to aphids. A growing body of evidence shows that aphids secrete effector proteins that suppress PTI and other plant defence pathways in plants. Mp10 is one of these effector proteins and was first identified from the green peach aphid Myzus persicae. Immunogold-labeling experiments of aphid feeding sites showed the presence of Mp10 in the cytoplasm of mesophyll cells nearby aphid stylet tracks, but not further away from these tracks. We now have direct evidence that Mp10 suppress PTI downstream of the receptor-like kinase BAK1. Knocking down Mp10 expression by RNA interference in the aphid resulted in a decline of aphid fecundity. Interestingly, we found that Mp10 is conserved amongst plant-feeding hemipterans and likely was present in a common ancestor before the divergence of clades that include aphids, whiteflies and leafhoppers.

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On the verge of a hot, juicy dinner: A heat- inducible phloem protein that compromises aphid feeding

Kloth KJ2*, Busscher-Lange J1, Wiegers GL1, Kruijer W1, Buijs G1, Meyer RC, Albrectsen BR2, Bouwmeester HJ1, Dicke M1 and Jongsma MA1.

1Wageningen University, The Netherlands 2 Umea University, Sweden

*Corresponding author: [email protected] Oral presentation

One of the most effective host-plant resistance mechanisms to aphids is to obstruct phloem ingestion. In several plant species, phloem ingestion can be restricted almost instantly after sieve element penetration. Phloem proteins are considered to play a role in this phenomenon by plugging the food channel in the aphid stylet bundle. The diversity and functions of phloem proteins are yet poorly understood. To find novel resistance genes, we screened Myzus persicae behavior on 350 Arabidopsis natural accessions with a video tracking platform. Genome-wide association mapping with 214k SNPs revealed a strong association between aphid feeding behavior and polymorphisms in PHLO, a gene encoding a heat shock-like protein. Characterization of knockout and near-isogenic lines, showed that PHLO reduces the duration and the rate of phloem ingestion. The effects were stronger during high ambient temperature, and we observed that knockouts suffered from reduced seed set during heat stress. In transgenic lines expressing fluorescent fusion proteins, PHLO localized to the sieve element margins. We consider that this chaperone-like phloem protein may create protein agglomerations which interfere with sap translocation into the aphid food channel and affect phloem flow rates during heat stress.

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Nitric oxide as a signaling molecule in bioactive pea response to pea aphid infestation

Morkunas I1, Woźniak A1*, Formela M1, Bednarski W2, Marczak L3, Narożna D4, Dancewicz K5, Mai VC1,6, Borowiak-Sobkowiak B7, Floryszak-Wieczorek J1 and Gabryś B5

1 Department of Plant Physiology, Poznań University of Life Sciences, Poland 2 Institute of Molecular Physics, Polish Academy of Sciences, Poland 3 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland 4 Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poland 5 Department of Botany and Ecology, University of Zielona Góra, Poland 6 Department of Plant Physiology, Vinh University, Vietnam 7 Department of Entomology and Environmental Protection, Poznań University of Life Sciences, Poland

*Corresponding author: [email protected] Oral presentation

The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e. S- nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L.cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. This study is the first to demonstrate that application of NO on pea seedlings reduce A. pisum performance and feeding activity. At the same time, we showed here the dynamics of defense (e.g. changes in the redox status, phenylpropanoid metabolism and β-glucosidase activity) induced by exogenous NO in P. sativum response to pea aphid attack. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non- pretreated leaves. A decrease in the level of O2− was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 hpi. Directly after the pretreatment with GSNO an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors limited of the A. pisum population growth rate. Reduction of pea aphid performance was caused a consequence of reduced phloem sieve element contact. Results of these studies suggest sequence in the dynamic events that occur as a result of NO application and A. pisum infestation of P. sativum.

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An aphid effector associates with a host trafficking protein to promote virulence

Rodriguez PA2, Escudero-Martinez C1,2 and Bos, JIB1,2*

1 Cell and Molecular Sciences, The James Hutton Institute, UK 2 Division of Plant Sciences, School of Life Sciences, University of Dundee, UK

*Corresponding author: [email protected] Oral presentation

Aphids are major economic pests, causing significant economic losses to many crops worldwide. To improve pest control strategies against aphids, it is essential to understand how these insects interact with host plants at the molecular level. Host-feeding insects need to extensively manipulate host cell processes to suppress immunity and/or promote nutrient release. This typically involves the secretion and delivery of saliva inside host cells, which contains a cocktail of proteins and other molecules that are involved in promoting virulence. However, knowledge on the activities of these proteins or molecules, also called effectors, is limited. We have identified and characterized aphid effectors from economically important species to understand how they interfere with cellular processes. Using yeast-two-hybrid screens and co-immunoprecipitation approaches we have identified several aphid effector-host protein interactions. One example is Myzus persicae effector Mp1, which associates with host Vacuolar-Protein-Sorting-Associated Protein 52 (VPS52). Functional analyses of natural Mp1 and VPS52 variants from different aphid and plant species, respectively, showed a strong link between effector virulence activity and association with VPS52. Moreover, we found that the Mp1-VPS52 association was highly specific to Myzus persicae-host interactions. Also, co-expression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. Over-expression of VPS52 resulted in reduced aphid fecundity. In addition, aphid infestation led to reduced VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our data show that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner.

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Characterization and effect of glandular trichomes on plant aphid interactions in tomato

Blanco L* and de la Peña E

Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM) "La Mayora" UMA-CSIC, Spain

*Corresponding author: [email protected] Oral presentation

Tomato, Solanum lycopersicum is one of the most important crops worldwide, but its agricultural production is impaired by different insect pests that affect its growth and productivity. Some species of aphids as Macrosiphum euphorbiae are of particular importance as they not only limit plant growth but also are important vectors of viruses and other plant pathogens. In many areas of the world a significant part of aphid management still relies on chemical control, a situation that requires the further development of alternative production methods and IPM. In this sense, there are two complementary ways of action: (1) developing crop varieties resistant to insects and; (2) improving biological control strategies using natural enemies of insect pests. In either case, it is necessary to understand the role of plant defenses as they mediate not only the interaction of plants with insect herbivores, but also with natural enemies. Glandular trichomes have a key role in plant defense against insect pests. They are involved in the production of defensive compounds (e.g. acylsucroses) that often mediate the interaction with aphids and also with their natural enemies. In this study we addressed how trichome density and leaf acylsucroses vary among different genotypes of tomato, and to what extent are these traits induced by aphid herbivory. The performance of aphids on different genotypes of tomato that vary in the type and density of glandular trichomes was also analyzed. Finally, the main signaling pathways related with these plant defensive traits were characterized.

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Genetics of virulence in pea aphid on Medicago truncatula hosts differing in R gene complement

Kanvil S1, Selby M1, Iqbal J1, Lopez-Cobollo R1, Chong W1, Tanny T1, Oteo G1, Wood A1, Tabassum N1, Rashid U1, Nowakowska S1, Kanja C1, Lyu C1, Hui S, Simon J-C2, Bos J3, Powell G1 and Turnbull C1*

1 Imperial College London, UK 2 INRA Rennes, France 3 University of Dundee, UK

*Corresponding author: [email protected] Oral presentation

Pea aphid is much studied as a model for speciation and adaptation to different legume host species, but less is known about how its genetic variation impacts on compatibility within a single host species represented by genotypes possessing different complements of R genes. Here, we tested clones of pea aphid from different host race groups on Medicago truncatula genotypes carrying or lacking the previously mapped QTL, RAP1, that confers strong race-specific resistance to certain clones. These patterns of virulence/avirulence and resistance/susceptibility broadly conform to the classic gene-for-gene model. Using laboratory crosses of virulent x avirulent parents to generate several F1 and F2 populations, we show segregation patterns for virulence that are consistent with Mendelian inheritance of a relatively small number of genes with large apparent effects. A single heterozygyous locus, dominant for virulence, appears to explain a substantial proportion of variation, but additional loci are likely to contribute to progeny phenotypes. Impact of host genetic background was also found, especially when comparing the M. truncatula genotype Jemalong A17, in which RAP1 was originally discovered, with near-isogenic lines (NILs) derived from a cross of Jemalong A17 with the susceptible (rap1) genotype DZA315.16. Although RAP1 appears to contribute the majority of resistance, other loci are also involved, such that resistance is significantly diminished in NILs carrying RAP1 compared with their Jemalong A17 progenitor. We highlight the potential of these aphid populations as resources to reveal the molecular basis of virulence and avirulence, thus providing better understanding of aphid-plant interactions, potentially leading to design of more robustly resistant crop plants.

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A wheat specific integrated-domain NLR gene as role player in the wheat-Diuraphis noxia interaction

Venter E* and Nicolis VF

University of Johannesburg, South Africa

*Corresponding author: [email protected] Oral presentation

Diuraphis noxia (Kurdjumov) is an economically important pest of wheat and barley in many countries, including South Africa. Crop yields are negatively affected during aphid infestation due to chlorosis and necrosis of the leaves, eventually leading to the death of susceptible cultivars. The effective use of resistant wheat cultivars is undermined by the emergence of resistance breaking D. noxia biotypes, threatening the cultivation of wheat. The aim of this study was to characterise a defence related nucleotide-binding leucine-rich repeat (NLR) gene, Adn1. Adn1 is a coiled-coil NLR residing in a family of NLR genes targeted by tae-miR-7757. This miRNA binding site is located in the NB-ARC domain of the NLR genes. Adn1 contains a C-terminal WRKY integrated domain that conforms to the integrated domain as effector decoy hypothesis. Knockdown of this gene in the resistant cultivar Tugela DN (harbouring Dn1) using virus induced gene silencing (VIGS), resulted in a susceptible phenotype in response to RWA-SA1. The expression pattern of Adn1 indicated regulation in response to aphid infestation. Phylogenetic analyses further indicated that Adn1 has a structure unique to hexaploid wheat. This study reports the role of an ID-NLR following the integrated decoy hypothesis applied to plant-pest interactions.

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Small RNAs from whiteflies are transferred to tomato phloem during feeding van Kleeff P, Galland M, Schuurink R and Bleeker P*

University of Amsterdam, The Netherlands

*Corresponding author: [email protected] Oral presentation

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host- defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT- PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

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Metabolic responses to variable host plants play a key role in different degrees of polyphagy in the Bemisia tabaci species complex

Malka O1*, Feldmesser E2, Santos-Garcia D1, Sharon E1, Van Brunschot S3, Easson MLAE4, Gershenzon J4, Vassão DG4, Seal S3, Colvin J3 and Morin S1

1 Department of Entomology, Faculty of Agriculture, the Hebrew University of Jerusalem, Israel 2 Department of Biological Services, Weizmann Institute of Science, Israel. 3 Agriculture, Health and Environment Department, Natural Resources Institute, University of Greenwich, UK 4 Max Planck Institute for Chemical Ecology, Germany

*Corresponding author: [email protected] Oral presentation

The cryptic polyphagous phloem-feeder Bemisia tabaci can feed on a broad range of host plants. However, the adaptation strategies utilized for handling the insect’s diverse diet remain poorly understood. By using six species of B. tabaci and four host plants (eggplant, cassava, kale and pepper), we investigated how B. tabaci adults cope behaviorally and metabolically with the environmental unpredictability presented by the experimental set of host plants. All six species were colonized on eggplant plants for at least two generations. Adults were transferred from the eggplant colony to sucrose diet for 72 hr and then to one of the four tested host plants. After 24 hr, adults were collected for RNAseq analysis. Transcriptional changes associated with B. tabaci responses to host switching revealed several insights. First, large set of genes involved in the metabolism of carbohydrates, fats, amino acid, xenobiotic and vitamins showed differences in their expression pattern between species but, within species, these genes were constitutively expressed, and showed no response to the host plant utilized. Second, within each species, sets of genes associated with environmental information processing and various organismal systems, responded to host switching. These include genes encoding for proteins involved in signal transduction, membrane transport, immune system, endocrine system, sensory system and digestive system. Based on these results, we assume that differences in the expression pattern of various metabolic pathways may hold the key to understanding the differences in host adaptation among closely related B. tabaci species. Moreover, the level of polyphagy might be related to the level of efforts to manage energetic costs, with species using the same set of genes for different metabolic pathways being less polyphagous. Also, within species, transcriptional plasticity of genes associated with information processing and organismal systems are likely to play a central role in short-term (24 hr) adaptation to new host plants. Our transcriptional results were supported by both behavioral and metabolomics data.

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Role of plant cell wall and cell wall-derived oligogalacturonides in defense against Myzus persicae

Gravino M1*, Mugford ST2, Chen Y2, Cervone F1, De Lorenzo G1 and Hogenhout SA2

1 Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Università di Roma Sapienza, Italia 2 Department of Cell and Developmental Biology, John Innes Centre, UK

*Corresponding author: [email protected] Poster 78

As sessile organisms, plants are continually exposed to pathogens, to which, in most cases, they resist by activating the innate immune system. A first barrier against pathogens is represented by the plant cell wall. Emerging evidence indicates that plant cells harbor sophisticated mechanisms for sensing the alteration of cell wall integrity during biotic stress. For instance, they perceive endogenous molecules generated and/or released as a consequence of the cellular damage caused by pathogens, e.g. the so-called damage-associated molecular patterns (DAMPs). A well- known class of DAMPs is represented by oligogalacturonides (OGs), pectin-derived oligosaccharides made of α-1,4-linked galacturonic acid that derive from the partial hydrolysis of homogalacturonan. OGs have a pivotal role in defense against fungal and bacterial pathogens; however, their role in defense against insects is not yet clear. Aphids are sap-feeding insects of the order Hemiptera and are among the most destructive pests in agriculture. Myzus persicae (green peach aphid) is a species with a broad host range and colonizes hundreds of plants species in over 40 plant families, including brassicas. We use Arabidopsis thaliana as a model to better understand the role of OGs and cell wall in the response of plants to M. persicae. Current research is focused on how the following impact plant-aphid interactions: (1) cell wall modifications, i.e. pectin methyl esterases and their inhibitors (PMEIs); (2) OG perception/signaling, i.e. Wall- Associated Kinase 1 (WAK1), Glycine Rich Protein 3 (GRP3), Somatic Embryogenesis Receptor-like Kinases (SERKs); and (3) OG accumulation, i.e. polygalacturonases and their inhibiting proteins (PGIPs). It is expected that this work will help towards the identification of plant resistance mechanisms to aphids.

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Transcriptome analysis of the salivary glands of grain aphid, Sitobion avenae

Zhang Yong1, Fan Jia1, Sun Jingrui1, Frédéric Francis2, Chen Julian1*

1 State Key Laboratory of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, PR China 2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liège, Belgium

*Corresponding author: [email protected] Poster 79

Aphid emits saliva in the process of probing and feeding on host plant phloem, and aphid saliva plays important roles in aphid-host interactions, such as assisting aphid digestion, detoxification, eliciting or suppressing plant defenses. Grain aphid, Sitobion avenae, is one of the most important pests of cereals worldwide causing serious economic loss by direct feeding and transmitting Barley yellow dwarf virus (BYDV). In this study, we performed the transcriptome analysis of salivary glands of S. avenae using Illumina HiSeq 2500. A total of 33,079 assembled unigenes were identified in the salivary glands of alate and apterous adult aphids. Of the all obtained unigenes, 15,833(47.86%) and 10,829(32.73%) unigenes showed high similarity to the known proteins in Nr and Swiss-Prot databases respectively. 526 unigenes were predicted to encode secretory proteins, including some digestive and detoxifying enzymes and potential effectors. The RT-PCR and RT- qPCR results showed that all of the 15 most highly expressed putative secretory proteins specifically expressed in salivary glands. Interestingly, 11 of the 15 most highly expressed putative secretory proteins were still not matched to function-known proteins. We also detected the expression of 9 interested putative secretory proteins in aphid different tissues, including some digestive and detoxifying enzymes, effectors and Ca2+ binding proteins. The results showed that glutathione-S-transferase 1 was specifically expressed in salivary glands, angiotensin converting enzyme and reticulocalbin-2 were highly expressed in salivary glands, trehalase and calumenin had high expression in both salivary glands and alimentary canals. These findings provide a further insight into the composition of S. avenae saliva, identification of potential effector molecules and understanding of the roles of saliva in induced resistance in wheat.

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Different effect mechanisms of aphid pre- infestation on transmission and acquisition of Tomato Yellow Leaf Curl Virus in host plants and whiteflies

Tan XL, Chen JL, Benelli G, Desneux N, Yang XQ, Liu TX and Ge F

Chinese Academic of Agricultural Sciences, China

*Corresponding author: [email protected] Poster 80

Herbivore infestation may affect plant defense, which is regulated by jasmonate and salicylate mediated signaling pathways, and may also influence feeding dynamics of subsequent herbivores and transmission and acquisition of the virus. We hypothesize that such a ‘plant–vector–virus’ relationship might be influenced by Myzus persicae pre-infestation. The variations of Tomato Yellow Leaf Curl Virus concentration in host plant tissues and in whiteflies were similar which was increased firstly then decreased, suggesting positive relationships of instantaneous pre-infestation by aphids with TYLCV transmission and acquisition. The underlying mechanisms were further confirmed by feeding behavior and expression of key genes in SA signaling and JA signaling by real- time quantitative PCR before transmission and acquisition. A similar trend was also found in phloem ingestion in the transmission process and whole feeding behavior of whiteflies in the TYLCV acquisition stage according to the electrical penetration graph test. Transcript levels of key genes were up-regulated in SA signaling and down-regulated in JA signaling with increased aphid pre-infestation times before transmission. However, variations of those genes differed before acquisition. Our results proved tomato plant–whitefly–TYLCV interactions were influenced by aphid pre-infestation: SA and JA pathways co-regulated TYLCV concentration in transmission stage, and the whitefly feeding behavior didn't dominate the proceeding. In comparison, whitefly acquisition of TYLCV was simultaneously dominated by the JA pathway and whitefly feeding behavior. These findings enhance our understanding of plant–arthropod herbivore–virus interactions, which are important for ecological pest and pathogen management programs.

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Using dsRNA of the BtGST2 detoxification gene to develop Bemisia tabaci resistant plants

Eakteiman G*, Moshitzky P, Moses-Koh R, Malka O, Mestre-Rincon N and Morin S

Department of Entomology, Hebrew University of Jerusalem, Israel

*Corresponding author: [email protected] Poster 81

Bemisia tabaci, the sweet-potato whitefly, is one of the most devastating pests worldwide, causing enormous economical lost in agriculture. As other phloem-feeding insects, B. tabaci is mainly controlled by synthetic insecticides. Insecticide resistance is one of the major problems agriculture faces nowadays and developing alternatives to insecticides is necessary. As a highly polyphagous pest, B. tabaci utilizes an induced general detoxification system capable of dealing with a broad range of plant toxins. Our main goal was to develop an RNA interference technology that will allow the production of pest-resistant crops capable of silencing insect detoxification genes. Artificial feeding assay was performed by exposing B. tabaci adults to double-strand (ds) RNA of the BtGST2 (dsBtGST2) gene, a known general detoxifier in B. tabaci. In addition, transgenic Arabidopsis thaliana plants expressing dsBtGST2 under a phloem specific promoter were produced and feeding and development assays were conducted. Finally, gene expression, in all the experiments, was analyzed by Real Time PCR. Artificial diet showed significant down-regulation in BtGST2 gene expression in the mid-gut of adults compared to the control group. Individuals developing on transgenic A. thaliana plants showed significant developmental delay when compared to those developing on wild type (WT) plants. In parallel, BtGST2 gene expression in the mid-gut showed significant down-regulation in individuals fed on transgenic plants compared to individuals fed on WT plants. Slowing the development of B. tabaci on host plants by RNAi can increase the efficiency of its control by natural enemies, which will lead to minimizing the use of synthetic insecticides. Future experiments will assess the influence of combined down-regulation of two detoxification genes (BtGST2 & BtCYP6like5) on the ability of B. tabaci to utilize plant hosts.

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Metabolism of activated plant chemical defenses by phloem feeders

Vassão DG1*, Malka O2, Easson MLAE1, Paetz C1, Stanisic A1, Reichelt M1, Springate S3, Colvin J3, Gershenzon J1 and Morin S2

1 Max Planck Institute for Chemical Ecology, Germany 2 Hebrew University of Jerusalem, Israel 3 University of Greenwich, UK

*Corresponding author: [email protected] Poster 82

The widely successful feeding habit of hemipterans is at least partially dependent on their ability to efficiently circumvent plant chemical defenses. While these insects are typically not considered to cause extensive disruption of plant tissues while feeding, there is evidence that activated (two- component) plant chemical defenses can still be triggered. In face of such exposure to plant- derived toxins, detoxification strategies would consequently confer advantages to these insects. While investigating the whitefly Bemisia tabaci feeding on glucosinolate-containing Brassica plants, we discovered a novel biochemical detoxification mechanism based on sequential addition of multiple glucose moieties. This addition is regio- and stereo- specific, blocks activation by plant enzymes, and appears to be a quantitatively major metabolic pathway for glucosinolate metabolism in this insect. We have additionally determined that cyanogenic glucosides, another important class of plant activated defenses, are analogously metabolized, and have also observed these reactions in representatives of several different types of phloem feeding insects of the suborder Sternorrhyncha. The presence of high concentrations of carbohydrates in the phloem sap diets of these insects minimizes the energy costs of detoxification, but can cause difficulties in osmotic adjustment. This previously uncharacterized detoxification strategy may thus represent an adaptation to multiple plant chemical defense classes as well as a way to reduce osmotic pressures by the formation of polymeric glucose units.

164 | S6 INSECT- PLANT DIALOG

Feeding by the green peach aphid (Myzus persicae) elicits rapid cytosolic calcium elevations in Arabidopsis thaliana mediated by BAK1, GLR3.3/3.6 and TPC1

Vincent TR1, Avramova M1, Canham J1, Higgins P2, Bilkey N2,3, Mugford ST2, Pitino M2, Toyota M3,4,5, Gilroy S3, Miller T1, Sanders D1 and Hogenhout SA2*

1 Department of Metabolic Biology, John Innes Centre, UK 2 Department of Cell and Developmental Biology, John Innes Centre, UK 3 Department of Botany, University of Wisconsin, USA 4 Department of Biochemistry and Molecular Biology, Saitama University, Japan 5 Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Japan

*Corresponding author: [email protected] Poster 83

Plants defend themselves against biotic threats, including the green peach aphid (Myzus persicae), through activation of innate immunity, which is thought to involve a transient rise in cytosolic calcium ion concentration. However, the mechanisms underlying this calcium rise and its potential role in plant-insect interactions are not clear. Here, we demonstrate rapid and highly-localised plant calcium elevations around the feeding sites of M. persicae. These elevations correlate with aphid probing of the epidermal and mesophyll cells shortly after aphid settlement and are dependent on the plant defence co-receptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE 1 (BAK1), which is known to play a role in the induction of early plant defence against pathogens and aphids. The elevations are also dependent on the plasma membrane GLUTAMATE-LIKE RECEPTORS 3.3 and 3.6 (GLR3.3 and GLR3.6) and the vacuolar TWO-PORE CHANNEL 1 (TPC1) that respectively releases extracellular and vacuolar calcium into the cytosol.

S6 INSECT- PLANT DIALOG | 165

A secreted whitefly effector and its putative in planta target van Kleeff PJM1*, de Vos M2, Dekker H3 and Schuurink RC1

1 Plant physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands 2 Keygene N.V, The Netherlands 3 Mass spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands

*Corresponding author: [email protected] Poster 84

It has recently become clear that herbivores can influence plant-defences through saliva-secreted effectors. Effectors can manipulate plant defenses to benefit the herbivore and its progeny. We study the manipulation of plant defenses by effectors within the saliva of the phloem-feeder Bemisia tabaci (whitefly). We have collected RNAseq data for adult salivary glands, midgut, thorax (enriched in salivary glands), abdomen (enriched in midguts), whole body as well as nymphs and eggs. Using the predicted whitefly proteins from the RNAseq assembly, we have identified 2 secreted - putative - effector proteins in artificial diet. Whitefly fecundity bioassays on N. tabacum transiently expressing these putative effectors showed that one of them (S2) increases the oviposition rate of whiteflies. Phytohormone measurements revealed that S2 increases SA levels and reduces JA levels when transiently expressed in N. tabacum. Currently we are conducting Y2H and pull-down experiments to identify the in planta target(s) of these two proteins. The identification of whitefly effector proteins and their in planta target proteins could lead to new breeding targets for herbivore resistant plants.

166 | S6 INSECT- PLANT DIALOG

Investigating the mechanisms behind host race adaptation in Acyrthosiphon pisum with help of the redox-state in legumes

Paulmann M1,2*, Oelmüller R2, Gershenzon J1, Furch A2 and Kunert G1

1 Max Planck Institute for Chemical Ecology Jena, Germany 2 Friedrich-Schiller-University Jena, Germany

*Corresponding author: [email protected] Poster 85

In insect-plant interaction plants have evolved striking defenses to protect themselves from aphid attack. Aphids, in turn, adapt to plant defenses, and the adaptations required to overcome the defense system of a given plant may be highly specific. One strong example is the pea aphid species Acyrthosiphon pisum, which consists of at least 15 different host races, each specialized on a different subset of host plants of the legume family but all able to feed on the universal host plant Vicia faba. Understanding the mechanisms behind the development of aphid host races could give novel insights into the process of sympatric speciation, and may contribute to strategies for pest management in agricultural systems. In our group we mainly explore the influence of three host races on their universal host plant V. faba and their native hosts Medicago sativa, Pisum sativum and Trifolium pratense. Previous work suggests that different host races manipulate plant-defense signaling to their own advantage, and perform better on their native hosts due to their ability to modulate the salicylic acid- and jasmonic acid-signaling pathways. Especially the modulation in jasmonic acid signaling was due to an effect upstream of jasmonic acid precursors. Thus pea aphid host races might influence the very early defense reactions of plants. Plant defenses earlier than phytohormone formation include reactive oxygen species (ROS). The accumulation of ROS is tightly linked with the formation of ascorbate and glutathione, both molecules being important to control the redox-status of the plant. Thus, we are investigating whether the different pea aphid host races manipulate plant-defense signaling already at the level of the ROS production and the formation of the antioxidants ascorbic acid and glutathione.

S6 INSECT- PLANT DIALOG | 167

Struggle to survive: aphid – plant relationships under low light stress. A case of Acyrthosiphon pisum Harris and Pisum sativum L.

Dancewicz K1, Paprocka M1, Morkunas I2 and Gabryś B1*

1 Department of Botany and Ecology, University of Zielona Góra, Poland 2 Department of Plant Physiology, Poznań University of Life Sciences, Poland

*Corresponding author: [email protected] Poster 86

Light is primary source of energy and also plays signaling and regulatory roles in developmental processes and defence responses of plants. The aim of the study was to determine the performance, settling preferences, probing behaviour and feeding activity of Acyrthosiphon pisum on Pisum sativum grown in complete darkness (NL), with light at minimum level required for photoperiodic reaction (LL) and under full light (FL) conditions. The effect of A. pisum infestation on metabolic status and defense responses of peas under FL, LL, and NL conditions was also determined. The lack of or insufficiency in light supply and aphid infestation had a profound effect on plant metabolic status. However, the potency of metabolic responses to these factors depended on the variation in light intensity level-aphid infestation combination, with light intensity level having a priority in this interaction. The intrinsic rate of increase and net reproduction of aphids were more than four and 16 times lower, respectively, on LL and NL than on FL plants. In contrast to aphids on FL plants, the majority of A. pisum rejected LL and NL plants during settling and failed to locate phloem on these plants. Probing was not impeded on LL and NL plants but probes were significantly shorter than on FL plants and consisted mainly of non-phloem activities. In aphid-infested plants under FL conditions, a significant decrease in cholorphyll a, chlorophyll b, carotenoids, and phenolics content and β-glucosidase activity occurred. Minimum light offered to plants was sufficient for plant survival but not sufficient for the nutrient production for aphids. Under the biotic stress of aphid infestation, the light-deprived plants with low phlotosynthetic pigments content invested in supporting basic metabolism, which was manifested in low β-glucosidase activity and low content of phenolics.

168 | S6 INSECT- PLANT DIALOG

Barley transcriptome analyses upon interaction with different aphid species identifies thionins contributing to resistance

Escudero-Martínez C*, Morris JA, Hedley PE and Bos J

James Hutton Institute and Dundee University, UK

*Corresponding author: [email protected] Poster 87

Aphids are phloem-feeding insects that cause yield loss on a wide range of crops, including cereals such as barley. While most aphid species are limited to one or few host species, some are able to reproduce on many plants belonging to different families. Interestingly, aphid probing-behaviour can be observed on both host and non-host species indicating that interactions take place at the molecular level that may impact host range. Here, we aimed to gain insight into the interaction of barley with aphid species differing in their ability to infest this crop by analysing transcriptional responses. First, we determined colonization efficiency, settlement, and probing behaviour for the aphid species Rhopalosiphum padi, Myzus persicae and Myzus cerasi, which defined host, poor- host and non-host interactions, respectively. Analyses of barley transcriptional responses revealed gene sets differentially regulated upon the different barley-aphid interactions and showed that the poor-host interaction with M. persicae resulted in the strongest regulation of genes. Interestingly, we identified several thionin genes strongly up-regulated upon interaction with M. persicae, and to a lesser extent upon R. padi interaction. Ectopic expression of two of these genes in Nicotiana benthamiana reduced host susceptibility to M. persicae, indicating thionins contribute to defences against aphids. The next step will be to further implicate these genes (LEA and thionin family) in aphid host range using barley CRIPR-Cas9 knockout for aphid functional assays. Our work will thereby provide novel insights into host and non-host defences in a monocot crop to aphids.

S6 INSECT- PLANT DIALOG | 169

Competitive displacement between two invasives species of Bemisia tabaci and preference to oviposition in different hosts

Watanabe LFM*, Bello VH, Marchi BR, Moraes LA, Marubayashi JM, Yuki VA, Pavan MA and Krause-Sakate R

Faculdade de Ciências Agronômicas de Botucatu – FCA/UNESP, Brazil

*Corresponding author: [email protected] Poster 88

Bemisia tabaci is considered one of the most important pests worldwide. In Brazil, the invasive Middle East-Asia Minor1 cryptic species (MEAM1, known as biotype B) has been predominant in the field since the 1990s. Another invasive species, the Mediterranean (MED, known as the Q biotype) was reported in 2014 in the South of Brazil and more recently in São Paulo State and is a new concern to the Brazilian agriculture. In order to evaluate the competitive displacement between MED and MEAM1 in tomato cv Santa Clara and pepper hybrid Magali R, 10 couples from each whitefly species were placed together in these hosts, separately, and 100 insects were analyzed every 25 days (for tomato) and 30 days (for pepper) plants in order to indentify the species. On pepper Magali R at the 4th sampling (120 days after the start of the experiment), only MED was detected, whereas for tomato Santa Clara, only MEAM1 species was detected (at 100 days). Oviposition of MED and MEAM1 was compared on cotton, bean cv Pérola, tomato cv Santa Clara and pepper hybrid Magali R. After 48 hours, in cotton and tomato, MED laid more eggs than MEAM1: 72,4 and 59,8 eggs laid by MED and 46,8 and 25,8 eggs laid by MEAM1, respectively. Bean and pepper were oviposited with same efficiency by MED and MEAM1. The oviposition test could not explain the competitive displacement observed by MED and MEAM1 in tomato Santa Clara and pepper Magali R. The viability of eggs and nymphs is in course.

170 | S6 INSECT- PLANT DIALOG

Oxidative stress in aphids due to interaction with the host plants

Durak R*1, Molon M2, Durak T3 and Chrzanowski G4

1 Department of Zoology, University of Rzeszow, Poland 2 Department of Biochemistry and Cell Biology, University of Rzeszow, Poland 3 Department of Ecology, University of Rzeszow, Poland 4 Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities, Siedlce, Poland

*Corresponding author: [email protected] Poster 89

Formation of the reactive oxygen species (ROS) is a part of reactions resulting from the normal metabolism of the insect cells. Additionally, insects feeding on plants are exposed to ROS generated by the plant in response to biotic stress. Maintaining of the ROS balance is crucial for insect organism functioning as well as for understanding of the oxidative stress resulting from the insect-host plant interaction. Insects are equipped with a range of anti-oxidant and detoxifying enzymes as well as those responsible for oxidoreduction, enabling them defence against ROS generated by cells under oxidative stress and keeping them in balance. The main factor responsible for an increase in the ROS and oxidative stress level in animal cells is high temperature. Another cause of an oxidative stress, especially in phytophagous aphids, is a change of the host plant. The goal of this study was the analysis of the oxidative stress indicator levels and elucidation of the mechanism responsible for reduction of the ROS, which are generated due to the change of the host plant, and are managed by the antioxidant system in aphid tissues. The study was carried out using an oligophagous aphid species, Cinara tujafilina (del Guercio 1909). The aphids were reared on Thuja orientalis L.”Aurea Nana” in an environmental chamber, and then transferred onto two different plant cultivars during the experiments. We observed that the change the host species induces oxidative stress in C. tujafilina. The highest level of H2O2 was detected in the cells of females after 48 h from their transferral onto a new plant. The highest level of antioxidative enzyme activity (SOD, CAT) was recorded in aphids infesting the “Justynka” cultivar. The level of the enzyme activity was dependent on the time elapsed since the host change. We also found a very high level of an enzyme related to glutathione metabolism, S- glutathione transferase (GST). We therefore discussed the results in terms of the biochemical adaptations of C. tujafilina for infestation of new host plants.

S6 INSECT- PLANT DIALOG | 171

Characterization of the role of the acrostyle – organ within aphid stylets, which bears receptors of plant viruses – in plant-aphid interactions

Deshoux M, Blanc S & Uzest M*

INRA, UMR 385 BGPI, France

*Corresponding author: [email protected] Poster 90

Aphids are sap-feeding insects with piercing-sucking mouthparts. They are serious pests in agriculture particularly because they transmit plant viruses to most important crops worldwide. The majority of these viruses are transmitted in a non-circulative manner where viruses are retained on specific receptors located in the aphids’ mouthparts. The acrostyle, an “organ” located at the tip of aphids’ maxillary stylets discovered a few years ago, bears the receptors of Cauliflower mosaic virus and likely those for numerous agronomically important non-circulatively transmitted viruses. Apart from transmitting plant pathogens, the acrostyle, which is conserved among aphid species, has likely a role for the insect itself. At the confluence of the salivary and food canals, in contact with cell content, phloem sap and aphid saliva, the acrostyle may have a specific role during the aphid feeding process, leading to plant/insect compatible interactions. In attempts to define the physiological functions of the acrostyle, the main goal of my PhD project will be to search for putative partners from plant (defense molecules) and from aphid saliva (effectors) that could interact temporarily with the cuticular proteins/peptides at the surface of the organ. The main approaches that will be developed and the most recent advances will be presented.

172 | S6 INSECT- PLANT DIALOG

The effect of lead on the generation of signal molecules in pea seedlings during pea aphid infestation

Woźniak A1*, Drzewiecka K2, Kęsy J3, Motała R4, Borowiak-Sobkowiak B5, Dancewicz K6, Gabryś B6, Bednarski W7 and Morkunas I1

1 Poznań University of Life Sciences, Poland 2 Poznań University of Life Sciences, Poland 3 Nicolaus Copernicus University, Poland 4 The Institute of Plant Protection National Research Institute, Poland 5 Poznań University of Life Sciences, Poland 6 University of Zielona Góra, Poland 7 Institute of Molecular Physics, Polish Academy of Sciences, Poland

*Corresponding author: [email protected] Poster 91

Under natural conditions we may frequently observe the effect of many stress factors acting simultaneously or successively. The first aim of the study was the determination of the interrelations between the level of lead and the level of the generated signalling molecules and then the examination of how the cross-talk of lead and A. pisum regulated the level of these signalling molecules. Moreover, the second aim of the study was to verify whether the presence of lead in the pea seedlings organs and the induction of signalling pathways, dependent on the different concentrations of this metal, and trigger other defence responses (the generation of semiquinone radicals and ROS) to phytophage. The third aim was to examine the influence of lead on demographic parameters of pea aphid population and the feeding activity of A. pisum with the use of the method of electronic recording of feeding (EPG). The research was conducted on seedlings of P. sativum L., in hydroponic cultures on Hoagland medium, with addition of lead (hormesis effect and toxic effect). Only adult insects of A. pisum were used. The main focus of the research with insects was on the leaves. The level of lead was determined by means of flame atomic absorption spectrometry (FAAS) and electrothermal atomic absorption spectrometry (ETAAS). The level of signaling molecules was determined with the following methods: HPLC (salicylic acid, SA) and GC (abscisic acid, ABA). The concentrations of free radicals and paramagnetic centres were measured using electron paramagnetic resonance (EPR) and the hydrogen peroxide (H2O2) content by the spectrophotometric method. Experiments conducted within this study are basic research and provide new information for contemporary plant biology concerning the phenomenon of hormesis in the system of an abiotic factor (lead, in the substrate) – plant (the signalling network) – phytophage (insect with piercing-sucking apparatus). S6 INSECT- PLANT DIALOG | 173

Hemipteran vector manipulates host plant metabolism to fulfil its nutritional needs

Killiny N and ME Rogers*

Department of Entomology & Nematology, University of Florida, IFAS, Citrus Research & Education Center, Lake Alfred, FL, USA

*Corresponding author: [email protected] Poster 92

The citrus industry is currently threatened by huanglongbing, also known as citrus greening disease. The bacterial pathogen, Candidatus Liberibacter asiaticus is phloem restricted and transmitted by the Asian citrus psyllid, Diaphorina citri. In addition to citrus varieties, this vector can colonize other species from the family Rutaceae such as orange jasmine (Murraya paniculata), and curry leaf tree (Bergera koenigii), which may act as reservoir hosts in the field. Using gas chromatography-mass spectrometry, we studied the effect of infestation with D. citri on the phloem sap composition of Valencia sweet orange (Citrus sinensis), orange jasmine, and curry leaf tree. Additionally, the honeydews secreted from the D. citri nymphs, which have the most feeding activity and higher transmission rate for the bacterium, were investigated. Our findings show that while the overall effect on phloem saps is similar, some specific varietal differences were observed. Amino acids and organic acids were the most affected compounds by the infestation with D. citri. The increase in γ-aminobutyric acid, L-proline indicated that infested plants were under stress and the increase in glycine and L-serine indicated an increase in photorespiration. The increase in citric acid and succinic acid may indicate an induction of the tricarboxylic acid cycle in D. citri-infested plants. In addition, the honeydew profiles were similar among the three hosts with slight differences, which could be attributed to the differences in the phloem saps. The trace amounts of amino acids in the honeydew indicated that amino acids were the growth-limiting nutrients for D. citri and their increase in the phloem sap of their host plants upon infestation may enhance the development and reproduction of D. citri. Understanding the specific interactions between the vector and the host plants will help in finding a weak link of the chain that we can target to better control the vector-borne diseases.

174 | S6 INSECT- PLANT DIALOG

Research of metabolic enzyme CYP3A4 related approach when peach aphid in the process of transform host from pepper to faba bean

Jing-Yun Zhu, Yi Zhang, Xing-Xing Wang, Shi-Ze Zhang and Tong-Xian Liu*

State Key Laborabory of Crop Stress Biology on the Arid Areas, and the Key Laboratory of Crop Pest Management on the Losses Plateau of Ministry of Agriculture, Northwest A&F University, Yangling, Shanxi,China

*Corresponding author: [email protected] Poster 93

The peach aphid, Myzus persicae (Hemiptera: Aphididae) is a severe agricultural pest worldwide because of its rapid adaptation to new host and serious damage to a wide range of crops through phloem feeding, honeydew excretion and transmission of plant viruses. To study the adaptability of M. persicae, the aphids were shifted from hot pepper to faba bean and were reared on faba bean plants for more than 15 generations before experiment. The method of gene silencing was used to explore the CYP3A4 gene’s function, which associated with peach aphid’s adaptability. The results showed that the metabolic enzyme CYP3A4 gene was up-regulated during the process of peach aphid adapt to faba bean. The expression level of CYP3A4 gene had significant difference between first generation and ninth generation, then the expression level was stable after ninth generation. The expression level of CYP3A4 gene on the aphids injected with DsRNA CYP3A4 was lower compared with control. Further investigations on the CYP3A4 gene function are needed.

Pest-Disease

7 | Management

176 | S7 PEST- DISEASE MANAGEMENT

Management of the Asian citrus psyllid and citrus greening disease

Rogers ME*, Langdon KW, Serikawa RH and Ebert TE

Department of Entomology & Nematology, University of Florida, IFAS, Citrus Research & Education Center, Lake Alfred, FL, USA

*Corresponding author: [email protected] Keynote Lecture

Following the 2005 discovery of citrus greening disease in Florida, citrus pest management programs are now focused largely on controlling the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. Citrus growers have increased their use of insecticides for ACP control with the goal of slowing the rate of spread by ACP of the phloem-limited bacterium, Candidatus Liberibacter asiaticus, which causes greening disease. It has been speculated that insecticide induced mortality occurring before ACP are able to penetrate the phloem with their mouthparts would reduce the likelihood of pathogen inoculation of a healthy plant. In electropenetrography (EPG) studies, it was found that soil-applied neonicotinoid insecticides could indeed prevent phloem-related feeding behaviors whereas most foliar-applied insecticides failed to provide such feeding disruption. Further studies utilizing EPG and artificial diets demonstrated that the disruption in feeding that occurred when ACP fed on systemically-protected citrus plants was due to feeding deterrence and not insecticide-induced mortality. The implications of these findings in terms of managing ACP populations and considerations for insecticide resistance management and monitoring will be discussed.

S7 PEST- DISEASE MANAGEMENT | 177

Kaolin affecting the host plant finding ability of Diaphorina citri in field conditions

Miranda MP*, Tomaseto AF, Zanardi OZ, Volpe HXL, Vizoni MC, Lopes JP and Martini FM

Fund for Citrus Protection (FUNDECITRUS), Brazil

*Corresponding author: [email protected] Oral Presentation

The Asian citrus psyllid, Diaphorina citri, is a major citrus pest worldwide due to its ability to transmit the bacteria associated with Huanglongbing disease. D. citri uses both visual and olfactory cues to find its host plants, however, visual seem to be the most important. Kaolin is a white nonabrasive fine-grained mineral that when sprayed on the plants forms a particle film that interferes in the host recognition by the insects. Therefore, the purpose of our study was to assess how kaolin may affect the host plant finding ability of D. citri in field conditions. This study was performed in a 1.5 year-old sweet orange grove during summer season in two situations: absence and presence of young shoots (≈15cm). In both experiments, a wettable powder (WP) kaolin formulation (Tessenderlo Kerley, Inc./NovaSource) was sprayed twice, 10 days and 4 h before psyllids release, at 5 and 3% w/v, respectively. Adult psyllids (10-15 days old) were marked with fluorescent powder and assessments (number of psyllids/plant) were performed 1, 2 and 3 days after releasing; an additional assessment, 7 days after psyllids release, was performed in the experiment with the presence of young shoots. In all assessment periods, D. citri plant finding ability was clearly disrupted in plots where kaolin was applied. At the final assessment, the treatment with kaolin resulted in a reduction of 99 and 98% in the number of psyllids per plant compared to untreated plots in the situation of absence and presence of young shoots, respectively. Moreover, there was no difference in the number of psyllids per plant at 3 and 7 days after releasing. In both experiments, most psyllids (≥80%) were found on the first two rows. Therefore, our results showed that kaolin could be a valuable strategy for D. citri management in the citrus grove edges.

178 | S7 PEST- DISEASE MANAGEMENT

Ecology and management of Bactericera cockerelli in potato crops

Vereijssen J1*, Agnew NM1, Barnes AM1, Furlong J1, Logan DP1, Thompson SE1, Butler RC1, Finlay KJ2 and Yen AL2

1 The New Zealand Institute for Plant & Food Research Limited, New Zealand 2 Biosciences Research, Agriculture Victoria, Department of Economic Development, Jobs, Transport and Resources, Australia

*Corresponding author: [email protected] Oral Presentation

The tomato potato psyllid, Bactericera cockerelli (Šulc), was first found in New Zealand in 2006. It wasn’t until 2009, however, that the psyllid was identified as the vector of the new-to-science plant pathogenic bacterium Candidatus Liberibacter solanacearum (CLso). CLso is the putative agent of zebra chip disease in potato tubers. The insect-pathogen complex has caused severe economic losses in the capsicum, tomato, potato and tamarillo industries in New Zealand. The initial industry response was to control the vector using insecticides; however, this was unsustainable both environmentally and economically. A more fundamental understanding of vector and pathogen ecology is critical for effective and sustainable vector management in potato crops. We collated a list of non-crop host plants and assessed the role of these plants in the ecology of B. cockerelli in agricultural areas. All life stages of B. cockerelli were present year-round on the non-crop perennial host plants assessed (Lycium ferocissimum and Solanum aviculare). We also detected CLso in wild-collected Datura stramonium and S. pseudocapsicum outside the cropping season. Although the incidence of non-crop plants infected with CLso in the environment may be low, these plants may be a potential reservoir for the pathogen and vector in the absence of a suitable crop host, providing an inoculum source for infection of subsequent crops. Previous pest management research focused on reduced spray programmes based on degree days and action thresholds customized for a particular potato-growing area. This research was terminated in the South Island and instead a grower-defined research programme commenced in the 2016-17 season focusing on end-of-season and post-desiccation management of B. cockerelli, to decrease incidence and severity of zebra chip disease. Despite B. cockerelli first being detected in New Zealand ten years ago, there are still questions around its ecology and successful management strategies.

S7 PEST- DISEASE MANAGEMENT | 179

Zebra chip in Oregon and Washington: a retrospective towards managing the disease

Rondon S*

Oregon State University, USA

*Corresponding author: [email protected] Oral Presentation

The Columbia Basin of Oregon and Washington and Idaho in the United States Pacific Northwest were affected by Zebra Chip (ZC) in 2011. While potato psyllids had been consistently found in the region by early to mid-July each year, no ZC disease caused by Candidatus Liberibacter solanacearum (Lso) was confirmed until late 2011. Given the great importance of potato production in the region, this disease poses a significant threat to potato production. An extensive trapping program started in 2012 to determine the distribution and expansion of this pest. Phenology models are currently providing a good inside related to the phenology of psyllids in the region. Moreover, samples collected were molecular differentiated and two haplotypes were found in potato fields: western and northwestern haplotypes, however, where those haplotypes come from and where do they survive the winter was unknown. Thus, we conducted a study in the spring, fall and winter of 2012, 2013 and 2014 to determine if psyllids could be found overwintering. Solanum dulcamara L., a.k.a. bittersweet nightshade, a perennial weed of wet habitats and riverbanks, was found to serve as a green bridge for psyllids in the off season. Other weeds hosts are currently being tested to determine their role in the survival of this insect. This information provides a good inside in the complexity of controlling this pest.

180 | S7 PEST- DISEASE MANAGEMENT

Evaluation of sugarcane varieties for differential resistance to insect vector of sugarcane white leaf disease

Hanboonsong Y*, Roodee J and Soothorn S

Entomology Division, Faculty of Agriculture, Khon Kaen University, Thailand

*Corresponding author: [email protected] Oral Presentation

The leafhopper, Matsumuratettix hiroglyphicus (Matsumura) (Homoptera: Cicadellidae), is an important vector of sugarcane white leaf (SCWL) phytoplasma causing SCWL disease. SCWL disease decreases the sugarcane production in Thailand and has recently spread to other Asian countries. At present, there is no effective method or sugarcane resistant variety to control this disease. Therefore, the aim of this study was to evaluate sugarcane varieties for differential resistance to insect vector by assessment of leafhopper vector feeding behavior on six sugarcane varieties (four sugarcane commercial varieties and two sugarcane wild type species) over an 8 h period using the electrical penetration graph technique (EPG) and insect life history parameters (survival and reproductive rates). Plants in which the insects showed the longest ingestion time in phloem cells and the least number of probing events with longer non-probing phase and the highest survival and reproductive rates are considered susceptible varieties. Cluster analysis from both EPG and insect life history data, can group sugarcane varieties into three groups including susceptible, moderately resistant and resistant. Insect vector transmission test confirmed this classification. This screening method is fast and could be used to quickly screen a large number of sugarcane plant varieties to identify those that are disease resistant.

S7 PEST- DISEASE MANAGEMENT | 181

Transcriptomics of altered aphid phenotype on Vat+ resistant plants

Sattar S* and Thompson GA

Penn State University, USA

*Corresponding author: [email protected] Oral Presentation

Aphis gossypii (cotton-melon aphid) is a major agricultural pest affecting a wide range of economically important plants. In melon, genetic resistance towards these phloem-feeding pests is due to the presence of Vat (virus aphid transmission), a single dominant gene belonging to the R (resistance)-gene family. Aphids feeding on resistant melon plants carrying the Vat gene show an altered phenotype as compared to aphids feeding on susceptible plants. These aphids are lighter in color and smaller. They have a longer pre-reproductive period and shortened reproductive and post-reproductive periods with significantly reduced reproductive rates. The goal of this study is to relate these phenotypic changes induced by Vat–mediated resistance at the molecular level. Global transcriptional changes in A. gossypii that occur within the first 48 hours of aphids interacting with resistant Vat+ or susceptible Vat- host plants were used to relate altered gene expression with gene ontology and phenotype. Initial poly (A) RNA sequencing studies of mixed- aged aphids revealed a total of 262 aphid transcripts with differential expression; 198 genes were up-regulated during the susceptible interaction and 98 genes were up-regulated during the resistant interaction. Annotation of these differentially expressed aphid transcripts was assigned into several GO categories, including growth and development, reproduction, stress homeostasis, negative regulation and apoptosis. Since reduced reproductive potential is a hallmark of Vat- mediated resistance in aphids, current research focuses on in-depth analysis of transcripts affecting aphid reproduction, including RNAi-mediated functional validation.

182 | S7 PEST- DISEASE MANAGEMENT

Searching for strategies to control the green

peach aphid on potato crops

Álvarez Adriana E.1* and Machado-Assefh Cristina R.1, 2

1 Universidad Nacional de Salta, Facultad de Ciencias Naturales, Argentina 2 Consejo Nacional de investigaciones Científicas y Técnicas (CONICET-CCT Salta), Argentina

*Corresponding author: [email protected] Oral Presentation

Potato is an important vegetatively propagated intensive world crop threatened by many phytophagous insects; among them, the generalist aphid Myzus persicae constitutes a threat because it is the most efficient vector of potato viruses. Potato viral diseases are important phytosanitary problem that causes losses in yield and quality of tubers. Currently, aphids are controlled with synthetic insecticides and taking into account the increase in production that is necessary for food security, the treatment of this pest with insecticides is not environmental or economically feasible. In our group, we seek to contribute to the development of new alternative strategies for integrated pest management. We work at field scale searching to uncover weeds and wild plants surrounding the potato crops that are hosts for aphid vectors or reservoirs for potato viruses PVY and PLRV. In addition, at lab scale we focus on, 1) searching for new and unexplored sources of aphid resistance in wild germplasm, being the Argentinean center of origin and diversity of potato wild relatives; 2) studying plant-insect interactions to deepening our understanding of the processes occurring in order to assist the development of control tools, specifically we study the role of aphid endosymbiotic bacteria and plant leaf senescence on the host plant preference and acceptance by Myzus persicae, 3) evaluating natural products from autochthonous plant sources with antifeedant and biocidal effect. Finally, it should be discussed that in general, crop diseases and pests are among the biggest problems facing food security and therefore, there must be and integrative address of them in order to find new control strategies.

S7 PEST- DISEASE MANAGEMENT | 183

The effects of host resistance on whitefly- Tomato yellow leaf curl virus interactions: Implications for viral epidemics and management

Srinivasan R1*, Marchant W1, Legarrea S2

1 University of Georgia, USA 2 University of Amsterdam, Netherlands

*Corresponding author: [email protected] Oral Presentation

Tomato yellow leaf curl virus and its vector the Sweetpotato whitefly, (Bemisia tabaci Gennadius), are a chronic problem in southeastern United States, particularly in Florida and Georgia. TYLCV- resistant tomato genotypes, with semi-dominant (Ty) genes introgressed from wild Solanum species, are often planted to limit yield loss. These genes do not provide complete resistance (hypersensitive response); typically, infected plants display less severe symptoms. However, transmission assays indicated that these resistant genotypes also were infected at the same percentage as TYLCV-susceptible genotypes. Back transmission assays indicated that though resistant genotypes displayed mild symptoms post infection they still functioned as effective inoculum sources i.e. whiteflies acquired TYLCV from resistant genotypes and effectively (>80%) inoculated non-infected plants. Experiments were further conducted to assess how resistant genotypes affected virus accumulation and modulated the preference and fitness of non- viruliferous and viruliferous whiteflies temporally (3, 6, and 12 weeks post virus inoculation). Virus quantitation (qPCR) revealed that TYLCV accumulation was reduced at all time periods in resistant than susceptible genotypes. The amount of virus acquisition by whiteflies also followed a similar trend –whiteflies acquired fewer viral copies from resistant than susceptible genotypes. Non-viruliferous whiteflies exhibited a preference for TYLCV-infected plants over non-infected plants. The preference switched to non-infected plants in the case of viruliferous whiteflies. Vector fitness benefits were more obvious on infected plants than non-infected plants, and these effects were prominent up to six weeks post inoculation especially with TYLCV-susceptible genotypes. Results suggest that using TYLCV-resistant genotypes could reduce the overall inoculum level and limit virus spread than susceptible genotypes. A population genetics approach is currently being undertaken to assess whether the continuous use of such resistant genotypes would generate enough selection pressure at the virus genome level leading to development of highly-virulent strains of the virus. Results will be discussed. 184 | S7 PEST- DISEASE MANAGEMENT

Whiteflies: Developing host plant resistance in watermelon from wild sources

Simmons AM and Levi A

U.S. Department of Agriculture, ARS, U.S. Vegetable Laboratory, USA

*Corresponding author: [email protected] Oral Presentation

The whitefly (Aleyrodidae) Bemisia tabaci causes serious damage to horticultural crops, including watermelon (Citrullus lanatus var. lanatus) and it is known to transmit many plant viruses. This whitefly is highly polyphagous, with over 1,000 known species, and can adapt to the environment. Yet, there is a wide range in acceptance and performance of B. tabaci among host plant species. With a relatively short generation time and high rate of oviposition, whitefly populations can quickly build on suitable host plants like watermelon. Commercial watermelon cultivars share a narrow genetic base and are highly susceptible to whiteflies. Citrullus colocynthis is a perennial desert watermelon species with a broad genetic base and viable sources of resistance to insect pests and diseases of watermelon. This watermelon species is indigenous to arid regions of Northern Africa, the Mediterranean, and Southwest Asia. Experiments were conducted with Citrullus genotypes collected in different geographic regions, and a source of resistance to whiteflies (Bemisia tabaci; MEAM1) was identified based on antibiosis, non-preference, and plant tolerance. Experiments were conducted in the laboratory, greenhouse, and field. Experiments in the laboratory included a vertical Y-tube monitoring technique for the rapid testing of whitefly response to phytochemicals, as well as for the collection of data on whitefly oviposition and survival. Citrullus genotypes showing resistance to whiteflies are being developed into germplasm lines. Traditional and molecular approaches are being used to incorporate whitefly resistance into watermelon cultivars.

S7 PEST- DISEASE MANAGEMENT | 185

Calophya terebinthifolii and Calophya lutea: Host specificity of two potential biological control agents of Brazilian peppertree in Florida, USA

Prade P*, Overholt WA, Cuda JP and Minteer CR

Entomology and Nematology, University of Florida, USA

*Corresponding author: [email protected] Oral Presentation

In weed biological control programs, host specificity studies are essential for determining the suitability of an insect as a biological control agent. It is important to demonstrate the candidate insect has a narrow host range, ideally limited to the target plant in the proposed are of introduction. Brazilian peppertree (Schinus terebinthifolia Raddi, Anacardiaceae) is one of the most aggressive invasive plants in Florida. Two gall inducing psyllids, Calophya terebinthifolii Burckhardt & Basset and Calophya lutea Burckhardt (Hemiptera: Calophyidae) were discovered in Brazil damaging Brazilian peppertree leaves. The objective of this study was to determine the host specificity of C. terebinthifolii and C. lutea by monitoring oviposition and gall formation on non- target species. Insects were exposed to 89 plant species belonging to 50 families. The number of eggs and nymphs was evaluated 5 and 14 days after oviposition. Both species oviposited only on five non-target species in the Anacardiaceae family, and oviposition was 2.81-fold greater on Brazilian peppertree when compared with that on non-target species. More importantly, gall development and subsequent adult emergence occurred only on Brazilian peppertree. Eggs that were laid on non-target species desiccated, or first instar nymphs were unable to induce gall formation on the plant, leading to death of the immatures. The host specificity exhibited by C. terebinthifolii and C. lutea suggests that both psyllids would be safe to release for biological control of Brazilian peppertree in Florida.

186 | S7 PEST- DISEASE MANAGEMENT

The genotype of Pereskia aculeata (Cactaceae) influences performance of its biological control agent, Catorhintha schaffneri (Coreidae) in South Africa

Egbon IN1,2*, Paterson ID2 and Compton S2,3

*Corresponding author: [email protected] Oral Presentation

1 Department of Animal and Environmental Biology, University of Benin, Nigeria 2 Zoology and Entomology Department, Rhodes University, South Africa 3 Ecology and Evolution Department, Faculty of Biological Science, University of Leeds, UK

Identifying origins of alien plants is often difficult, but the ability to pinpoint their source populations is important when collecting natural enemies for use as biocontrol agents. This is especially important when variation exists within the target weed species which may influence agent performance and establishment. This study investigated whether intraspecific variation among clones of Pereskia aculeata, an invasive cactus in South Africa of South American origin, influenced the performance of the tip wilter biocontrol agent Catorhintha schaffneri. The development of and damaged caused by newly-hatched nymphs on ten P. aculeata genotypes sourced from five countries and cultured insects collected from Santa Catarina in Brazil was compared. Stage-specific duration, survival, and body weight were monitored. Host suitability and susceptibility were compared using Maw’s Host Suitability and Dobie’s Susceptibility indices. Development time and survival varied greatly between genotypes, with nymphs developing more quickly and most likely to survive on invasive South African genotypes and the native Brazilian genotypes from which they were originally collected. In contrast, no nymph survived to adult stage on genotypes from Misiones (Argentina) and Pedernales (Dominican Republic). Wet weight of newly emerged adult females was slightly heavier among the invasive genotypes than the native sympatric and allopatric host genotypes in the following order: invasive genotypes > native sympatric host > native allopatric hosts > native allopatric hosts from outside the native range of the insect. However, male weight initially fell slightly out of sync with the female trend. These findings suggest the invasive genotypes of P. aculeata in South Africa are comparably suitable like those from the native regions where the plant and insect originated, but the insect performs badly on genotypes beyond its native range. S7 PEST- DISEASE MANAGEMENT | 187

Investigating plant-aphid-environment interactions: Rhopalosiphum padi - barley interactions under plant water stress

Leybourne D1, 2, 3*, Valentine T3, Bos J1, 2 and Karley A3

1 Department of Plant Sciences, School of Life Sciences, Dundee University, UK 2 Cell and Molecular Sciences, The James Hutton Institute, UK 3 Ecological Sciences, The James Hutton Institute, UK

*Corresponding author: [email protected] Oral Presentation

The bird cherry-oat aphid, Rhopalosiphum padi, is a significant pest of European cereals. It is estimated that the overall yield loss to crops ranges from 10-80%, as a result of localised feeding damage and the transmission of plant viruses. Differing levels of water availability and changes to atmospheric CO2 levels can positively or negatively affect one, or both, of the organisms in the plant-aphid system. Climate change is set to enhance the aphid pest status as changes in global temperature and precipitation levels alter their geographical range, level of activity and population abundance. Examining how interactions at the plant-aphid interface alter under different environmental conditions can identify novel approaches to enhance crop tolerance to compounding biotic and abiotic stressors. Hordeum spontaneum 5 (HsP5), a wild relative of barley, possesses partial aphid resistance. Comparisons of aphid performance while feeding on HsP5 and the modern barley cultivar, H. vulgare cv. Concerto, showed that aphid growth rate and fecundity were significantly decreased when feeding on HsP5. Analysis of HsP5 and cv. Concerto leaf architecture has indicated genotypic differences in trichome density and epicuticular wax composition; analysis is ongoing to fully characterise the composition of leaf structural and nutritional compounds. Electrical Penetration Graph (EPG) monitoring of aphid feeding behaviour will be used to elucidate the potential mechanism(s) of partial-resistance. The impact of water stress on aphid performance will also be assessed using spectroscopic, microscopic and biochemical methods to examine water stress effects on plant leaf architecture, biochemical composition and nutritional quality. We will assess the impact in parallel with effects on aphid fecundity, growth rate and feeding behaviour. Additionally, changes in expression of key plant and aphid genes in response to water availability will be examined.

188 | S7 PEST- DISEASE MANAGEMENT

Isoclast™ Active as a new tool for managing virus vectors and virus transmission

Mezei I1*, Gomez LE2 and Abad R3

1 Dow AgroSciences Hungary 2 Dow AgroSciences LLC, USA 3Dow AgroSciences Ibérica, Spain

*Corresponding author: [email protected] Oral Presentation

There are more than 2000 known phytopathogen virus species and many of them cause important diseases of various plants that humans grow for food. In addition, new plant viruses are being discovered every day. The majority of plant viruses that cause disease in agricultural crops rely on biotic vectors for transmission and survival. The largest classes of plant virus-transmitting vectors are insects belonging to the orders Hemiptera (whiteflies, aphids, leafhoppers and planthoppers) and Thysanoptera (thrips). The different modes of viral transmission by vectors include non-persistent, semi-persistent and persistent viruses and the mode and effectiveness of transmission is all different between the different type of viruses and their vectors. The effective and fast control of vectors is crucial to prevent or at least mitigate virus transmission and the damage caused by the viruses. Isoclast tested in several lab and open field trials effectively controlled virus vectors such as aphids resulting in significant prevention of virus transmission. This effect is achieved by a very fast feeding cessation followed by the pest mortality, which results in low virus infection in the field and significant yield increase in comparison to the untreated.

S7 PEST- DISEASE MANAGEMENT | 189

Laboratory evaluation of a mycoinsecticide (BotaniGard® ES) on Riptortus pedestris (Hemiptera: Alydidae)

Lim UT 1* and Mahbubur RM 2

1 Department of Bioresource Sciences, Andong National University, Republic of Korea 2 Bangladesh Agricultural Research Institute, Bangladesh

*Corresponding author: [email protected] Oral Presentation

Riptortus pedestris (Hemiptera: Alydidae) is considered one of the most economically important pest of soybean in Korea. A mycoinsecticide BotaniGard® ES (Beauveria bassiana GHA) was evaluated as a biocontrol agent against R. pedestris. Both adult and nymph were treated with three conidial concentrations (1×109, 1×108, and 1×107 spore/ml), and its virulence was assessed under three relative humidities (55, 75, and 95% RH). Mortality of both adult and nymph was affected significantly by the conidial concentration, but not by RH. At 1×109 spore/ml, LT50 values for either adult or nymph were not always significant among the RH levels. However, at any RH level, LT50 values for both adult and nymph were always different among the conidial concentrations. The fungal infection rate of adult and nymph on 21st day of application varied 31- 70% and 60-78% without much significance among the treatments, respectively. There was no significant difference in both mortality and infection rate between field-collected and laboratory- reared populations. From field verification in our previous study, only nymph population decreased in all treated fields regardless of dose and frequency of spray, and no reduction in adult populations was observed. Nymphs collected from treated plots showed infection rate of 35-68% while 0% in untreated plots. Application of BotaniGard® ES also reduced damage on soybean pods and seeds. The potentials of the mycoinsecticide and its future application are discussed.

190 | S7 PEST- DISEASE MANAGEMENT

Behavioural responses of Hyalesthes obsoletus to volatiles of plants sprayed with resistance inducers

Riolo P*, Minuz RL, Mancini V, Ruschioni S, Romanazzi G and Isidoro N

Dipartimento di Scienze Agrarie, Alimentari e Ambientali – Università Politecnica delle Marche, Italy

*Corresponding author: [email protected] Oral Presentation

Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae) is considered to be the principal vector of ‘Candidatus Phytoplasma solani’, which is the causal agent of the grapevine yellows “Bois noir (BN)” in European vineyards. This planthopper is a polyphagous species living mainly on wild and cultivated herbaceous plants often hosting the phytoplasma. The life history of H. obsoletus, its affiliation to wild host plants, and its erratic feeding behavior on grapevine impede the effective control of this planthopper. Among alternatives to synthetic pesticides, a good interest was gained by the application of resistance inducers to control plant pathogens and insect herbivores. In a recent study it was evaluated the activity of some elicitors in inducing recovery of BN infected grapevines, and encouraging results were provided by benzothiadiazole (BTH) and by two different formulations containing glutathione plus oligosaccharides (GO1 and GO2). In this study, Y-tube olfactometer bioassay was designed to investigate H. obsoletus behavioral responses to volatile emitted by grapevine shoots sprayed with these three commercial formulations containing BTH, GO1 and GO2. Each diluted formulation was applied at 3 different times (0, 2 and 7 days before bioassays). A volume equivalent to 1,000 l/ha of deionized water was applied to control plants to a run-off point, at the same times of treatments. The number of planthoppers tested was 32 for every treatment (total n=288 specimens). Our study showed that BTH-treated grapevine shoots were significantly repellent for H. obsoletus when applied one week before insect bioassays. Moreover, planthoppers were significantly attracted to volatiles emitted by grapevine shoots sprayed with GO2 formulation the same day of bioassays, when compared to the control. No behavioral responses have been elicited by treatments with GO1. The results of this study could be used as part of a sustainable and environmentally friendly strategy to control H. obsoletus adults in vineyard agroecosystems.

S7 PEST- DISEASE MANAGEMENT | 191

Bacillus thuringiensis translocation inside citrus plants and insecticidal activity against Diaphorina citri, vector of HLB causal agents da Cunha T 1,2*, de Oliveira Dorta S 1,2, Monnerat R3, Miranda M 4, Machado MA 1 and de Freitas Astúa J3

1 Centro APTA Citros Sylvio Moreira-IAC, Brazil 2 University of São Paulo, ESALQ/USP, Brazil 3 Brazilian Agricultural Research Corporation, EMBRAPA, Brazil 4 Fundo de Defesa da Citricultura, Fundecitrus, Brazil

*Corresponding author: [email protected] Oral Presentation

Effectiveness of Bacillus thuringiensis (Bt) insecticidal Cry toxins have been shown to control insect pests either in spray products or expressed in transgenic crops. The discovery of Bt strains as plant endophytic opened new perspectives for studies to control phloem-sucking insects, such as the Asian citrus psyllid (ACP) Diaphorina citri. Such insect is the vector of Candidatus Liberibacter spp., the causal agents of Huanglongbing (HLB), the main citrus disease in the world. In previous studies, we identified Bt strains effectively control D. citri nymphs. The aims of this study were to confirm the mortality caused by the Bt strains previously identified as pathogenic to D. citri, to demonstrate their translocation inside sweet orange seedlings, and to select the best Bt strains to control ACP. All the bioassays were performed confining ten 3rd instar D. citri nymphs on Citrus sinensis (L.) Osbeck seedlings (six replicates/treatment and replicated twice). The treatments consisted of suspensions of different strains of Bt spores, in addition to the distilled water control. Each Bt strain suspension or the control treatment was placed on the substrate, close to the crown of the plantlet. The nymphs’ mortality was evaluated every 24 hours up to 120 hours post-inoculation. The presence of the bacteria was confirmed in young leaves through isolation in NYMS medium and PCR detection with specific primers. The Bt strains translocation was confirmed in all of the C. sinensis seedlings used in the bioassays. Eight Bt strains were evaluated and three of those showed nymphs mortality percentage above 60%. The results revealed that the concentrations of 106 and 107 spores ml-1 yielded the best nymph control, reaching up to 80% of mortality.

192 | S7 PEST- DISEASE MANAGEMENT

Effect of the amount of source habitat on the rice damage caused by a mirid pest Stenotus rubrovittatus: constructing a spatial predictive model of rice damage and the potential hazard map using land use data

Tabuchi K*, Furihata S, Okudera S and Takahashi A

NARO Tohoku Agricultural Research Center, Japan

*Corresponding author: [email protected] Oral Presentation

Recent studies have shown that surrounding non-crop habitat of agricultural field, such as the amount or configuration of the source habitat of pests within a species-specific spatial scale affects the abundances of pests occurring at each field, crop damages and yields. For a sorghum plant bug, Stenotus rubrovittatus (Hemiptera: Miridae) which is one of the major rice pests in Japan, we tested whether the potential hazard of occurrence of pecky rice damage could be predicted by a spatial model based on land use data. From three-year field study, we examined the relationship between the area of land use within a radius of 300m around each focal paddy field and the abundance of S. rubrovittatus against the number of pecky rice damage in Maesawa region in northern Honshu Island, Japan. We also tried mapping to visualize potential hazard by using a model with a GIS software. From a linear mixed model analysis and model selection by AIC values, a spatial predictive model using the area of source habitat without the abundance of S. rubrovittatus was determined as a best model. Therefore, it is indicated that potential hazard of pecky rice damage will be predicted just after when the spatial arrangement design of arable fields in a certain year is determined. The predicted value of pecky rice damage against the observed value was not highly sensitive for quantitative prediction, however, the model worked accurately enough for predicting whether the brown rice grade is first or not which is most important for local farmers. The future prospect of the prediction of crop damage using land use information will be discussed.

S7 PEST- DISEASE MANAGEMENT | 193

The role of an integrated-domain NLR in wheat resistance against the different Diuraphis noxia biotypes occurring in South Africa

Venter E* and Mkhize N

Department of Botany and Plant Biotechnology, University of Johannesburg, South Africa

*Corresponding author: [email protected] Poster 94

The occurrence of Diuraphis noxia hampers wheat production. This aphid is negating the effect of resistance breeding programmes through the development of resistance breaking biotypes. There are currently four biotypes in South Africa (RWA-SA1-4) with varying levels of virulence against the different Dn-genes deployed in breeding programmes. We have identified a WRKY-domain carrying nucleotide-binding leucine-rich repeat (NLR) gene named Adn1 that plays an important role governing the resistance response in Dn1 carrying wheat cultivars against the RWA-SA1 biotype of D. noxia. The aim of this study was to elucidate the role of Adn1 in imparting resistance against the different D. noxia biotypes in South Africa. Knockdown of Adn1 in several resistant wheat cultivars was performed using virus-induced gene silencing (VIGS). The effect of this was evaluated through RT-qPCR, determining the induction of the oxidative response, the effect on aphid biology, and general plant response. The results indicate that Adn1 plays a significant role in determining the resistance response of the different wheat lines. Knocking down of Adn1 completely abolished the resistance response in the cultivars. Thus, we hypothesise that Adn1 is a critical part of the wheat resistance mechanism against pests.

194 | S7 PEST- DISEASE MANAGEMENT

Food and behavioral characterization of bedbugs in predator-prey interaction

Campos JM1*, Martínez LC1, Rueda AP1, Rolim GS1, Paulo PD1, Fereres A2, Zanuncio JC1 and Oliveira EE1

1 Universidade Federal de Viçosa- UFV. MG, Brazil 2 Instituto de Ciencias Agrarias-CSIC, Spain.

*Corresponding author: [email protected] Poster 95

Species of the genus Podisus are widely distributed and are considered a major natural enemy with promising potential for use in biological control programs. Its occurrence has been registered in the control of eucalyptus, cotton, soybean and tomato defoliation caterpillars. Studies of the bioecology associated with alternative prey types have been performed as reported with Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae), Musca domestica L., 1758 (Diptera: Muscidae), or Bombyx mori L., 1758 (Lepidoptera: Bombycidae), and artificial diets, in order to produce the largest number of individuals. However, a more thorough analysis of the effect of this ingestion can be made by observing the path that the stylet traverses. The feeding behavior of Podius nigrispinus (Heteroptera: Pentatomidae) in its prey (T. molitor) was investigated using the Electrical Penetration Graph (EPG) technique. Based on this study that is still underway, the likely waveforms related to the predator feeding behavior in the prey are: non-probing (np), penetration phase (1), salivation phase (2) and ingestion phase (3). With this study, it is expected to better understand the behavior of predators used in the biological control of agricultural and forest pests, to add value in the mass creation of these natural enemies and to establish new strategies in integrated pest management.

S7 PEST- DISEASE MANAGEMENT | 195

Neem: Mode of action on rose aphids (Macrosiphum rosae) and consequences for application

Bartelsmeier I1*, Kilian M2 and Dicke M2

1 Bayer AG, Crop Science Division (Monheim, Germany) 2 Laboratory of Entomology, Wageningen UR (Wageningen, The Netherlands)

*Corresponding author: [email protected] Poster 96

Natural insecticides based on neem tree (Azadirachta indica) seed kernel extracts are a promising alternative to synthetic pesticides. However, they do not provide a strong knock-down effect and their mode of action needs to be understood in more detail for controlling aphids successfully. The commercial neem product NeemAzal®-T/S inhibited the population growth of rose aphids (Macrosiphum rosae), led to a delayed development and to a higher mortality of young nymphs. Further, reproduction of females was reduced, when these females grew up on NeemAzal®-T/S treated plants. No effect was found on host plant choice, on feeding and on adult aphids. For a successful application of NeemAzal®-T/S, the described effects need to be considered next to potential influencing factors such as environmental temperature and application timing.

196 | S7 PEST- DISEASE MANAGEMENT

Bioefficacy of hexane extract of Ocimum sanctum on survival, development and reproductive bioactivities of Dysdercus koenigii Fabricius (Heteroptera: Pyrrhocoridae)

Gupta KK* and Kayesth S

Deshbandhu College, University of Delhi, India

*Corresponding author: [email protected] Poster 97

Dysdercus koenigii Fabricius (Heteroptera: Pyrrhocoriedae) commonly known as red cotton bug is a serious pest of cotton worldwide. Both adults and nymphs damage the standing crop by sucking sap from developing cotton bolls. Present research work focused on impact of Ocimum sanctum on growth, development and reproductive fitness of Dysdercus. Ocimum leaves were extracted with hexane by ‘cold extraction method’. The laboratory reared newly emerged fifth instar nymphs were exposed to the extract at concentrations ranging from 0.00625% to 10% by ‘thin film residual method’ for a period of 24h. Survival, growth and development of treated nymph were studied for seven days. The adults emerged from the treated nymphs were used to study their reproductive performance. Our results indicated that hexane extract of Ocimum leaves was highly toxic at concentrations 1.25% or above. The extract also accounts for delayed toxicity as evident by increased mortality during subsequent 7 days of exposure. Decreased growth index of the treated nymphs indicated adverse effect of the extract on the growth. Different types of developmental anomalies reported in the treated nymphs include incomplete moulting and death of the nymphs, supernumerary nymphal instars, adultoids, adults with wing deformities, and adults with exuviae attached to the body. Treatment with concentration 0.05% or below did not impair survival growth and development significantly but abbreviated reproductive potentials of the adults. Both the males and the females exhibited altered courtship behaviour. Consequently, the mating success and sustenance of mating decreased. The mated females laid lesser number of eggs. The hatching success of the eggs was also decreased. GCMS analysis of the extract revealed presence of phytochemicals with biological activities such as insecticidal, JH mimics, JH intermediates. These phytochemicals individually or synergistically affect various life processes and therefore, can be used as potential botanicals for management of heteropteran pests.

S7 PEST- DISEASE MANAGEMENT | 197

Evaluation of spatial, temporal and geographic variation of within-field distribution of the sugarcane Aphid, Melanaphis sacchari Zehntner in grain sorghum

Royer TA1*, Lindenmayer JC1, Giles KL1,Zarrabi AA1, Elliott NC2, Bowling R3, Knutson AE3, Seiter N4, Brown S5 and McCornack B6

1 Oklahoma State University, USA 2 USDA ARS, USA 3 Texas A&M Agrilife, USA 4 University of Arkansas, USA 5 Louisiana State University, USA 6 Kansas State University, USA

*Corresponding author: [email protected] Poster 98

Grain sorghum, Sorghum bicolor (L.) is grown on more than 2.6 million hectares in the United States, and is worth an estimated $1.7 billion US annually. Since 2013, the sugarcane aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae) has become a key pest of grain sorghum in the southern Great Plains and southeastern U.S., affecting nearly 1.3 million hectares. Current management practices are confined to the use of a select few insecticides that are effective. Other management tactics, while being evaluated, are to date, of limited value. The objective of this research is to develop efficient sampling protocols, based on a statistical analysis of the spatial, temporal and geographic variation of the aphid’s distribution in the field that will ultimately link to established economic injury levels for M. sacchari. This paper describes results from data collected from 161 fields located across six states using a stratified sampling protocol. Spatial distribution patterns for M. sacchari count data within fields were described which allowed for determination of a 3-plant sample unit that accounted for 90% of the population variation. Strong/significant relationships between means and variances permitted preliminary calculations of sample sizes. This research should result in customized sampling recommendations that accurately assess M. sacchari populations based on their growing regions and allow producers to reduce or eliminate unnecessary insecticide applications and conserve natural enemies.

198 | S7 PEST- DISEASE MANAGEMENT

Discovery and characterization of a novel class of pesticidal bacterial toxins for potential control of phytophagous stinkbugs (Hemiptera: Pentatomidae) through genetic engineering of plants

Bressan A*, Lehtinen D, Baek J, Magalhaes LC, Qin X, Dunn EW, Vaknin D, Joo DM and Razvan DV

Bayer CropScience, R&D Trait Research, USA

*Corresponding author: [email protected] Poster 99

Genetic engineering has allowed the development of insect resistance traits for several crops. Most sources for insect resistance traits have originated from gene-encoding toxins derived from Bacillus thuringiensis (Bt) to target primarily lepidopteran and coleopteran pests. Hemipteran pests can pose a serious threat to several agricultural commodities worldwide because of the direct feeding damages on the plants and the transmission of plant pathogens. Furthermore, Bt- derived technology available in the market does not control such group of pests. Our goal is to identify new lead genes that can be expressed in plants through genetic engineering technologies, and that can control hemipteran insects, including phytophagous stinkbugs. We have analyzed the sequenced genomes of our microbial collection, which exceeds 100,000 gram-positive and gram- negative microbes, and sequenced metagenomes from a number of different microbiomes. Using our proprietary discovery platform, we have identified ORFs that putatively encode novel insecticidal toxins. Those ORFs were cloned and expressed through recombinant DNA technology in E. coli. The purified putative toxins were tested in in vitro insect-feeding bioassays and were found to suppress growth and cause mortality in a variety of target hemipteran species, including stinkbugs. The newly characterized toxins are currently deployed to transform plants to make them resistant to stinkbugs.

S7 PEST- DISEASE MANAGEMENT | 199

Employing host plant resistance (HPR) in the control of cowpea aphids in Northern Ghana

Kubi GT1*, Kusi F, Asare A1 and Botchey M2

1 University of Cape Coast, Ghana 2 Savanna Agricultural Research Institute (SARI), Ghana

*Corresponding author: [email protected] Poster 100

The incidence of phloem-sucking insect pests on food crops continues to be a major production setback in the agricultural sector. Their direct damages coupled with indirect transmission of viral pathogens lead to more than 90% yield losses in extreme cases. In Northern Ghana, cowpea is considered the second most important cultivated grain legume. The crop is well noted for its drought tolerance and adaption to marginal soils but yield expectancy is hardly realized due to vast array of biotic stress of which the cowpea aphid (Aphis craccivora) plays key damaging roles. The indiscriminate use of agro-chemicals as a fundamental means of aphids control is not only expensive for the peasant farmer but also has adverse environmental concerns. Cultivating aphid- resistant cowpea varieties thus presents an environmentally friendly and economically sound approach. Seven newly improved Striga gesnerioides resistant cowpea lines obtained from the University of Cape Coast, were screened for aphid-resistance using both phenotypic data scores and SSR markers. Preliminary results obtained selected two resistant and two susceptible lines to aphids. Based on this, cross breeding of these lines were done with a known aphid-resistant variety, SARC 1-57-2 with the aim of improving the field resistance of the susceptible lines and also determine if the source of resistance in both lines were the same as the resistant donor. If different genes are found controlling resistance, then gene pyramiding will be our ultimate goal to merge the two distinct genes.

200 | S7 PEST- DISEASE MANAGEMENT

AgBiome: Harvesting the plant microbiome to uncover novel insecticidal products

Raines TM*, McGuire A, Tran H and the AgBiome Team

AgBiome, Inc, USA

*Corresponding author: [email protected] Poster 101

AgBiome is a biotechnology company applying advanced knowledge of the plant-associated microbiome to create innovative products for agriculture. Our discovery platform allows us to efficiently capture and screen the most diverse and unique microbes for agriculturally relevant applications. To drive our successes, we use our in-depth knowledge of the crop microbiome and an extensive network of field-sampling partners to build an ever-expanding proprietary strain collection. This collection sources our discovery of new biologicals and trait genes for use in insect control. Unlike many programs which only use metagenomic surveys, the AgBiome platform is focused on true isolated microbial strains and their fully sequenced genomes. Each environmental isolate is screened in high-throughput bioassays against a broad range of insect pests. We have utilized this effective discovery platform to find novel solutions for the control of hemipteran species and have discovered numerous active microbial strains and identified over ten novel insecticidal proteins with distinct modes of action. By integrating sequence data with performance data across multiple assays, the AgBiome team will continually improve their ability to quickly identify active insecticidal leads that are attractive product candidates.

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Characterization and introgression of potato- psyllid-Lso resistance in tomato

Avila CA1,2*, Kurpis J2, Viloria Z2, Marconi T2

1 Department of Horticultural Sciences, Texas A&M AgriLife Research, USA 2 Texas A&M AgriLife Research and Extension Center at Weslaco, USA

*Corresponding author: [email protected] Poster 102

The tomato-potato psyllid (TPP), Bactericera cockerelli, vectors the phloem-limited bacteria Candidatus Liberibacter solanacearum (Lso), the causative agent of economically important diseases including the tomato vein-greening and potato zebra-chip. In the absence of commercial resistant cultivars to both the vector and the pathogen, growers have dramatically increased pesticide use to control the vector. Since no adequate resistance level is found in cultivated tomato, we screened wild tomato relative species for resistance. We identified, six accessions with high levels of resistance to the TPP (survival < 10 %) and two accessions resistant to Lso. Identified TPP and/or Lso resistance sources included accessions of S. habrochaites, S. pennelli, S. huayalasense, S.chmielewskii, S. corneliomulleri, and S. galapagense. To accelerate introgression of both TPP and Lso resistance, we are taking advantage of recombinant inbred lines (RILs) developed from the cross of S. habrochaites or S. pennellii with cultivated tomato (S. lycopersicum). These RILs representing the genome of S. habrochaites (LA1777) or S. pennellii (LA0716) in the cultivated tomato background. The S. habrochaites RIL parent LA1777 is an excellent TPP resistance source since no living adults and nymphs were found on infested plants after 10 days. However, when we screened S. habrochaites RILs, we did not find single lines with the same resistance level as the LA1777 source, although significant reduction on insect fecundity and survival was observed on RILs with introgressions in chromosome 3, 5, 6, and 8 as compared to susceptible control. These results indicate that resistance in LA1777 is controlled by more than one gene. Furthermore, gene expression analysis shows that RIL with introgression in chromosome 8 upregulate salicylic acid defensive signaling in response to TPP feeding, while no up-regulation occurs in susceptible plants. Partial resistance levels on individual RILs provide a good protection level against the insect and can be used for breeding purposes.

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Pear psylla Cacopsylla bidens (Šulc) management by natural volatile compounds

Ibdah M1*, Rachmany D1, 2, Shaltiel-Harpaz L2, 4, Gerchman Y3, Holland D1

1 NeweYaar Research Center, Agricultural Research Organization, Israel 2 Tel Hai College, Israel 3 University of Haifa in Oranim, Israel 4 Migal Galilee Research Institute, Israel.

*Corresponding author: [email protected] Poster 103

Pear Psylla is the most important problem insect pest of pear in all pear-growing regions, in Asian, European, and USA. Pear psylla damages pears in several ways. Loss of crop and tree vigor, and sometimes loss of trees, can occur from pear decline disease, caused by a phytoplasma organism that psylla injects into pear trees. Pear decline has varying effects on the trees depending on variety, rootstock, quality of the growing site, and pear psylla numbers. Psylla control is a major obstacle to efficient integrated pest management and naturally volatile compound as an insecticide and are an attractive option. Yet, to date there are several way to management the Pear psylla e.g. biological, cultural and chemical control. We have identified two pear accessions (Py.760-261 and Py.701-202) from the local Newe Ya’ar live collection as having resistance to pear psylla. GC-MS volatile metabolic profiling has been performed for the two pear accessions. We have identified several volatile compounds accumulates in the leaves of these cultivars, but not in the commercial Spadona pear variety. Laboratory experiments and applications of some these volatile compounds were very effective against psylla eggs, nymphs and adults. Unlike other insecticide, the discovery of this volatile compound shows high resistant to Pear psylla than the commercial insecticide. To the best our knowledge, this is the first report on the accumulation and application of the volatile compound in Pear leaves against pear Psylla. Our new invention provides a new concept for inhibiting Pear Psylla growth, by spraying the volatile compound at very low concentration into the pear trees. This approach could be applicable for a variety of other fruits, crops specifically those, which show dramatically Psylla pest problems.

S7 PEST- DISEASE MANAGEMENT | 203

Combining trap crop and insecticide to control tarnished plant bug in strawberry

Dumont F* and Provost C

Centre de recherche agroalimentaire de Mirabel, Canada

*Corresponding author: [email protected] Poster 104

The polyphagous tarnished plant bug (TPB), Lygus lineolaris (Hemiptera: Miridae) causes severe damages in strawberry field in Quebec. Currently, only chemical insecticide successfully controls this major pest. Alternatively, TPB aggregate in trap crop (alfalfa, buckwheat, mustard), but do not remain long enough on these plants to significantly reduce damages on strawberries. However, the attractiveness of the trap crop gives the opportunity to gather TPB in an area of the field where chemical treatments could be applied more efficiently. The aim of this study was to test the effectiveness of the combination of trap crop and chemical treatments to control TPB during both summer and autumn. Randomized block design included treatment with no trap crop, buckwheat or mustard row implemented close to strawberry plants. Half blocks were treatment with insecticide (cypermethrin) sprayed on strawberry plants (in treatment without trap crop) or directly on trap crop. In autumn, TPB was attracted in mullein plants subsequently treated with chemical (cypermethrin) or biological (Beauveria bassiana) insecticide. Blocks with trap crop (either buckwheat or mustard) attracted more TPB than blocks without trap crop. The use of insecticide only reduced TPB population slightly. In spite of a superior efficiency of the buckwheat over the mustard, more TPB was observed on strawberry plants close to trap crop and more fruit damages were recorded than in no trap crop blocks. In autumn, more than 20 % of the TPB population was observed on mullein plants before hibernation, which gives the opportunity to increase winter mortality. The efficiency of the combined trap crop and insecticide approach relies on the use of adapted insecticide dosage and intervention thresholds.

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Neonicotinoid insecticide resistance in populations of the potato psyllid (Bactericera cockerelli) in Texas

Szczepaniec A* and Varela K

Texas A&M AgriLife Research, USA

*Corresponding author: [email protected] Poster 105

The potato psyllid (TPP), Bactericerca cockerelli, a vector of a pathogen causing the “zebra chip” (ZC) disease in potatoes, is by far the greatest threat to profitability of potato production in Texas. TPP has many generations each year and easily migrates over large distances using air currents. It has been managed primarily by use of insecticides at planting and later throughout the potato- growing season, with as many as 10-12 insecticide applications during the season to suppress TPP. Another important factor contributing to the high likelihood of resistance development is lack of any regulation of insecticide use in Mexico, just south of the largest potato-growing region in Texas. TPP are highly mobile and influx of resistant psyllids from Mexico to Texas is likely. Thus, TPP from all of the major potato-growing regions of Texas was tested for resistance to neonicotinoid insecticides, the main class of pesticides used to suppress TPP at planting. We found evidence of resistance to the neonicotinoid insecticides in all of the TPP populations, although the level of resistance differed among regions. TPP populations from Weslaco exhibited lower neonicotinoid sensitivity than psyllids collected from the Texas Panhandle. These data suggest that neonicotinoid insecticides are unlikely to provide sufficient suppression of TPP in Texas.

S7 PEST- DISEASE MANAGEMENT | 205

Silencing of Diuraphis noxia virulence gene through RNA interference using a novel siRNA delivery method

Swiegers HW and Botha AM*

Genetics Department JC Smuts Buidling Stellenbosch University, South Africa

*Corresponding author: [email protected] Poster 106

Resistance in wheat is often counteracted by Diuraphis noxia (Russian wheat aphid) through the development of a new biotype. The virulent biotype SAM (South African Mutant) was developed in the laboratory from biotype SA1 under selection pressure from resistant wheat and is thus used as a model to study virulence is these aphids. Subsequently, the genomes and salivary proteomes of SA1 and SAM were compared to discover genes potentially involved in the development of virulence in D. noxia. In this study, one of these genes, a cuticle protein - DnRR1, was investigated along with C002 as C002 has been silenced in other aphid species. Both the target genes were shown to be upregulated in SAM compared to SA1. Hereafter, silencing through RNA interference of the candidate genes in SAM was attempted using a novel method. This method involved the injection of siRNA into wheat leaves on which SAM was contained and allowed to feed. Synthetic dnRR1- or c002-siRNA was used in this case and buffer injections or leaves with no injection were used as controls. Feeding on dnRR1- or c002-siRNA reduced the survival rate of SAM feeding on resistant wheat, ‘Gamtoos-R’, significantly. Silencing of dnRR1 was observed at 48h post injection when feeding on dnRR1-siRNA, but interestingly c002 was upregulated when SAM fed on c002- siRNA compared to the controls. Catalase and peroxidase activity in SAM was also determined to be upregulated when the aphid fed on dnRR1- or c002- siRNA. These results demonstrate potential for these genes to serve as targets for the control of aphids via plants producing aphid gene-specific dsRNA. RR1 is highly conserved and could thus not only target aphids, but other insects as well.

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Re-evaluation of plant introductory lines for Russian wheat aphid Diuraphis noxia (Kurdjumov) (Hemoptera: Aphididae) resistance

Bapela TM1*, Tolmay VL1 and Tsilo TJ1,2

1 Agricultural Research Council, Small Grain Institute, South Africa 2 Department of Life and Consumer Sciences, University of South Africa, South Africa

*Corresponding author: [email protected] Poster 107

Russian wheat aphid Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae) has economically dominated as an aggressive pest of wheat in South Africa (S.A) since its discovery in 1978. Leaf rolling, leaf striping, purple discolouration and stunted growth characterize the damage symptoms from RWA infestation. Due to ineffectiveness of other control measures, host plant resistance serves as an effective and reliable method for RWA control. Resistance-breaking biotypes, upon existing resistant cultivars, necessitate identification and deployment of resistance against new biotypes. Eleven wheat accessions used at the ARC-Small Grain Institute’s (S.A) pre- breeding programme, namely PI 47545, PI 137739, PI 140207, PI 222666, PI 222668, PI 245462, PI 262660, PI 294994, PI 347006, PI 366515 and PI 372129 were tested and re-evaluated using two S.A RWA biotypes i.e. RWASA1 and RWASA2. In addition, five check genotypes i.e. Gariep, Yumar, PAN 3144, CItr 2401 and Hugenoot were also used in the study. Test entries PI 294994, PI 347006, PI 372129 were resistant to RWASA1. PI 47545, PI 222666, PI 262660 were moderately resistant to RWASA1 and only PI 137739 was moderately susceptible to RWASA1. PI 294994, PI 347006, PI 372129, PI 366515, and PI 222666, were resistant to RWASA2, although PI 47545, PI 262660 and PI 137739 were found moderately resistant to RWASA2. These lines will be tested further for RWASA3 and RWASA4 resistance and the ribonucleic acid (RNA) profile of the four biotypes will determined. This is done to identify and characterise aphid salivary effectors enabling the four D.noxia biotypes to induce damage on potential accessions. However, these lines show high potential for being used in pre-breeding and breeding programs.

S7 PEST- DISEASE MANAGEMENT | 207

Evaluation of intercrop plants for the cultural control of tomato yellow leaf curl disease

Hu FY, Mou DF and Tsai CW*

Department of Entomology, National Taiwan University, Taiwan

*Corresponding author: [email protected] Poster 108

Tomato yellow leaf curl disease is a devastating viral disease which leads to 20%-100% reduction in the yield of tomato worldwide. The disease is associated with a group of tomato yellow leaf curl viruses that belong to the genus Begomovirus in the family Geminiviridae. Bemisia tabaci transmits tomato yellow leaf curl viruses in a persistent-circulative mode. Tomato yellow leaf curl Thailand virus (TYLCTHV) is now the most predominant virus in Taiwan. To control plant viruses transmitted by insects in a nonpersistent mode, intercropping with appropriate barrier plants can intercept the insect vectors thus reduces the transmission efficiency of the viruses. However, there are few studies related to the cultural control of persistently transmitted plant viruses by intercropping. The objectives of this study were to select non-host plants of TYLCTHV as intercrop plants to intercept the virus-transmitting B. tabaci and to examine whether feeding on the non- host plants reduces the virus transmission efficiency. The host preferences of B. tabaci (TYLCTHV- infected and non-viruliferous) to five candidate plant species (cucumber, Chinese kale, okra, corn, and soybean) compared to tomato were examined. Cucumber and okra were more attractive to the non-viruliferous B. tabaci than tomato, whereas soybean and corn were less attractive than tomato. The TYLCTHV-infected B. tabaci preferred cucumber to tomato but disliked corn. Therefore, we chose cucumber, Chinese kale, and okra to examine whether feeding on these plants after the acquisition of TYLCTHV reduces the virus transmission rate. Results showed that the virus transmission rate decreased when B. tabaci fed on these plants for 6, 12, and 24 hours after virus acquisition. These plant species are potential non-host plants to be used in the intercrop system for the cultural control of tomato yellow leaf curl disease. Further research is needed to determine the effect of intercropping in the fields.

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Possible threat of Halyomorpha halys (Hemiptera, Pentatomidae) to agriculture in Serbia

Konjevic A*

Department of Plant and Environmental Protection, Faculty of Agriculture, University of Novi Sad, Serbia

*Corresponding author: [email protected] Poster 109

Invasive brown mramorated stink bug Halyomorpha halys (Hetroptera, Pentatomidae), originated from Asia, is becoming cosmopolitan species inhabiting USA and Europe. Fast spreading caused that more than a decade later BMSB is recorded in many European countries: Switzerland, Lichtenstein, Austria, Germany, France, Italy, Hungary, Greece, Romania, Russia and Bulgaria. The first finding in Serbia dates in October 2015, when it was recorded in urban zones of Belgrade, and small town Vrsac, at the Serbian-Romanian border. During 2016 H. halys has been observed in infested areas in order to obtain data about the population and possible establishment in the country. Results showed that H. halys found suitable conditions for its survival in moderate climate conditions, and its spreading is proved by new positive localities and plant hosts detected. By the end of vegetative season of 2016, hosts list was expanded by two important agricultural crops, tomato and soybean. The first nymphs of H. halys in soybean were recorded in late August 2016. In the same period low abundance was observed on tomato plants in the area of Vrsac town, with no evidence of significant economic loss of the yield. Interesting fact is that the BMSB specimens were together with, at this time common, also invasive species Nezara viridula, present in Serbia since 2008. There was no evidence of antagonism among them, just as it was observed on hibiscus plants and high abundance of both H. halys and Pyrrhocoris apterus. Even though the abundance of H. halys population was relatively high in urban hibiscus plants, there was no evidence of economic losses in tomato and soybean production due to low abundance of specimens in agricultural crops. Therefore, no specific treatments were conducted to control this species in 2016, but economic important damages could be possible in the years to come.

S7 PEST- DISEASE MANAGEMENT | 209

Resistance reaction of selected elite bread wheat lines to multiple, international biotypes of Russian wheat aphid (Hemiptera: Aphididae)

Tolmay VL*, Vitou J, Edwards O, Puterka GJ, Baker C, El-Bouhssini M, Turanli F and Cakir M

Agricultural Research Council-Small Grain Institute, South Africa

*Corresponding author: [email protected] Poster 110

Russian wheat aphid (RWA) (Diuraphis noxia) is the most important pest of wheat in South Africa annually causing severe damage to crops in the summer-rainfall production region. South Africa is a world leader in the development of cultivars with resistance to this pest. The first resistant cultivar ever released in the world was released by ARC-Small Grain Institute (ARC-SGI) in 1992 and to date South African producers have had access to more than 35 different resistant cultivars. However the occurrence of resistance breaking biotypes has been reported from both the USA and South Africa where resistant cultivars have been utilised commercially, prompting a renewed interest in the identification of resistance genes that are effective against multiple biotypes. This poster reports on the characterisation of seedling resistance in elite South African, RWA resistant germplasm using multiple, international biotypes of RWA.

210 | S7 PEST- DISEASE MANAGEMENT

Comparative toxicity of benzoylurea insecticides on Leptopharsa gibbicarina (Hemiptera: Tingidae) nymphs

Martínez LC, Plata-Rueda A and Campos JM*

Universidade Federal de Viçosa, Brazil

*Corresponding author: [email protected] Poster 111

The lace bug, Leptopharsa gibbicarina Froeschner (Hemiptera: Tingidae) a pest and vector of Pestalotiopsis disease of the oil palm (Elaeis guineensis Jacquin) in the Americas. Effective methods for pest management are needed urgently. Bioassays were conducted to compare the toxicity to L. gibbicarina of the insecticides: diflubenzuron, flufenoxuron, lufenuron, novaluron, teflubenzuron, triflumuron. The toxicity of each insecticide to the nymphs of L. gibbicarina was determined as: (1) the LC50 and LC90 under laboratory conditions, after exposure of six concentrations of each insecticide applied in a topical solution by each insect, (2) survivorship and, (3) mortality under semi-controlled field conditions after applications of insecticides into the leaves in the oil palm tree. The mortality of L. gibbicarina was higher with diflubenzuron, novaluron, teflubenzuron, triflumuron, while flufenoxuron and lufenuron were less effective. Higher mortalities were obtained with concentrations of 1.25, 2.5 μL mL-1 for determining LC50 values and 5, 10 μL mL-1 for determining LC90 values during 72 h. The evaluated concentration of the benzoylurea insecticides led to low survival time of lace bug nymphs. The mortalities of L. gibbicarina had similar tendencies under laboratory and semi-controlled field conditions. Our study showed that chitin of the nymphs was clearly inhibitor in vivo and confirmed the effects directly into the insect integument. Diflubenzuron, novaluron, teflubenzuron, and triflumuron caused substantial mortality in L. gibbicarina nynmphs and, thus, can be used rotationally in integrated pest management programs (IPM) against this pest in the oil palm plantations.

S7 PEST- DISEASE MANAGEMENT | 211

Entomopathogenic fungi as a potential control agent against the lace bug, Leptopharsa gibbicarina (Hemiptera: Tingidae) in oil palm

Plata Rueda A, Campos JM and Martínez LC*

Universidade Federal de Viçosa, Brazil

*Corresponding author: [email protected] Poster 112

Pestalotiopsis is a disease that causes damage to the leaves of oil palm (Elaeis guineensis Jacquin) and defoliation in commercial plantations. A primary means of infection and spread of the disease is by Leptopharsa gibbicarina Froeschner (Hemiptera: Tingidae) as an insect-vector. We tested the pathogenicity, adherence and hydrophobicity of 2 Beauveria bassiana (Balsamo) Vuillemin isolates and 2 Purpureocillium lilacinum (Thom) Luangsa-ard isolates against adults of L. gibbicarina. The strains were screened for relative pathogenicity, and the median lethal time eliciting 50% mortality (LC50) was calculated. Adults of L. gibbicarina infected with P. lilacinum and B. bassiana were observed under Scanning Electron Microscopy (SEM) to counted and characterize morphologically the process of adhesion and the conidia development of these fungi, prior to and after the death of the host. Conidial surface hydrophobicity was assessed using aqueous-solvent partitioning assays, which determine the ratio of conidia distributed between the aqueous and organic phases. In general, the P. lilacinum strains were more virulent with lower LT50 values than B. bassiana strains. The LT50 values ranged from 8.3 to 13.9 days. The median lethal concentrations (LC50) of all the isolates were also determined. The strains were also tested for survival under laboratory nest conditions. Scanning Electron Microscope observations revealed that P. lilacinum and B. bassiana adhered to the body surface of both adults with differentiation in mouthparts, hemelytra, and legs. Strains of B. bassiana displayed decreased conidia hydrophobicity and loss of water mediated dispersal that P. lilacinum. In conclusion, the present study suggests that these strains might be candidate for a programme of biological control of L. gibbicarina.

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Pathogenicity of Bacillus thuringiensis to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Dorta SO1,2, Balbinotte J1, Lopes JRS1*, Monnerat RG4, Machado MA2, Freitas-Astúa J3

1 Universidade de São Paulo, ESALQ-USP, Brazil 2 Centro APTA Citros Sylvio Moreira-IAC, Brazil 3 Embrapa Mandioca e Fruticultura, Brazil 4 Embrapa Recursos Genéticos e Biotecnologia, Brazil

*Corresponding author: [email protected] Poster 113

The discovery that Bacillus thuringiensis (Bt) strains are able to endophytically colonize different plant species has opened new perspectives for studies on phloem feeding insects. The pathogenicity of Bt strains to the Asian citrus psyllid (ACP) Diaphorina citri, the main vector of causal agents of citrus huanglongbing (HLB), has been suggested. Here, we confirm such pathogenicity after the evaluation of recombinant Bt isolates harboring 10 individual cry or cyt toxin genes against ACP nymphs. A series of bioassays was performed in seedlings of Citrus sinensis (L.) Osbeck using 3rd instar nymphs of ACP (five seedlings with ten nymphs/ treatment). Negative controls, without Bt, were added to each test. Toxins were evaluated in at least two independent bioassays. Seedlings were inoculated by drench application of a suspension containing Bt crystals and spores, and the ACP mortality was evaluated daily for five days. Bt isolation from dead nymphs and young leaves, and PCR with specific primers for each cry or cyt gene were performed and confirmed the involvement of the Bt on psyllid mortality. Among ten recombinant isolates tested, one of them stood out, causing up to 93% mortality after 120 h of inoculation. The promising gene was cloned and is being used to genetically modify citrus plants with the objective to obtain transgenic resistance against D. citri and mitigate the negative impact of HLB on citrus crops.

S7 PEST- DISEASE MANAGEMENT | 213

Botanical extract of Synandrospadix vermitoxicus has antifeedant and toxic effects on Myzus persicae

López-Isasmendi G1,2, Machado-Assefh CR1,2, Uriburu ML3 and Álvarez AE1*

1 Universidad Nacional de Salta, Facultad de Ciencias Naturales, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-CCT), Argentina 3 CONICET-INIQUI, , Argentina

*Corresponding author: [email protected] Poster 114

The green peach aphid, M. persicae (Homoptera, Aphididae), is a major pest for potato crops (Solanum tuberosum) mainly for its role as vector on virus transmission. Currently, aphids are controlled with synthetic insecticides which cause environmental damage and insect resistance. Our goal is to find new natural products from plant sources with antifeedant and/or toxic effects on M. persicae. Synandrospadix vermitoxicus (Araceae) is an autochthonous species from Northwestern region of Argentina. The tubers of S. vermitoxicus are used locally on animal injuries to protect against insect larvae but its extracts have not been studied against aphids. Here, we evaluated the ethanolic extract in three essays, 1) antifeedant activity by studying the preference of aphids on a dual choice test with leaves of potato plants treated with the extract at 2.5; 5; 10 mg.mL-1 on Tween-20, 0.05%; The number of aphids settled on each treated leaf was recorded from 5 min until 4 h after experiment started. Data was analyzed by binomial test and the settling inhibition index; 2) toxicity over M. persicae, by administrating it for 4 days through artificial diets at 50; 100; 1000 µg.mL-1, and the number of adults and average number of nymphs per adult was calculated. Data was analyzed by ANOVA and Duncan test, 3) qualitative and quantitative compositions of the S. vermitoxicus botanical extract looking for the principal classes of secondary metabolites. Leaves treated with 10 mg.mL-1 of the extract resulted on a strong settling inhibition already at 5 min and increases with time. Aphids fed on artificial diets supplemented with the extract at 1000 µg.mL-1 had negatively affected their survival and fecundity. The composition of the extract included an array of flavonoids, terpenoids and sugars. The blend of the botanical extract showed to have bioactive properties against M. persicae.

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Evaluation of potential new sources of melon host plant resistance to the whitefly, Bemisia tabaci

Natwick ET1*, Wintermantel WM2, Gilbertson RL3, Blanco SG2 and McCreight JD2

1 University of California ANR Cooperative Extension, USA 2 United States Department of Agriculture-Agricultural Research Service, USA 3 University of California Davis, USA

*Corresponding author: [email protected] Poster 115

Melon (Cucumis melo L.) genotypes that support fewer numbers of whitefly could reduce the frequency or the amount of insecticide applications required to keep the insects in check, as was the case with cotton where measurable resistance to whitefly in some genotypes reduced the number of sprays, thus reducing production costs while ensuring a marketable product (Chu et al. 1998. J. Cotton Sci. 2:1–9). Melon germplasm has been reported to have low-level resistance to sweetpotato whitefly Biotype B, Bemisia tabaci MEAM1 (SPWF-B), e.g., PI 313970 and TGR 1551. We evaluated these and other potential sources of low-level resistance to SPWF-B in field (Holtville) and greenhouse (Salinas) tests. Evaluation of eight potential sources of SPWF-B resistance in fall 2013 and spring 2014 identified PI 145594 as a potential of resistance and PI 116482 as a potential source of extreme susceptibility. In addition, PI 122847 was identified as a potential source of SPWF-B resistance in an ad hoc comparison with susceptible ‘Top Mark’ in 2014. The resistance reactions of 11 putative sources were evaluated in spring field tests in 2015 and 2016 for numbers of adults and immatures in insecticide and no insecticide treatments. Greenhouse evaluation of putative SPWF-B resistance sources utilized no-choice tests for: oviposition, egg-to-adult survival, and insect growth index. Field evaluations found PI 313970 and PI 122847 as potential antixenosis resistance sources based on SPWF-B adults and immatures levels. Greenhouse tests indicated: TGR 1937, PI 161375 and PI 122847 are potential resistance sources based on oviposition; TGR 1551 = TGR 1937 > PI 313970 are potential resistance sources based on reduced egg-to-adult survival; and TGR 1551, PI 123689 are potential resistance sources based on the insect growth index.

S7 PEST- DISEASE MANAGEMENT | 215

Indirect effect of supplementary UV radiation on Bemisia tabaci settlement

Prieto-Ruiz I*, Garzo E and Fereres A

Instituto de Ciencias Agrarias-CSIC, Spain

*Corresponding author: [email protected] Poster 116

The increment of ultraviolet (UV) radiation both A (315-400 nm) and B (280-315 nm) that reaches the earth surface has increased substantially in the past decades; this increment has altered the relationship between insects and plants since UV radiation has a key role in both the herbivore life processes and plant chemistry and morphogenesis. Here we evaluated the indirect effect of supplementary UV radiation on the settlement of Bemisia tabaci on eggplant under glasshouse conditions, its effects on the plant and its implications for pest management. We placed eggplants at one-true leaf stage (21 days) inside whitefly-proof cages (1 x 1 x 1 m) in a glasshouse under different UV radiation treatments, UVA+/UVB+, UVA-/UVB+ or UVA-/UVB- as control. Plants were exposed to supplemental UV and PAR radiation supplied by fluorescent and sodium vapor lamps during 30, 60 or 90 minutes each day for 21 days. Afterwards, two hundred adults of B. tabaci MED species were released in each cage and set of exposed plants and counted 2, 6, 24 and 48 hours after infestation. Once concluded, eggplants were analyzed for chemical content and morphology variations. In parallel, eggplants were grown until harvest to assess the effects of supplemental UV light on yield components. Our results demonstrated that a supplemental UVA+/UVB+ combined with PAR radiation of 30 or 60 minutes reduced the amount of whiteflies settled in the plant without reducing the morphological characteristics of the plants such as fresh weight, stem length, number of leaves and leaf area. However, UVA+/UVB+ during 90 minutes or UVA-/UVB+ radiation for any time reduced whiteflies settlement as well as plant development. Furthermore any supplemental UV radiation treatments of 90 minutes significantly increased carbon concentration on the leaves, while decreasing nitrogen. We conclude that a supplementary combination of UVA+/UVB+ and PAR radiation of 30 minutes each day during 21 days could be an effective strategy to reduce whitefly infestation in glasshouses.

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Effects of the plant strengthener BION® on the fitness and feeding behavior of the cotton aphid Aphis gossypii (Glover) on melon plants

Moreno-Delafuente A1,3*, Garzo E2,3, Viñuela E1,3, Fereres A2, 3 and Medina P1,3

1 Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid (UPM), Spain 2 Instituto de Ciencias Agrarias-CSIC, Spain 3 Unidad Asociada IVAS (CSIC-UPM): Control de insectos vectores bajo sistemas de agricultura sostenible

*Corresponding author: [email protected] Poster 117

Plant strengtheners are modern agricultural products that enhance plant resistance to biotic and abiotic stresses. Among plant strengtheners, the active ingredient of the plant activator BION® (acibenzolar-S-methyl; Syngenta, Spain) has been developed as an inducer of systemic acquired resistance that promotes plant resistance against several plant pathogens and pests. In this study, we evaluated the effect of BION® in the development, reproduction and feeding behavior of Aphis gossypii on melon plants (Cucumis melo L., Cucurbitaceae) with the goal of assessing the possible persistence of BION® in those plants. Aphis gossypii was exposed to melon plants at 0 (fresh residue), 4 and 7 days after sprayed BION® (50 g/ha), using water-sprayed plants as control. For the fitness experiment, mortality of nymphs and adult aphids from first-generation; pre- reproductive time (d); number of progeny produced in a period equal to the pre-reproductive time (Nd); intrinsic rate of natural increase (rm); mean generation time (Td) and mean relative growth rate (RGR); were calculated on each treatment. Nymphal mortality in fresh residue treatment was 100 %, so no population parameters could be analyzed subsequently. Nymphal and adults mortality of four-day-old residue (21%, 54%, respectively) were higher than control (11%, 15%) and seven-day-old residue (4%, 20%). There was a significant decrease in Nd, rm and RGR in four-day-old residue treatment compared with the rest of the treatments, but no statistical differences between control and seven-day-old residue treatment were found. In addition to the fitness experiments a feeding behavior study is being conducted by means of the electrical penetration graph (EPG) technique using synchronized adults of A. gossypii exposed to melon plants at fresh residue and four days after BION® spray-off, in comparison with those fed on untreated plants. The possible toxicity of BION® will be discussed on the basis of our findings.

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Weeds as host for aphids in potato crop fields in Salta province, Argentina

Said-Adamo, Mm1, Machado-Assefh, CR1,2, Cortéz SD1,2 and Álvarez, AE1*

1 Universidad Nacional de Salta, Facultad de Ciencias Naturales, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-CCT), Argentina

*Corresponding author: [email protected] Poster 118

Among diseases that affect potatoes, viruses are particularly important because viral particles can remain in the tubers, and since the potato crop reproduces vegetatively, viruses can persist for a long time causing severe losses due to decreased yield or quality. The most important potato viruses are the PVY (Potato virus Y) and PLRV (Potato leafroll virus). These viruses are efficiently transmitted by Myzus persicae and Macrosiphon euphorbiae, which are highly polyphagous aphids being able to infest a large diversity of plants belonging to more than 40 botanical families. Many vectors hosts have also been described as PLRV and PVY reservoirs (Solanaceae, Brassicaceae, Amaranthaceae, Nolanaceae and Portulacaceae). In Argentina, little is known about the plant species surrounding the potato fields that are hosts for vectors or reservoirs for plant viruses. Successive samplings were carried out at 3 potato production sites in the Salta province (Argentina) during 2015 and 2016. Samples of weeds and wild plants with aphids’ colonies were taken from the areas around the production lots. Each plant sample was cleaned with a brush to remove the aphids, and processed for its determination in the botanical lab using keys. Herbarium specimens were preserved for each of the species of plants collected. The aphids collected were preserved in 70% alcohol for later identification. We determined 28 weeds and wild plants that were hosts for aphid vectors of potato viral diseases. Macrosiphum euphorbiae and Myzus persicae, among other aphid species, were detected in the 3 sites studied.

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AgBiome: In vitro sucking pest bioassay development for novel insecticidal gene discovery

McGuire A*, Tran H and the AgBiome Team

AgBiome, Inc, USA

*Corresponding author: [email protected] Poster 119

AgBiome is a biotechnology company discovering novel solutions to agriculturally relevant problems by mining the plant-associated microbiome. With the world’s largest collection of fully sequenced microbes, we have discovered >3500 potential insect control genes. At the heart of our success are the bioassays we have developed to assess insecticidal activity. Nezara viridula and Aphis glycines are two challenging species of agricultural pests on which we have focused our efforts. During the process of developing a bioassay from an idea to a reproducible platform, we overcame difficulties such as selecting an appropriate pierceable membrane, optimizing an artificial diet and establishing assay parameters. Through innovation and teamwork, we created robust in vitro bioassays that allow us to quickly identify and develop active proteins.

S7 PEST- DISEASE MANAGEMENT | 219

Population dynamics of Planococcus citri (Risso) and Pseudococcus viburni (Signoret) (Hemiptera: Pseudococcidae) on persimmon (Diospyros kaki Thunb).

Vercher R*, González-Cavero S and Mañó P

Universitat Politècnica de València, Spain

*Corresponding author: [email protected] Poster 120

Diospyros kaki Thunb. cultivation became widespread in Spain and in others Mediterranean countries in the last years. In the Valencian Region, several citrus pests started to attach persimmon, with important lost in production. It is the case of Pseudococcidae (Hemiptera) that are causing high percentages of lost and customs restriction (quarantine concern). First step to stablish appropriate Integrated Pest Management (IPM) programs is to know diversity, behaviour, abundance, and population dynamics of pests. Studies of the population dynamics of Planococcus citri (Risso) and Pseudococcus viburni (Signoret) adults were carried out in Valencian Region (eastern Spain) in persimmon orchard plots from 2014 to 2016. Results indicated three Pseudococcidae species were found in persimmon orchards: Planococcus citri Risso, Pseudococcus viburni Signoret and Pseudococcus longispinus Targioni Tozzetti, where P. citri was the most abundant specie. Male population dynamics showed P. citri was present from middle May to November, while Ps. viburni occurred in colder period (spring and autumn-winter). Female population dynamics was different, adults specimen appeared in persimmon fruits from July to December. Ps. longispinus (not specific pheromone were available), females only were found in fruits from December.

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Classical biological control of Diaphorina citri Kuwayama in citrus crop in Brazil

Sá LAN, Prado SS*, Gulfier N, Diniz AJF, Veiga ACP, Girardi EA, Morgan DJW and Herreid J

Embrapa Meio Ambiente, SP, Brazil

*Corresponding author: [email protected] Poster 121

Brazil is the world’s largest producer of oranges, amounting to about 30% of the world’s production of sweet orange and 50% of orange juice. The country’s share in this commodity chain is about US $14.6 billion annually, generating approximately 350 thousand jobs. Brazilian citrus is threatened by yet another exotic pest, which arrived in 2004: the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of the citrus disease huanglongbing (HLB or citrus greening), caused by the bacteria Candidatus Liberibacter spp. Current chemical control practices for this psyllid involves ineffective and expensive application of insecticides in orchards. This research aims to develop and /or adapt technologies that focus on the biological control of D. citri, and HLB. The exotic bioagent Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) was imported from California-USA through California Department of Food and Agriculture (CDFA) to "Costa Lima" Quarantine Facilities of Embrapa Meio Ambiente in December 2016. The bioagents were reared in cages on D. citri which were cultured on curry leaf plants Bergera (Murraya) koenigii (Sapondales: Rutaceae). The production of the first generation of D. aligarhensis in Brazil occurred between Jan 16th and Feb 1st 2017 (611 adults). Colonies of these adults of D. aligarhensis have been established to perform competition experiments with another exotic parasitoid present in Brazil, Tamarixa radiata (Hymenoptera: Eulophidae), in order to fulfill the requirements prior to approval for release of D. aligarhensis in citrus orchards.

S7 PEST- DISEASE MANAGEMENT | 221

Alien scale insects of Europe: an updated checklist

Germain JF1* and Pellizzari G 2

1 Anses, laboratoire de la santé des végétaux, unité entomologie et plantes invasives, France 2 Dipartimento di Agronomia, Animali, Alimenti, Risorse Naturali e Ambiente DAFNAE Università di Padova, Italy

*Corresponding author: [email protected] Poster 122

An inventory of all alien terrestrial arthropods to Europe was established in 2010 through a consortium of researchers of biological invasions in Europe (DAISIE : Delivering Alien Invasive Species Inventories for Europe). The alien arthropod fauna is highly diverse with a total of 257 families but only 11 of these families contribute more than 30 species, including Scale Insects (Hemiptera: Coccomorpha) which constitute the second group in number of species. For obvious reasons, such information requires regular updates to reflect the dynamic nature of biological invasion. Based on a literature survey, an additional species list of alien scale insects is given here and main biogeographic patterns are presented. Alien scale insects in Europe belong mainly to three different families (Diaspididae, Pseudococcidae, and Coccidae). They come mainly from Asia, Central and South America but for about one third the origin remains unknown. Two European countries present a particularly high number of alien species (France, and Italy) lagging far behind Spain, Great Britain and Portugal. Some alien scale insects are phytophagous and often harmful for crops, some others are only present on ornamentals. Some monophagous species are only known in botanical gardens or strictly associated with their host-plant. Global trade is the major pathway of introduction and the main habitats colonized are cultivated lands, greenhouses, gardens and parks.

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Quantifying the impact of predator guild on California red scale in Spanish citrus

Bouvet, JPR12*, Urbaneja A2, Pérez-Hedo M2 and Monzó C2

1 Instituto Nacional de Tecnología Agropecuaria (INTA); Estación Experimental Agropecuaria (EEA) Concordia, Argentina 2 Instituto Valenciano de Investigaciones Agrarias (IVIA); Unidad Asociada de Entomología Agrícola UJI-IVIA: Centro de Protección Vegetal y Biotecnología, Spain

*Corresponding author: [email protected] Poster 123

The California red scale (CRS) Aonidiella aurantii (Hemiptera: Diaspididae) is key pest in many citrus areas worldwide. Biological control (BC) is frequently considered insufficient to keep populations under economic injury levels and therefore its control must mostly rely on insecticides. CRS chemical control may negatively affect citrus natural enemies of this and other pests. In the case of predators, the lack of information about this group of natural enemies has led to the misconception that they are not as successful as parasitoids. In this work, we evaluated the importance of the citrus predator guild on the regulation of CRS populations, through field experiments and molecular gut-content analysis, when insecticides do not disrupt their action. Exclusion techniques were carried out for two years and for the three annual generations of CRS in two pesticide-free citrus orchards. Our results showed that natural mortality of CRS was about 60% comprising 80% of this mortality to natural enemies. Surprisingly less than 2% was attributed to parasitoid, with the rest caused by predators. We identified around 50 species of predators present in the vicinity of the studied CRS infested spots. We used polymerase chain reaction (PCR) to determine whether CRS was part of the diet. Prey DNA of the predator gut content in field collected specimens of the 22 most abundant species was detected. We observed that 10 of those species had fed upon this pest. These results demonstrate that 1) the role of predators in the regulation of phytophagous arthropods in citrus orchards is underestimated by the lack of specific studies and 2) there are more BC agents involved on the regulation of this pest than was previously thought. The conservation of these BC agents should therefore be one of the cornerstones of any integrated citrus pest management program.

S7 PEST- DISEASE MANAGEMENT | 223

Dynamic monitoring (B versus Q) and further resistance status to insecticides of Q-type Bemisia tabaci in China

Huixin Zhen, Wen Xie*, Shaoli Wang, Qingjun Wu, Xiaomao Zhou, Youjun Zhang

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, China

*Corresponding author: [email protected] Poster 124

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) causes serious economic losses in many countries and has developed substantial resistance to commonly used insecticides. To determine whether the ratio of B. tabaci B to B. tabaci Q has continued to change in China, we collected specimens in 2013 and 2014 from most provinces in the country. Resistance to insecticides was also assessed in Q-type B.tabaci. B. tabaci Q remained much more abundant than B. tabaci B throughout China in 2013 and 2014, representing 82% of the specimens in 2013 and 88% in in 2014. B. tabaci B was mainly found in the southeast coast and Yangtze river basin. Abamectin remained highly toxic to B. tabaci adults, with LC50 values < 0.2 mg/l in 2013 and 2014. Thiamethoxam showed lower toxicity to HN and HB strains, with LC50 values > 500 mg/l in 2014. Although cyantraniliprole also remained highly toxic to pre-adults of B. tabaci, the resistance ratio (relative to the sensitive Lab-Q strain) increased to 27.80 for eggs and to 35.79 for larvae. B. tabaci Q continues to dominate in China. As of 2014, abamectin, cyantraniliprole, and pyriproxyfen were still effective against B. tabaci but moderate resistance to cyantraniliprole and pyriproxyfen was detected in some areas

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HPIS 2017

List of Participants

226 | LIST OF PARTICIPANTS

Name e-mail Page Abt, Isabelle [email protected] O-102, P-120, P-121 Aguirre-Rojas, Line [email protected] Albittar, Loulou [email protected] P-111 Allan, Sandra [email protected] P-66 Almeida, Rodrigo [email protected] O-34, O-35, P-38, P-44, P-125 Álvarez, Adriana Elisabet [email protected] P-61, P-64, O-182, P-213, P-217 Andre, Bart [email protected] Antolínez, Carlos Andrés [email protected] P-71 Arias, María [email protected] Avila, Carlos [email protected] P-122, P-201 Backus, Elaine [email protected] P-42, O-57, P-73 Badillo, Ismael [email protected] P-122 Bahar, Ofir [email protected] O-106 Bapela, Theresa [email protected] P-206 Bartelsmeier, Imke [email protected] P-195 Beal, Dylan [email protected] O-35, P-44, O-103 Behi, Fatma [email protected] P-67 Bello, Vinicius [email protected] P-85, P-130, P-169 Berthelot, Edwige [email protected] P-117 Blanco, Lidia [email protected] O-154 Bleeker, Petra [email protected] O-157 Boissinot, Sylvaine [email protected] O-97, P-124, P-128 Boissot, Nathalie [email protected] Bos, Jorunn [email protected] O-153, O-155, P-168, O- 187 Bosco, Domenico [email protected] O-36, P-39, O-103, P-119 Bosque-Pérez, Nilsa [email protected] O-96, O-100 Botha-Oberholster, Anna-Maria [email protected] O-23, P-86, P-205 Boulain, Helene [email protected] P-25 Bouvery, Nathalie [email protected] P-119 Bouvet, Juan Pedro [email protected] P-222 Brault, Veronique [email protected] O-97, P-124, P-128 Brentassi, Maria Eugenia [email protected] P-64 Bressan, Alberto [email protected] P-198 Brown, Judith [email protected] O-108 Burger, Francois [email protected] P-86 Campos, Juliana [email protected] P-194, P-210, P-211 Carvalho, Claudine [email protected] O-105, P-138 Chen, Julian [email protected] O-148, P-160, P-161 Clemente Orta, Gemma Maria [email protected] P-129 Coates, Laurynne [email protected] P-139 Corkill, Rebecca [email protected] P-62, O-150 Cornara, Daniele [email protected] P-47 Coustau, Christine [email protected] O-144 Dáder, Beatriz [email protected] P-123 de Cunha, Tatiane [email protected] O-191 de Freitas Bueno, Adeney [email protected] LIST OF PARTICIPANTS | 227

De Marchi, Bruno [email protected] P-26, P-85, P-169 De Paulo, Paula [email protected] P-60, P-194 Del Estal, Pedro [email protected] Deshoux, Maëlle P-121, P-171 Diergaarde, Paul [email protected] Drucker, Martin [email protected] O-95, P-109, P-117, P- 123 Dumont, Francois [email protected] P-203 Durak, Roma [email protected] P-170 Eakteiman, Galit [email protected] P-162 Egbon, Ikponmwosa [email protected] O-186 Elliot, Simon [email protected] O-105, P-138 Escudero-Martínez, Carmen [email protected] O-153, P-168 Espinosa, Pedro J [email protected] Esquivel Fariña, Arnaldo [email protected] P-133 Esteves, Mariana [email protected] P-40, P-41 Eubanks, Micky [email protected] O-99 Eveillard, Sandrine [email protected] P-126 Fan, Jia [email protected] O-148, P-160 Fecury Moura, Monika [email protected] P-113 Fereres, Alberto [email protected] O-37, P-43, P-47, O-51, P-60, P-71, P-134, P-135, P-194, P-215, P- 216 Francis, Frederic [email protected] P-68, P-69, P-160 Franco-Lara, Liliana [email protected] P-31, P-141 Gabrys, Beata [email protected] O-152, P-165, P-167, P- 172 Garms, Brian [email protected] Garzo, Elisa [email protected] P-47, P-215, P-216 Gauthier, Kevin [email protected] O-102, P-120, P-121 Gegechkori, Arnold [email protected] O-19, P-29 Germain, Jean- Francois [email protected] P-221 Ghanim, Murad [email protected] O-107, P-140, O-145 Glawe, Grit [email protected] Gravino, Matteo [email protected] P-159 Hanboonsong, Yupa [email protected] P-42, P-79, P-114, O-180 Hogenhout, Saskia [email protected] O-20, P-62, O-104, P-110, P-131, O-150, P-159, P- 164 Hu, Fang-yu [email protected] Jacquot, Emmanuel [email protected] O-102, P-120, P-121 Jairin, Jirapong [email protected] O-54 Jiménez, Jaime [email protected] O-51 Kaewmanee, Chananat [email protected] P-114 Kang, Zhiwei [email protected] O-58 Kaur, Navneet [email protected] P-112 Khelifa, Mounia [email protected] P-117, P-137 Kliot, Adi [email protected] O-104 Kloth, Karen [email protected] O-151 Komada, Karen [email protected] 228 | LIST OF PARTICIPANTS

Konkevic, Aleksandra [email protected] Kordan, Bozena [email protected] P-65 Krause-Sakate, Renate [email protected] P-26, P-85, P-113, P-130, P-169 Kuhn, Taciana Melissa [email protected] O-56 Kunert, Grit [email protected] O-146, P-166 Le Goff, Guillaume [email protected] P-81 Leszczynski, Bogumil [email protected] P-63 Levy, Amit [email protected] O-107, P-140 Leybourne, Daniel [email protected] O-187 Li-Long, Pan [email protected] O-93 Lim, Un Taek [email protected] O-189 Liu, Shu-Sheng [email protected] O-50, O-93 Liu, Tong-Xian [email protected] O-58, P-161, P-174 Lopes, Joao [email protected] P-40, P-41, O-56, P-134, P-135, P-136, P-212 López Gómez, Manuel [email protected] López, Agustín Ignacio [email protected] P-61 López-Moya Gómez, Juan José O-94 Lucatti, Alejandro [email protected] Ma, Ruiyan [email protected] Maixner, Michael [email protected] Malka, Osnat [email protected] O-21, O-158, P-162, P- 163 Maluta, Nathalie Kristine [email protected] P-134, P-135 Marchal Rubio, Francisco Javier [email protected] Marcos, María Ángeles [email protected] O-149 Martínez Ferrer, María Teresa [email protected] Martínez Puras, Victorino [email protected] Martínez, Luis Carlos lc.martinez@outlook P-194, P-210, P-211 Martínez-Torres, David [email protected] P-27, P-28, O-76 Marzachi, Cristina [email protected] O-103 Masten Milek, Tatjana [email protected] P-88 Mathers, Thomas [email protected] O-20, O-150 McGuire, Amber [email protected] P-200, P-218 Medina, Pilar [email protected] P-216 Mezei, Imre [email protected] O-188 Miranda, Marcelo [email protected] O-177, O-191 Miranda, Miguel Ángel [email protected] O-37 Mituti, Tatiana [email protected] P-134 Monsion, Baptiste P-128 Moraes, Leticia [email protected] P-85, P-130, P-169 Moreno , Aranzazu [email protected] P-43, P-47, O-51, P-71 Moreno, Ana Beatriz [email protected] O-94 Moreno-Delafuente, Ana [email protected] P-216 Morente Díaz, Marina [email protected] O-37, P-43, P-47 Morin, Shai [email protected] O-21, P-82, P-89, O-158, P-162, P-163 Morris, Jacqueline [email protected] O-78 Mugford, Sam [email protected] O-20, P-62, O-150, P- 159, P-164 LIST OF PARTICIPANTS | 229

Mwafaq, Ibdah [email protected] P-202 Natwick, Eric [email protected] P-132, P-214 Navas-Castillo, Jesús [email protected] O-92 Ontiveros, Irene [email protected] Pacheco, Inaiara [email protected] P-115 Pavon, Ricardo [email protected] Pierre, Eric [email protected] Pla, Sandra [email protected] Plat, Vincent [email protected] Plata Rueda, Rosa Angelica Plata. [email protected] P-210, P-211 Plaza, María [email protected] P-43 Pons Guillouard, Inés [email protected] O-77 Poveda, Pablo [email protected] Prade, Patricia [email protected] O-185 Prado, Simone [email protected] P-83, P-220 Prator, Cecilia [email protected] P-125 Prieto, Inés [email protected] P-215 Purcell Sandy [email protected] P-44 Raines, Tracy [email protected] P-200 Ramos, Anderson [email protected] P-136 Rashed, Arash [email protected] P-116, P-118 Razvan, Dumitru [email protected] P-198 Reynaud, Bernard [email protected] P-72 Rezende, Jorge Alberto Marques [email protected] P-133, P-134 Riolo, Paola [email protected] P-45, P-46, O-190 Rivera, Estefania [email protected] Rogers, Michael [email protected] P-173, O-176 Roldan, David [email protected] Rondon, Silvia [email protected] O-197 Royer, Tom [email protected] P-197 Ruschioni, Sara [email protected] P45-, P-46, O-191 Ryckebusch, Faustine [email protected] Sabaté, Jordi [email protected] Sadeh, Dganit [email protected] O-145 Sales, Tiago [email protected] P-40 Sanchez del Coso, Elena [email protected] Santos, Marcelo [email protected] O-59 Santos-Garcia, Diego [email protected] O-21, P-82, P-89, O-158 Sauvion, Nicolas [email protected] P-73 Schuurink, Robert [email protected] O-157, P-165 Senol, Özhan [email protected] P-24, P-30 Serteyn, Laurent [email protected] P-68, P-69, P-70 Shize, Zhang [email protected] P-174 Sicard, Anne [email protected] O-35, P-38 Simmons, Alvin [email protected] O-184 Sjölund, Jennifer [email protected] O-22 Skaljac, Marisa [email protected] P-80 Snoeren, Tjeerd [email protected] Srinivasan, Rajagopalbabu [email protected] O-183 Streito, Jean-Claude [email protected] 230 | LIST OF PARTICIPANTS

Su, Lujuan [email protected] Suzuki, Tatsuya [email protected] Swiegers, Hendrik [email protected] P-205 Szczepaniec, Adrianna [email protected] P-204 Tabuchi, Ken [email protected] O-192 Tan, Xiao-Ling [email protected] O-58, P-161 Tannieres, Melanie [email protected] Then, Christiane [email protected] O-95, P-109 Thompson, Gary [email protected] O-181 Tjallingii, Freddy [email protected] O-51, O-52, P-61 Tolmay, Vicki [email protected] P-206, P-209 Tomkins, Melissa [email protected] P-110 Torne Rifa, María [email protected] Tran, Hai [email protected] P-218 Torsten, Will [email protected] O-98 Trebicki, Piotr [email protected] O-100 Tsai, Chi-Wei [email protected] P-207 Turnbull, Colin [email protected] O-155 Tzin, Vered [email protected] O-147 Uzest, Marilyne [email protected] P-171 Vaello, Teresa [email protected] O-149 van Herwijnen, Zeger [email protected] van Kleeff, Paula [email protected] O-157, P-165 Vassão, Daniel [email protected] O-158, P-163 Venter, Eduard [email protected] O-156, P-193 Vercher, Rosa [email protected] P-219 Vereijssen, Jessica jessica.dohmen- P-87, O-178 [email protected] Villanueva Montiel, Francisco [email protected] Viñuela, Elisa [email protected] P-216 Walker, Gregory [email protected] O-53 Walsh, Doug [email protected] Wang, Xifeng [email protected] O-101 Wangkeeree, Jureemart [email protected] P-79 Watanabe, Luis Fernando [email protected] P-130, P-169 Wayadande, Astri [email protected] P-127 Webster, Kenneth [email protected] O-55 Weintraub, Phyllis [email protected] Wenninger, Erik [email protected] P-116, P-118 Wilson, Michael [email protected] O-18 Wintermantel, William [email protected] P-112, P-132, P-214 Wouters, Roland [email protected] P-131 Wozniak, Agnieszka [email protected] O-152, P-172 Wu, Qingjun [email protected] P-223 Xie, Wen [email protected] P-223 Xu, Baoyun [email protected] Zeilinger, Adam [email protected] O-35 Zhu, Jingyun [email protected] O-58, P-174