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D3.1 Report on Cultural, Biological, and Chemical Field Ref. Ares(2020)2181278 - 22/04/2020 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Deliverable Title Report on cultural, biological, and chemical field strategies for managing grapevine yellows, lethal yellowing and “huanglongbing” Deliverable Number Work Package D3.1 WP3 Lead Beneficiary Deliverable Author(S) IVIA Alejandro Tena Beneficiaries Deliverable Co-Author(S) ASSO Youri Uneau CICY Carlos Oropeza COLPO Carlos Fredy Ortiz IIF Martiza Luis SUN Johan Burger UP Kerstin Krüger Planned Delivery Date Actual Delivery Date 30/04/2020 22/04/2020 R Document, report (excluding periodic and final X reports) Type of deliverable DEC Websites, patents filing, press & media actions, videos E Ethycs PU Public X Dissemination Level CO Confidential, only for members of the consortium This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Table of contents List of figures 1 List of tables 5 List of acronyms and abbreviations 7 Executive summary 10 1. Strategies for managing “huanglongbing” in citrus 12 1.1. Africa and Europe: Trioza erytreae “huanglongbing” vector 12 1.1.1. Spain: native biological control agents of Trioza erytreae 12 1.1.2. Spain: classical biological control of Trioza erytreae 15 1.1.3. South Africa: conservation biological control of Trioza erytreae in 26 public areas 1.2. America: Diaphorina citri as vector of “huanglongbing” 30 1.2.1. Cuba: eradication and chemical control for “huanglongbing” 30 management 1.2.2. Guadeloupe: organic management of “huanglongbing” 34 2. Strategies for managing lethal yellowing in palms 41 2.1. Biological control agents of Haplaxius crudus, lethal yellowing vector in 41 America 2.1.1. Biological control agents of Haplaxius crudus in Mexico 41 2.1.2. Biological control agents of Haplaxius crudus in Cuba 45 2.2. Resistant varieties of coconut 46 3. Strategies for managing grapevine yellows 51 3.1. South Africa: biological control agents of Mgenia fuscovaria, a leafhopper 51 vector of aster yellows phytoplasma 3.2. South Africa: ecology and management of Mgenia fuscovaria 52 4. Antimicrobial peptides (AMPs) against grapevine yellows 59 5. References 65 ANNEX A: Sites sampled to study the parasitoid complex of Trioza erytreae in 71 South Africa ANNEX B: Protocol for coconut palm micropropagated plants acclimatization 74 ANNEX C: Seasonal plan for aster yellows management in the Western Cape 75 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 List of figures Figure 1: Parasitized nymphs of Trioza erytreae. Left: the red arrow shows 13 the presence of a parasitoid egg above a nymph of T. erytreae. Center: larvae of a parasitoid feeding on T. erytreae. Right: T. erytrae nymph with a parasitoid pupa inside. Figure 2: Details of the sampling protocol (stem-taps) to collect predators 14 in the field. Figure 3: Relative abundance of Trioza erytreae parasitoids collected from 19 individual parasitized nymphs at four sites in South Africa in 2017. Figure 4: Effect of Trioza erytreae size on the probability that an individual 20 of Syrphophagus cassatus emerge from the nymph. Figure 5: Dispersion of the parasitoid Tamarixia dryi in the 257 locations 23 sampled in the Canary Islands between 2018 and 2019. White dots: locations without Trioza erytreae and T. dryi. Red dots: locations with T. erytreae but without T. dryi. Green dots: locations with T. erytreae and T. dryi. Figure 6: Seasonal trend and parasitism rates of Trioza erytreae after the 24 release of the parasitoid Tamarixia dryi in the Canary Islands in 2019. A) Seasonal trend presented as percentage of each instar. B) Seasonal trend presented as mean (±SE) number of instars suitable of parasitism (from 2nd to 5th instar: N2-N5). C) Parasitism rates (mean ± SE). Left panels: citrus orchards in Tenerife, right panels: citrus orchards in Gran Canaria. Figure 7: Sampling sites in Gauteng and Mpumalanga in South Africa in 26 private gardens (yellow circles), experimental farms or research areas (green circles), and a commercial farm (red circle). Figure 8: Percentage of parasitism of T. erytreae at the University of 29 Pretoria Experimental Farm between June and September 2018. Figure 9: Daily minimum and maximum temperatures and rainfall at the 29 University of Pretoria Experimental Farm between June and September 2018. Figure 10: Percentage of parasitism of weekly sampling at the University of 30 Pretoria Experimental Farm and at a garden in Groenkloof (Pretoria) in September 2018. Figure 11: Typical blotchy mottle symptoms associated with HLB: 1, 31 grapefruit and 2, sweet orange. Figure 12: Citrus plants after the foliar application of kaolin. 32 1 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Figure 13: Incidence of HLB in citrus orchards with (A) and without (B) 33 eradication programs in Cuba from May 2017 to November 2019. Figure 14: Location of the four experimental citrus orchards in Guadeloupe. 35 Figure 15: Relative abundance of Diaphorina citri in citrus orchards of 38 assay 1. Months without data were not sampled. Figure 16: Relative abundance of Diaphorina citri in citrus orchards of 38 assay 2. Months without data were not sampled. Figure 17: HLB infection rates in the four experimental citrus orchards in 39 Guadeloupe in 2017, 2018 and 2019. Figure 18: Percentage of trees dead during the assay in the four 39 experimental citrus orchards in Guadeloupe in 2017, 2018 and 2019. Figure 19: Scheme of the IPM program developed by ASSO to be followed 40 in Guadeloupe to produce citrus. Figure 20: Scheme of the organic program developed by ASSO to be 40 followed in Guadeloupe to produce citrus. Figure 21: Derbide insect (Persis foveastis) naturally infected by fungi 42 found on a coconut leaf. Figure 22: Thiodina sp. jumping spider with adult insect in its mouth on a 43 coconut palm leaf. Figure 23: A Theridion sp. spider and a Haplaxius crudus insect trapped in 44 a web built by the spider on a coconut leaf (A). A close-up of the leafhopper trapped in the web (B). Two other leafhoppers already trapped in webs and the spiders nearby (C, D). Figure 24: Occurrence of lethal yellowing in Latin America and the 46 Caribbean (highlighted in yellow). Red arrows show the path, direction and current limits of spread. Figure 25: Preparation of coconut in vitro plants for shipment. Transfer from 48 glass containers (A) to plastic containers (B, C). When in vitro plants are ready (D) they are boxed (E) before shipment. Figure 26: LY-resistant coconut plants produced in vitro at CICY. After 49 arrival at the destination, within containers (A) and outside after washing (B). Bags with substrate mixture (C). In vitro plants placed within bags (D, E), ready for acclimatization in greenhouse (F, G) and after 15 days of acclimatization (H) (Photos by CIB, IIFT, CICY). 2 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Figure 27: Map of South African provinces and distribution of the aster 52 yellows phytoplasma in the Western Cape province in South Africa (red circles). Figure 28: An adult Mgenia fuscovaria feeding on a young grapevine shoot 53 in spring (A), symptoms of aster yellows phytoplasma in grapevine (B) and a close up of the symptoms in the leaves (C). Figure 29: Adult Mgenia fuscovaria, dorsal view (A) and female lateral view 54 (B). (M. Stiller, Agricultural Research Country-Plant Protection Research). Figure 30: Examples of food plants of Mgenia fuscovaria in vineyards. 54 Urtica sp. (Urticacea) (A), Lamium amplexicaule (Lamiaceae) (B), and Oxalis pes-caprae (Oxalidaceae) (C). Figure 31: Seasonal abundance of Mgenia fuscovaria in a vineyard infected 55 with aster yellows phytoplasma from November 2009 to December 2016 and following a prolonged draught from January 2017 to December 2019 (A). Temperature and precipitation during the past 30 years in Vredendal (source: en.climate- data.org) and grapevine phenology (B). Figure 32: Examples of alternative plant hosts of aster yellows 56 phytoplasma. Triticale (A), a cover crop in vineyards, maize (B) and young grapevine plants interplanted in vineyards, and Mesembryanthemum crystallinum (C) in the natural vegetation of the Western Cape. Figure 33: Monitoring of Mgenia fuscovaria with yellow sticky traps (A, B). 57 Figure 34: a) Vector map for the expression vector pRSF with GFP, WelQut 61 site (w) and the peptide b) example of His-tag purification of the lysate. Figure 35: SDS-PAGE gel showing the comparison between peptides 61 digested with WelQut and undigested peptides. Lane M – Color prestained protein standard ladder (11–245 kDa). Lane 1 – Control. Lane 2 – Vv-AMP1 digested. Lane 3 – SN-1 potato digested (peptide in red square). Lane 4 – SN-1 grape digested. Lane 5 – Vv-AMP1 undigested. Lane 6 – SN-1 potato undigested. Lane 7 – SN-1 grapevine undigested. Figure 36: Schematic representation of recombinant expression vectors. 63 (A) pRSF GFPwThrombin(long)-Vv-AMP1; (B) pRSF GFPwThrombin(short)-Vv-AMP1; and (C) pRSF GFPwNisP-Vv- AMP1. 3 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459 Figure 37: Tricine-SDS-PAGE (15% Tris-Tricine gel) analysis by 64 Coomassie blue staining of non-digested and digested protein samples. Lane M: 10-250 kDa molecular weight standard; Lanes 1-3: non-digested protein samples; Lanes 5-7: protease digested protein samples. The red arrow indicates the released Vv-AMP1 peptide (about 5.5 kDa). Figure 38: VvAMP1 activity against F. oxysporum. Micro- 64 spectrophotometric readings were recorded at 24 and 48 hours.
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  • The Ohio Journal of Science — Index 1951-1970
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