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
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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).
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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.
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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. Different concentrations of thrombin were tested 600 U (60), 300 U (30) and 150 U (15). The non-digested peptide was used as control (non-dig VVamp1). Figure 39: Schematic representation of recombinant expression vector with 64 accessory protein PLaC.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
List of tables
Table 1: Location and characteristics of the eight citrus orchards sampled 13 between 2017 and 2018 in Canary Islands. Table 2: Selection criteria and psyllid species used for host specificity 17 testing of Tamarixia dryi together with host plant species and experiment numbers. Table 3: Parasitism and hyperparasitism rates of Trioza erytreae in four 20 locations in South Africa. Table 4: Number of parasitized psyllids (mean ± SE) after exposure to 21 the parasitoid Tamarixia dryi during 48 hours. The number of replicates and the mean number of psyllids (± SE) per replicate are also provided. Psyllid hosts in the control treatment were not exposed to T. dryi. Table 5: Number of parasitized psyllids (mean ± SE) and their mortality 22 (mean % ± SE) after removal from their galls and exposure to the parasitoid Tamarixia dryi during 48 hours. The number of replicates and the mean number of psyllids (± SE) per replicate are also provided. Psyllid hosts in the control treatment were not exposed to T. dryi. Table 6: Locations with Trioza erytreae and its parasitoid Tamarixia dryi 23 in the Canary Islands between 2018 and 2019 after the release of the parasitoid. Table 7: Sampling sites, number of adults collected, and psyllids and 28 parasitoids emerged from psyllid nymphs on Citrus spp. in Gauteng and Mpumalanga from March (autumn) to mid-June (winter) 2018. Table 8: Citrus orchards selected to evaluate the temporal increasing of 32 HLB with and without eradication of infected trees. Table 9: Mean number of individuals of D. citri / shoot / tree observed 34 during the period 2018-2019 on trees treated with kaolin and untreated. Kaolin was sprayed in March, May and September. Table 10: Summary of the main characteristics of the experimental plots in 36 Guadeloupe. Table 11: Treatments applied in BIO and IPM management systems in 37 Guadeloupe. Table 12: Mean mycelial growth radius (mm ± standard deviation) of 13 43 Beauveria bassiana and two Metarhizium anisolpliae isolates cultured in vitro at three temperatures.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Table 13: Inventory of generalist predators in the yellow sticky traps in 45 Cuba from February 2019 to 2020. Table 14: Sites in Mexico with lethal yellowing resistant or presumably 47 resistant coconut genotypes and current status.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
List of acronyms and abbreviations
AMPs antimicrobial peptides Apr April ARC Agricultural Research Council ASSO ASSOFWI Aug August AY aster yellows B. bassiana Beauveria bassiana BLAST Basic Local Alignment Search Tool CIB Coconut Industry Board CICY Centro de Investigación Científica de Yucatán ‘Ca. L. asiaticus’ ‘Candidatus Liberibacter asiaticus’ ‘Ca. P. asteris’ ‘Candidatus Phytoplasma asteris’ ‘Ca. P. rubi’ ‘Candidatus Phytoplasma rubi’ ‘Ca. P. solani’ ‘Candidatus Phytoplasma solani’ C. deliciosa Citrus deliciosa C. paradisi Citrus paradisi C. reticulata Citrus reticulata C. sinensis Citrus sinensis C. volkameriana Citrus volkameriana C. carnea Chrysoperla carnea CLas ‘Candidatus Liberibacter asiaticus’ C. canariensis Convolvulus canariensis CRI Citrus Research International cv. cultivar Dec December DNase Deoxyribonuclease DTT Dithiothreitol E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid Feb February
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
g gram GFP Green fluorescent protein GY Grapevine yellows H. crudus Haplaxius crudus HLB Huanglongbing ICIA Instituto Canario de Investigaciones Agrarias HCl Hydrogen chloride i.e. idem est IIFT Instituto de Investigaciones en Fruticultura Tropical IPM Integrated Pest Management IPTG Isopropyl β- d-1-thiogalactopyranoside IVIA Instituto Valenciano de Investigaciones Agrarias Jan January kDa kilodalton L. Linnaeus LGD local green dwarf LY lethal yellowing M. anisopliae Metarhizium anisopliae Mar March M. fuscovaria Mgenia fuscovaria mg milligram ml milliliter mm millimeter mM millimolar NaCl sodium cloride Ni-NTA nickel-nitrilotriacetic agarose Nov November NTPs non-target psyllids Oct October PCR polymerase chain reaction P. trifoliata Poncirus trifoliata
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
P. pulvinatus Psyllaephagus pulvinatus Rnase ribonuclease rpm revolution per minute SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis SE standard error Sep September sp. species spp. multiple species SUN Stellenbosch University T. dryi Tamarixia dryi T. erytreae Trioza erytreae U units UP University of Pretoria V volt µl microliter µg microgram °C degree Celsius
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Executive summary
Different control methods against lethal yellowing (LY) in coconut palms (Cocos nucifera), citrus greening or “huanglongbing” (HLB) in citrus (Citrus spp.) and aster yellows (AY) in grapevine (Vitis vinifera) and their insects vectors were developed in the countries affected by the respective diseases or with the presence of their insect vectors. As with the definition of Integrated Pest Management (IPM), the methods developed and/or tested under the TROPICSAFE project were different also depending on the status and knowledge of the diseases in each country. To manage HLB in citrus in Europe, a classical biological control program was developed to introduce the parasitoid Tamarixia dryi in mainland Europe to reduce the spread of Trioza erytreae, the vector of HLB. The parasitoid was collected in South Africa, introduced, reared and released in the Canary Islands and Galicia in Spain, and in Portugal after demonstrating in the Canary Islands that its introduction should not have a significant negative environmental impact. In South Africa the parasitoid complex of T. erytreae was determined and it showed that there are three species of primary parasitoids, contrary to previous studies where two primary parasitoids were found. In Cuba and Guadeloupe HLB is transmitted by the psyllid Diaphorina citri and the disease is widely spread, therefore several strategies were tested: the use of kaolin to control the psyllid vector, the elimination of infected trees and the development of an organic management strategy. The elimination of infected trees in a wide area resulted the most promising strategy in these islands. To manage grapevine yellows (GY) disease in South Africa, an IPM program was developed based on the results obtained under TROPICSAFE (seasonal trend of the vector, natural enemies and host plant species of the associated phytoplasma), and on previous knowledge of the disease and its main insect vector, Mgenia fuscovaria. This program incorporates multiple strategies that include monitoring, weed management and chemical control. Moreover, four plant-derived antimicrobial peptides (AMPs) were selected to verify their potential use to control the disease in grapevine: VvAMP1, SN1pot, SN1vitis and VvScorpio. The four AMPs were cloned as fusions with green fluorescent protein (GFP) into bacterial expression vectors, and expressed in Escherichia coli. All four peptides were cleaved from GFP using thrombin, and purified using the his-tag purification method and the size exclusion chromatography. Despite some technical difficulties, adequate quantities of the peptides are now available for efficacy screening against the grapevine yellows- associated phytoplasma. The most promising strategy to manage LY in coconuts in America is the use of resistant varieties. This technique has been successfully used in Mexico in the past. Within TROPICSAFE, micropropagation protocols were developed to propagate LY-resistant germplasm in large scale, package and send to other partner countries. Resistant material was sent from Mexico to Cuba and Jamaica. Moreover, surveys to identify the main natural enemies of the cixiid vector of LY, Haplaxius crudus, were carried out in Cuba and Mexico. The low abundance of the vector did not allow to identify potential biological control agents that could be used in IPM programs.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Overall, the most promising management methods developed within the project are: i) the introduction of T. dryi in the Canary Islands (Spain) against T. erytreae; ii) the elimination of HLB-infected trees in Cuba; iii) the development of an IPM program against grapevine yellows in South Africa; iv) and the use of LY-resistant germplasm in America. These methods have been made available to WP5 (Economical sustainability and social sciences) partners for the evaluation of their socio-economical sustainability and applicability. No delays for this deliverable are reported.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
1. Strategies for managing “huanglongbing” in citrus
1.1. Africa and Europe: Trioza erytreae “huanglongbing” vector
1.1.1. Spain: native biological control agents of Trioza erytreae
Introduction Hymenopteran parasitoids are the main natural enemies of Trioza erytreae in its area of origin Sub-Saharan, Africa (Cocuzza et al., 2017). There, the parasitoid Tamarixia dryi (=Tetrastichus dryi) (Waterston) (Hymenoptera: Eulophidae) is widespread and has been reported as the most common and effective parasitoid of T. erytreae (Catling, 1969; McDaniel and Moran, 1972; van den Berg and Greenland, 2000; Tamesse et al., 2002). Psyllaephagus pulvinatus Waterston (Hymenoptera: Encyrtidae) is also an important parasitoid of the psyllid, especially in Cameroon (Tamesse et al., 2002). These two parasitoids are attacked by a complex of hyperparasitoids that reduce the efficacy of the two primary parasitoids as biological control agents of the psyllid (Mc Daniel and Moran, 1972; Tamesse et al., 2002). T. erytreae is also attacked by a complex of generalist predators that feed mostly on their eggs and nymphs. In general, chrysopids have been recorded as the most active predators followed by syrphids and coccinellis in Africa. A preliminary survey carried out in Madeira and Canary Islands also found coccinellids, spiders and lacewings preying on the psyllid (González-Hernández, 2003). Since T. erytreae arrived to Spain, its complex of natural enemies has never been studied in detail and it is unknown whether native parasitoids are attacking it, or whether the complex of predators that attack other citrus pests are now feeding on T. erytreae. The aim of this task was to determine the complex of parasitoids and predators that attack T. erytreae in the Canary Islands (Spain). These results will be useful to design conservation biological control strategies and to identify potential problems between native fauna and introduced biological control agents.
Material and Methods Citrus orchards. Eight commercial citrus orchards, which presented visual evidence of T. erytreae infestation during the previous seasons, were sampled in different areas of Canary Islands from 2017 to 2018. The sampling orchards ranged from 0.3 to 16 ha, their location, height, orientation and the varieties planted are shown in Table 1. These are the same orchards sampled in Deliverable 3.2. to determine the seasonal trend of the vector. Sampling protocols. To determine the parasitoid complex of T. erytreae, 50 shoots about 20 cm-long were randomly collected monthly in each of the eight orchards. Samples were bagged and transported to the laboratory inside a portable cooler. Each shoot was observed under a stereomicroscope to determine the presence of psyllid nymphs. These were carefully observed to determine the presence of eggs, larvae or pupae of parasitoids (Figure 1).
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The methodology described by Monzó et al. (2014) was adapted to determine the predator complex of T. erytreae. Predators were monitored monthly by stem-tap on one side of 20 randomly selected trees in each of the eight orchards. A 30 × 45 cm plastic tray was held horizontally about 30 cm underneath a randomly chosen branch, which was then struck sharply three times with a pipe (Figure 2). Predators falling on the tray were moved to dram containers by an entomological aspirator. The containers were filled with 80% ethanol and transported to the laboratory, where predators were identified and counted.
Table 1: Location and characteristics of the eight citrus orchards sampled between 2017 and 2018 in Canary Islands. Location Island Locality Citrus variety X Y Z Orientation Oranges La Laguna 363374 3156354 110 N - E (Washington Navel and Valencia Late)
Oranges La Orotava 349326 3141620 245 N (Navelina, Washington Tenerife Navel and Valencia Late) Oranges San Miguel 341411 3108187 470 S (Washington Navel)
Oranges Buenavista 317600 3138975 116 N - O (Navelina and Washington Navel)
Santa Brígida 451011 3100599 528 N - E
Gran Telde 454931 3098688 294 E Oranges Canaria Agaete 434170 3105777 245 N - O (Washington Navel)
Moya 442133 3107133 721 N
Figure 1: Parasitized nymphs of Trioza erytreae. Left: the red arrow shows 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.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Figure 2: Details of the sampling protocol (stem-taps) to collect predators in the field.
Results A total of 4,116 nymphs of T. erytreae suitable for parasitism (2nd to 5th instar) were observed in the eight citrus orchards between 2017 and 2018. None of these nymphs had signs of parasitism. A total of 7,914 potential predators of T. erytreae were collected between 2017 and 2018. Spiders (4,521 specimens; 57.1%) and ants (2,815 specimens; 35.6%) were the most abundant, but they were not identified because of their controversial role as predators of psyllids. The other 578 specimens belonged to the order Neuroptera (278 specimens; 3.5%), Coleoptera (268 specimens; 3.4), Hemiptera (28 specimens; 0.4%) and Diptera (4 specimens; 0.05%). Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), with 260 specimens, was the most abundant predator and it was present throughout the year. It was followed by the coccinelids Sthetorus wollastoni Kapur (95 specimens), Scymnus canariensis Wollaston (54 specimens) and Delphastus catalinae (Horn) (44 specimens) (Coleoptera: Coccinelidae). A total of 13 species of coccinelids were identified. All the hemipterans (28 specimens) were identified as the anthocorid Orius albidipennis (Say) (Hemiptera: Anthocoridae). Finally, four specimens of syrphidae were identified as Myathropa florea (Linnaeus) (Diptera: Shyrphidae).
Conclusion These results indicate that T. erytreae is not parasitized by any parasitoid species in the Canary Islands more than 15 years after its introduction. Therefore, the introduction of the parasitoid T. dryi from South Africa will not affect potential native parasitoids of T. erytreae. Moreover, its introduction will not be negatively affected by the potential competition with native parasitoids. Except ants and spiders, the number of predators collected was very low compared with other studies carried out in American citrus orchards and the psyllid Diaphorina citri (Qureshi and Stansly, 2009; Monzó et al., 2014). Therefore, the role of predators as biological control agents of T. erytreae seems very limited. Among them, only the generalist predator C. carnea was widely distributed throughout the year. Therefore, the introduction of the parasitoid T. dryi is highly justified in order to control T. erytreae populations.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
1.1.2. Spain: classical biological control of Trioza erytreae
Introduction The eradication of T. erytreae in mainland Europe is impossible because the psyllid has already spread through the west of the Iberian Peninsula, from Galicia in Spain to Lisbon in Portugal. Therefore, a series of strategies need to be implemented urgently. Nowadays, classical biological control is the most feasible approach because no native parasitoids have been recovered from T. erytreae during different surveys carried out in Europe (Cocuzza et al., 2017). The complex of parasitoids in South Africa and Swaziland (now Eswatini) was analyzed in detail during the 1960’s and 70’s (Catling, 1973; Mc Daniel and Moran, 1972) and in Cameroon twenty years ago (Tamesse et al., 2002). According to these studies, the two main primary parasitoids of T. erytreae in Southern Africa are T. dryi and P. pulvinatus. Both are solitary koinobiont parasitoids. The former is an ectoparasitoid, whereas the encyrtid is an endoparasitoid. These primary parasitoids are frequently attacked by a complex of hyperparasitoids (van der Berg and Greenland, 2000; Tamesse et al., 2002) that, accordingly to van der Berg and Greenland (2000), severely decrease the impact of the primary parasitoids. T. dryi was used in a classical biological control program in Reunion Island when T. erytreae was detected in 1974 (Etienne and Aubert, 1980; Aubert et al., 1980). In less than five years, T. dryi became established and controlled T. erytreae (Aubert and Quilici, 1988). Similarly, in 1982, T. dryi was imported into Mauritius. Since then, the parasitoid complex of T. erytreae has not been studied and several aspects of its biology are still unknown. Therefore, the parasitoid complex of T. erytreae was identified in several areas of South Africa and several aspects of the biology of the main parasitoids such as sex ratio, longevity and hyperparasitism were determined (Pérez-Rodríguez et al., 2019). Taking into consideration the results of the previous surveys and studies, the Instituto Valenciano de Investigaciones Agrarias (IVIA) in collaboration with the Instituto Canario de Investigaciones Agrarias (ICIA) applied for the permits to introduce T. dryi into the Canary Islands and mainland Spain. The Ministry of Agriculture allowed the introduction of the parasitoid in the Canary Islands in 2017 to evaluate its host-specificity before its release in the field. This is because, in the past, several introductions of parasitoids had undesirable negative effects on non-target insects (EPPO, 2014; Heimpel and Mills, 2017). Therefore, the host specificity of the parasitoid T. dryi was tested. For this purpose, 11 non-target psyllids (NTPs) were selected based on their phylogenetic proximity to T. erytreae and for ecological reasons such as sharing similar host resources (Urbaneja-Bernat et al., 2019). After these studies the parasitoid T. dryi was released in the Island of Tenerife in the spring of 2018 and its spread and efficacy were evaluated in the field to determine whether the parasitoid established and controlled the pysllid.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Material and Methods Parasitoid complex of T. erytreae in South Africa. Sampling took place in citrus producing areas in the four provinces of Gauteng, Limpopo, Mpumalanga and Western Cape in South Africa. A total of 65 citrus orchards, five public parks and 60 private properties were examined from September 21st to December 9th 2017. At all sites, the sampling date, the number of trees sampled, the variety of the trees and the presence of T. erytreae or its symptoms and of presence of parasitoids were recorded (Annex A). From the areas and the trees where T. erytreae was collected, the psyllids were transported to the laboratory to identify the presence of potential parasitoids, determine their relative abundance and parasitism rates in each location. From each tree, 3 to 20 leaves infested by T. erytreae were collected and transported in closed individual plastic bags to the laboratory where the number of alive psyllids and psyllids suitable for parasitism and parasitized was recorded after observation using a stereomicroscope. In order to identify all the parasitoids and calculate the rate of emerging parasitoids, psyllid nymphs were placed individually in 1 ml microtubes closed with cotton wool. Afterwards, the microtubes were kept in an incubator (LabconTM 2 LTGC 20, Laboratory Marketing Services cc, South Africa) under controlled conditions (12 hours light, 12 hours dark, 25°C, 60–70% relative humidity) and checked daily until the parasitoids emerged. Once emerged, their sex was determined and the identification at the species level was carried out. The morphological identification of T. erytreae and Tamarixia spp. parasitoids were confirmed by David Ouvrard (Natural History Museum, London) and Roger Burks (University of California, Riverside), respectively. In order to calculate the parasitism rate, each tree was used as a sampling unit because T. erytreae, as other psyllids, has an aggregative distribution pattern. The effects of host size on the secondary sex ratio of T. dryi and Tamarixia sp. and on hyperparasitism were also analyzed. After the parasitoids emerged, they were identified and their sex was determined. The psyllid nymphs have an oval shape and the host size was determined by calculating the area of an ellipse by multiplying r1 × r2 × π (r1: major radius, r2: minor radius). Both sex and hyperparasitism ratios were analyzed using general linear models assuming binomial errors. The assumed error structure was assessed by a heterogeneity factor equal to the residual deviance divided by the residual degrees of freedom. If an over- or an under dispersion was detected the significance of the explanatory variables was re-evaluated using an F-test after rescaling the statistical model by a Pearson’s χ2 divided by the residual degrees of freedom. All the data analyses were performed with the R freeware statistical package (Version 1.0.143). Host range testing of T. dryi. Host range tests were carried out at the University of Pretoria (South Africa) and the “Instituto Canario de Investigaciones Agrarias” (ICIA) (Tenerife, Spain). Two colonies of T. erytreae were established in each location using young lemon trees that were pruned before the infestation. Multiple criteria were used for the selection of 11 representative non-target psyllid species (NTP) for host specificity testing (Table 2). NTPs were collected from their respective host
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
plant species by cutting the twigs or the leaves where they were settled. Twigs and leaves were transported to the laboratory and those with nymphs were used in the experiments.
Table 2: Selection criteria and psyllid species used for host specificity testing of Tamarixia dryi together with host plant species and experiment numbers. Selection criteria Psyllid species and family Host plant species Experiment
Target pest species Trioza erytreae (Triozidae) Citrus limon 1*
Trioza alacris (Triozidae) Laurus nobilis 1, 2**
Trioza laurisilvae (Triozidae) Laurus azorica 1, 2 Close phylogenetic relatedness to T. Trioza sp. I (Triozidae) Withania aristata 1 erytreae Trioza sp. II (Triozidae) Convolvulus floridus 1, 2
Trioza sp. III (Triozidae) Convolvulus canariensis 1, 2
Native host plant Agonoscesna sp. (Psyllidae) Ruta pinnata 1, 2 related to citrus
Bactericera tremblayi Allium ampeloprasum 1 (Triozidae)
Euphyllura olivina (Psyllidae) Olea europea 1 High probability of occurrence in native Ctenarytaina eucalypti Eucalyptus globulus 1 vegetation (Psyllidae) surrounding the Glycaspis brimblecombei citrus groves Eucalyptus camaldulensis 1, 2 (Psyllidae)
Spondyliaspis plicatuloides Eucalyptus spp. 1 (Psyllidae)
* Naturally settled on leaves/galls. **Removed from galls and exposed to parasitoids on the leaves
In South Africa, the parasitoid T. dryi was obtained directly from parasitized nymphs of T. erytreae collected in a citrus orchard at the Experimental Farm of the University of Pretoria. In the Canary Islands, a colony of T. dryi sourced from South Africa was established using the colonies of T. erytreae. In both South Africa and Spain, one newly emerged female and male less than 24 hours old were introduced into a 2 ml glass vial, with a cotton wool cover and one drop of sucrose (South Africa) or honey (Canary Islands). After 24 hours the males were removed from the vial. The females were kept in the vials for additional two to three days before they were used in the experiments. Experiment 1. Host range experiment on naturally settled nymphs. To determine whether T. dryi parasitizes or kills the selected NTPs, psyllid nymphs were offered to single female parasitoids over a period of 48 hours (Table 2). A single female was released into a 100 ml glass vial containing one drop of honey and leaves infested with psyllids before closing the
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
vial with muslin. Each glass vial contained between one and three leaves infested with the corresponding NTPs. The number of alive, dead and parasitized psyllids were recorded after 48 hours. Experiment 2. Host range on nymphs removed from their galls and exposed to T. dryi. Experiments with psyllid species that settled and rolled leaves or produced galls that were apparently inaccessible to parasitoids were carried out as described above with healthy leaves (Table 2). To avoid breaking their fragile mouthparts, the rolled leaves or galls were opened and nymphs in the 3rd to 5th instar that were walking after being gently disturbed with a fine hair paint brush were carefully transferred to new healthy leaves. Nymphs were left for 24 hours to settle and feed on the new leaf. These new leaves were kept in 100 ml glass tubes with one drop of honey and closed with a muslin cover for ventilation. Then the same methodology described for the naturally exposed psyllid nymphs was followed. The mean number of nymphs exposed to parasitoids ranged between 10 and 23 nymphs and the experiment was replicated between 15 and 20 times (Table 4). Experiments 1 and 2 were carried out in parallel and the same controls were, therefore, used for both experiments (Tables 2). Dispersion of Tamarixia dryi and its effect on Trioza erytreae in Canary Islands. Dispersion. With the collaboration of ICIA and “Servicios de Sanidad Vegetal de Canarias”, 257 citrus orchards or gardens with citrus trees were sampled between September 2018 and March 2019 to determine the dispersion of T. dryi in the Canary Islands. The number of orchards or gardens are presented in Table 6 and in Figure 3. In each location, six shoots infested with T. erytreae, if present, were collected and transported to the laboratory within a plastic bag labeled with date and location. Once in the laboratory, shoots were observed under a binocular microscope and the following data were recorded: number of leaves / shoots, number of leaves with psyllids symptoms / shoots, number of nymphs alive or parasitized. Shoots were divided between shoots from fall and winter. Then, shoots were placed in a box and kept under controlled conditions (12 hours light: 12 hours dark, 25°C, 60–70% relative humidity) during 30 days and checked daily until the parasitoids emerged. With these data, the proportion of locations with T. dryi in the flush of fall and winter-spring was calculated. Parasitism. The effect of T. dryi on T. erytreae was studied in two pesticide-free lemon orchards located in the Canary Islands. Both plantations were infested with T. erytreae and were located in two different islands: Tenerife (x: 349442, y: 3140170) and Gran Canaria (x: 434131, y: 3105910). From March to September 2019, 12 infested trees were sampled once per week. In each tree, four 30 cm infested shoots belonging to a different cardinal orientation were randomly collected. All samples were placed in individual plastic bags, enclosed and transported in a cooler to the laboratory. Within the next 24 hours, four leaves per shoot were selected and examined under a stereomicroscope: one from the apical part of the shoot, two from the middle part and one from the last 30 cm of the shoot. All T. erytreae instar nymphs and stages were counted from eggs to adults. In order to calculate the parasitism rate of T. dryi, one T. erytreae infested leaf per tree was selected randomly. These leaves were not the ones considered previously for the analysis of the seasonal
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
trend and were infested with suitable psyllids for parasitism (from 2nd to 5th instar nymph), therefore they were not randomly selected. The number of suitable nymphs and of the parasitized nymphs out of the total was recorded. After counting, each leaf was embedded in a moist cotton, individualized in plastic tubes with a drop of honey and closed with a mesh cover. These plastic tubes were kept under controlled conditions (12 hours light: 12 hours dark, 25°C, 60–70% relative humidity) during 30 days and checked daily until the parasitoids emerged. Once emerged, they were identified to confirm that were T. dryi.
Results Parasitoid complex of T. erytreae in South Africa. A total of 580 parasitized T. erytreae individuals were collected during the survey. From these samples 334 parasitoids belonging to five species emerged. The parasitoids in the remaining 246 psyllids failed to develop. The parasitoid complex was composed of at least five species whose relative abundance varied with the sampling site (Figure 3). T. dryi was the most abundant primary parasitoid in Pretoria and Nelspruit (more than 95% of the emerged parasitoids). This parasitoid species was also present in Tzaneen. On the other hand, the primary parasitoids P. pulvinatus (79%) and Tamarixia sp. (65%) were the most abundant species in Nelspruit and Tzaneen, respectively. The most abundant hyperparasitoid was Aphidencyrtus cassatus Annecke (Hymenoptera: Encyrtidae), which was recovered in Nelspruit, Tzaneen and Pretoria. One specimen of an Aphanogmus sp. (Hymenoptera: Ceraphronidae) was recorded in Tzaneen.
Figure 3: Relative abundance of Trioza erytreae parasitoids collected from individual parasitized nymphs at four sites in South Africa in 2017.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Table 3: Parasitism and hyperparasitism rates of Trioza erytreae in four locations in South Africa. Parasitism assessment Number of Hyperparasitism Date Location Parasitism rate nymphs rate [mean ± EE (n)] examined [mean ± EE (n)]
09/28/2017 Nelspruit- CRI* 72 0.45 ± 0.19 (5) 0 (5)
09/29/2017 Nelspruit- ARC* 266 0.41 ± 0.21 (4) 0.09 ± 0.09 (4)
10/05/2017 Tzaneen 742 0.42 ± 0.07 (3) 0.08 ± 0.04 (3)
10/05/2017 Pretoria(University) 142 0.72 ± 0.12 (5) 0.02 ± 0.003(5)
*CRI: Citrus Research International (Nelspruit) *ARC: Agricultural Research Council (Nelspruit)
Parasitism rates ranged between 0.72 ± 0.12 in Pretoria and 0.41 ± 0.21 in Nelspruit (Table 3). Hyperparasitism rates were low and ranged between 0 and 0.09. The secondary sex ratio of the primary parasitoids T. dryi and Tamarixia sp. depended on T. erytreae size. In both species, females emerged from larger-sized hosts than males (T. dryi: F = −3.34; df = 1, 86; P < 0.001; Tamarixia sp.: F = −3.99; df = 1, 78; P < 0.001). Sex ratio in T. dryi turned female-biased around 0.40 mm2 and in Tamarixia sp. at around 0.90 mm2. Hyperparasitism also depended on the size of T. erytreae individuals. The hyperparasitoid A. cassatus tended to emerge from large hosts (F = 3.14; df = 1, 80; P = 0.002) (Figure 4). Hyperparasitism rates became higher than 50% when hosts were larger than 1.65 mm2.
Figure 4: Effect of Trioza erytreae size on the probability that an individual of Syrphophagus cassatus emerge from the nymph.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Host range testing of T. dryi. Experiment 1. Host range on naturally settled nymphs. When T. dryi females had access to their natural host T. erytreae, they parasitized between 1.5 and 2.1 nymphs in the 3rd to the 5th instar in 48 hours. No T. dryi eggs were observed on T. erytreae nymphs in the control treatments, indicating no accidental contamination. T. dryi females did not parasitize any of the 11 NTPs tested (Table 4).
Table 4: Number of parasitized psyllids (mean ± SE) after exposure to the parasitoid Tamarixia dryi during 48 hours. The number of replicates and the mean number of psyllids (± SE) per replicate are also provided. Psyllid hosts in the control treatment were not exposed to T. dryi. Parasitized Psyllid host Treatment Replicates Psyllids / plant psyllids Control 20 11.3 ± 0.2 - Trioza erytreae T. dryi 20 11.2 ± 0.4 1.9 ± 0.5 Control - - - Trioza alacris T. dryi 10 8.8 ± 1.9 0 Control 15 10.0 ± 0.0 - Bactericera tremblayi T. dryi 18 8.8 ± 0.5 0 Control 12 7.2 ± 0.5 - Glycaspis brimblecombei T. dryi 12 7.8 ± 0.5 0 Control 8 14.3 ± 1.3 - Aganoscesna sp. T. dryi 8 12.8 ± 1.5 0 Control 15 23.3 ± 1.5 - Trioza erytreae T. dryi 15 18.1 ± 1.2 2.1 ± 0.4 Control 20 17.1 ± 1.7 - Trioza laurisilvae T. dryi 20 15.6 ± 1.6 0 Control 20 10.8 ± 0.9 - Ctenarytaina eucalypti T. dryi 20 15.8 ± 0.9 0 Control 15 18.2 ± 2.4 - Trioza erytreae T. dryi 15 17.4 ± 2.3 2.0 ± 0.4 Control 9 11.8 ± 2.8 - Trioza sp.I T. dryi 9 12.6 ± 6.3 0 Control 15 12.7 ± 0.9 - Trioza sp. II T. dryi 15 11.0 ± 0.9 0 Control 15 11.6 ± 5.2 - Trioza sp.III T. dryi 15 10.4 ± 2.9 0 Control -- - Trioza erytreae T. dryi 6 6.8 ± 0.4 1.5 ± 0.3 Control -- - Euphyllura olivina T. dryi 4 6.0 ± 0.0 0 Control - - - Spondyliaspis plicatuloides T. dryi 10 6.5 ± 0.2 0
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Experiment 2. Host range of psyllid nymphs exposed to T. dryi. T. dryi females parasitized only one out of the 150 nymphs of Trioza sp. III settled on C. canariensis (Table 5). However, the immature parasitoid died in the first larval instar just after hatching. T. dryi females did not parasitize any of the other NTPs when removed from their galls and directly exposed to the parasitoid.
Table 5: Number of parasitized psyllids (mean ± SE) and their mortality (mean % ± SE) after removal from their galls and exposure to the parasitoid Tamarixia dryi during 48 hours. The number of replicates and the mean number of psyllids (± SE) per replicate are also provided. Psyllid hosts in the control treatment were not exposed to T. dryi. Psyllid host Treatment Replicates Psyllids / plant Parasitized psyllids Control 20 11.3 ± 0.2 - Trioza erytreae T. dryi 20 11.2 ± 0.4 1.9 ± 0.5 Control 16 9.8 ± 0.5 - Aganoscesna sp. T. dryi 16 10.0 ± 0.0 0 Control 15 23.3 ± 1.5 - Trioza erytreae T. dryi 15 18.1 ± 1.2 2.1 ± 0.4 Control 20 10.0 ± 0.0 - Trioza laurisilvae T. dryi 20 10.0 ± 0.0 0 Control 15 18.2 ± 2.4 - Trioza erytreae T. dryi 15 17.4 ± 2.3 2.0 ± 0.4 Control 15 10.0 ± 0.0 - Trioza sp. II T. dryi 15 10.0 ± 0.0 0 Control 15 10.0 ± 0.0 - Trioza sp. III T. dryi 15 10.0 ± 0.0 0.05 ± 0.05
Dispersion and effect of T. dryi on T. erytreae in Canary Islands. Dispersion. T. dryi was recovered in five out of the six islands where T. erytreae is present: Tenerife, La Palma, La Gomera, El Hierro and Gran Canaria 18 months after its release in the island of Tenerife (Table 6 and Figure 5). In this island, T. dryi was found in the 85.71% of the 42 citrus orchards infested by T. erytreae. The parasitoid was distributed throughout the north part of the island and from 18.4 to 788.5 meters above the sea level. The parasitoid spread from Tenerife and reached at least four more islands: La Palma, La Gomera, El Hierro and Gran Canaria. In these four islands, T. dryi was found in more than 50% of the orchards or gardens where T. erytreae was present. The parasitoid was not recovered in the Islands of Lanzarote and Fuerteventura, where the presence of the pysllid vector is very limited (Table 6 and Figure 5). Parasitism. One year after the release of T. dryi, the mean number of T. erytreae suitable for parasitism was very low and the few individuals recovered were parasitized by T. dryi (Figure 6). After June, T. erytreae suitable for parasitism could not be founded in the orchards. In Gran Canaria, the mean number of psyllids suitable for parasitism was higher than in Tenerife and reached the maximum in June. The parasitism rates remained over 0.7 from April to July. After July, the number of T. erytreae suitable for parasitism was extremely low and did not recover.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Table 6: Locations with Trioza erytreae and its parasitoid Tamarixia dryi in the Canary Islands between 2018 and 2019 after the release of the parasitoid. Number Tamarixia dryi number (percentages) Number of (percentages) of Island locations locations with Locations with Locations without sampled Trioza erytreae T. dryi T. dryi
Tenerife 81 42 (51.85%) 36 (85.71%) 6 (14.29%)
La Palma 28 25 (89.29%) 14 (56%) 11 (44%)
La Gomera 9 4 (44.44%) 3 (75%) 1 (25%)
El Hierro 14 4 (28.57%) 2 (50%) 2 (50%)
Gran Canaria 51 40 (78.43%) 30 (75%) 10 (25%)
Lanzarote 30 3 (10%) 03 (100%)
Fuerteventura 44 0 0 0
Total 257 118 (45.91%) 85 (72.03%) 33 (27.97%)
Figure 5: Dispersion of the parasitoid Tamarixia dryi in the 257 locations 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.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Figure 6: Seasonal trend and parasitism rates of Trioza erytreae after the 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.
Conclusion These results confirm that T. dryi is the main parasitoid of T. erytreae in its area of origin and it is a highly specific parasitoid, as it did not parasitize the 11 non-target psyllid species used in the host range test. Therefore, its introduction, release and establishment in Europe within the classical biological control program of T. erytreae should not affect other psyllid species, and no significant environmental impact is expected. On the other hand, the potential benefits of its establishment are very high, especially since the psyllid is not yet present in the main citrus producing areas (Mediterranean basin). If T. dryi is as efficient as
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
it has been demonstrated in the Canary Island, the parasitoid might delay the spread of T. erytreae in mainland Europe. The Spanish and Portuguese governments allowed the release of the parasitoid in mainland Europe in the fall of 2019. In collaboration with the different authorities, the parasitoids reared at ICIA were transported and released in three locations in Galicia and three locations in Portugal. Several national and regional media echoed these releases. Spain https://www.lavanguardia.com/vida/20191129/471949706262/ivia-introduce-insecto-tamarixia- dryi-para-frenar-el-greening-en-citricos.html https://sevilla.abc.es/agronoma/noticias/fitosanitarios/exito-control-biologico-plaga-citricos-el- algarve-andalucia/ https://www.phytoma.com/noticias/noticias-de-actualidad/primeras-sueltas-experimentales-de- tamarixia-dry-en-la-peninsula https://www.lasprovincias.es/economia/gobierno-decide-liberar-20191111014420-ntvo.html http://valenciafruits.com/primeras-sueltas-de-tamarixia-dryi-para-frenar-la-expansion-del-vector- del-greening/ https://www.revistamercados.com/inician-sueltas-experimentales-para-controlar-el-insecto- vector-del-greening/ https://fruittoday.com/primeras-sueltas-experimentales-de-tamarixia-dryi-en-la-peninsula-iberica/ https://www.agrodiario.com/texto-diario/mostrar/1646659/ivia-contribuye-seguimiento-suelta- insecto-frena-expansion-vector-greening-citricos https://www.agrodigital.com/2019/11/20/avances-en-la-gestion-de-la-psila-africana/ https://val.levante-emv.com/economia/2019/11/29/ivia---frena-expansion/1950146.html https://www.freshplaza.es/article/9162235/espana-autoriza-la-liberacion-de-un-insecto-contra-el- vector-del-hlb/ Portugal https://www.noticiasaominuto.com/pais/1367477/governo-inicia-programa-biologico-de-luta- contra-praga-que-afeta-citrinos https://www.vidarural.pt/destaques/luta-biologica-contra-a-trioza-erytreae-vai-avancar/ https://jornaleconomico.sapo.pt/noticias/ministerio-da-agricultura-inicia-programa-experimental- de-luta-biologica-contra-a-trioza-erytreae-521619 IVIA, under the TROPICSAFE project and in collaboration with the University of Pretoria and the University of Stellenbosch in South Africa, ICIA and “Servicio de Sanidad Vegetal de Canarias” in the Canary Islands, and the Spanish and Portuguese Governments, has developed a classical biological control program to introduce the parasitoid T. dryi from South Africa to Europe and reduce the spread of T. erytreae in Europe including: i) the identification of the parasitoid complex of T. erytreae in South Africa; ii) the collection of T. dryi in South Africa; iii) the introduction and rearing of T. dryi in Spain; iv) the evaluation of T. dryi the host specificity, as requested by the Spanish authorities before its release in the field; v) the release in the field; vi) The evaluation of its establishment, dispersion and effect on T. erytreae in the field.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
1.1.3. South Africa: conservation biological control of Trioza erytreae in public areas
Introduction Commercial citrus growers in South Africa are largely reliant on synthetic insecticide applications to manage citrus pests. Therefore, refuges outside commercial plantings for the psyllid hosts and primary parasitoids of T. erytreae may be critical. The aim of TROPICSAFE was to determine the presence, identity and abundance of parasitoids (parasitic wasps) of T. erytreae in non-commercial areas, private gardens and regional parks in two provinces of South Africa. In addition to the surveys carried out in spring and summer in 2017 (Pérez-Rodríguez et al., 2019), further surveys were carried out in autumn and winter in Gauteng and Mpumalanga in order to complement the previous surveys in South Africa. The rationale is that citrus trees in gardens and parks are less likely to be treated with insecticides and may, therefore, serve as refuges. Conservation biological control aims to improve the conditions for natural enemies, for example through managing habitats that favor natural enemies. The information on the occurrence of parasitoids in non-commercial areas will be useful in devising strategies and incorporating conservation biological control in a management plan for T. erytreae containment.
Material and Methods Qualitative and quantitative surveys of T. erytreae and its parasitoids took place from late autumn to early spring in two citrus producing provinces, Gauteng and Mpumalaga (Figure 7). Qualitative surveys were carried out in one commercial citrus orchard as control, two public parks, two research stations, and eight private gardens between 15 February and 29 September 2018. Quantitative surveys were undertaken in Gauteng at the University of Pretoria Experimental Farm in a citrus orchard and a private garden. At both sites, insecticides were not used.
Limpopo
North West Gauteng Mpumalanga
Free State KwaZulu-Natal
Northern Cape
Eastern Cape
Western Cape
Figure 7: Sampling sites in Gauteng and Mpumalanga in South Africa in private gardens (yellow circles), experimental farms or research areas (green circles), and a commercial farm (red circle).
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Qualitative sampling of Rutaceae (Citrus spp. and Murraya spp.) took place at each site by collecting leaves that showed signs of psyllid damage such as galls and chlorosis or that had nymphs present on their surface. The number of leaves collected for qualitative sampling varied between 3 and 20 per site, depending on the number of psyllids observed. Leaves from each plant were transferred to one plastic bag per plant, labeled with a code reporting site, plant number and date of sampling and transported to the University of Pretoria. Leaves were transferred to labeled Petri dishes with moist filter paper. The Petri dishes were kept inside insect cages to prevent the escape of insects and the infestation by other insect species. Insect cages were kept at 25 to 28°C, with 30 to 60% relative humidity and a cycle of 12 hours light and 12 hours dark. Petri dishes were checked daily for the emergence of adult insects for one month after the collection. Emerged adults were placed individually in a labeled 1.5 ml microtube with one drop of sucrose solution. The microtubes were closed with moistened cotton wool and kept in insect cages under controlled conditions (25 to 28°C, 30 to 60% relative humidity, 12 hours light and 12 hours dark). The tubes were checked daily until the insects died. Quantitative sampling took place in Pretoria at two sites, the University of Pretoria (UP) Experimental Orchard and a private garden. Both were sampled once per week to compare changes in psyllid and parasitoid abundance over the time. Quantitative sampling started after June 1st 2018. Sampling was carried out by randomly selecting five infested citrus trees and collecting leaves from the trees that showed signs of psyllid damage as well as leaves that had nymphs present on their surface at the experimental citrus orchard. Nymphs in the private garden were surveyed on a single tree in September 2018. Samples were transported to UP in closed plastic bags labeled with a code, reporting site, plant number, and the date of sampling. In the laboratory, nymphs were placed individually in 1.5 ml microtubes by cutting out the portion of the leaf with the psyllid. The tubes were closed with moistened cotton wool and placed inside an insect cage under controlled conditions (25 to 28°C, 30% to 60% relative humidity, 12 hours light and 12 hours dark). The microtubes were monitored each day for the emergence of adult insects. Upon emergence, 1 ml of sucrose solution was placed inside the microtube as needed until the insect died. If no insect had emerged one month after the collection date, the sample was discarded. Parasitoids were identified using the key by Tamesse (2009). The percentage of parasitism for each tree at one site and date were recorded.
Results Qualitative sampling of T. erytreae. All psyllid samples collected from Citrus spp. and Murraya spp. between 6 March 2018 (autumn) and 14 June 2018 (winter) were identified as T. erytreae (Table 7). Sampling resulted in the capture of 111 T. erytreae adults on leaves from plants in Gauteng. The emergence rate of psyllid adult or parasitoids was very low, possibly due to the low humidity experienced in the winter. Twenty-five parasitoids, identified as T. dryi, emerged from the samples (Table 7). The parasitoids have been submitted to an expert to confirm their identification. The largest number of adults collected from leaves and the largest number of nymphs were from a private garden in Germiston,
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Gauteng. The largest number of parasitoids emerged from nymphs were collected in Greenstone, Gauteng. No parasitoids emerged from T. erytreae collected from Murraya spp. (n = 48) in Germiston (Gauteng) or from nymphs collected on citrus in Mpumalanga.
Table 7: Sampling sites, number of adults collected, and psyllids and parasitoids emerged from psyllid nymphs on Citrus spp. in Gauteng and Mpumalanga from March (autumn) to mid-June (winter) 2018. Number of Number of Number of Number of adults adult T. Province Location nymphs parasitoids T. erytreae erytreae examined emerged captured emerged Bramhof 1 0 0 Craigavon 1 0 0 Fourways 2 2 0 0 Germiston 81 33 0 0
Gauteng Greenstone 18 3 15 (gardens) Groenkloof 5 0 5 Krugersdorp 2 0 0 Meyerspark 2 3 0 3 Orange Grove 3 0 3 Ridgeway 28280 0 Nelspruit 1 0 0 (research station 1) Mpumalanga Nelspruit 1 0 0 (research station 2) Commercial farm 1 0 0 Total 111 99 3 26
Quantitative sampling of T. erytreae. All samples with nymphs collected between June 6th and September 27th 2018 from a citrus orchard at the UP experimental orchard and at a private garden in Pretoria were identified as T. erytreae. The parasitoids emerged from T. erytreae were identified as T. dryi. The specimens were submitted to an expert who confirmed their identification. The abundance of parasitoids, measured as parasitism, was highest in spring in September 2018 (Figure 8). The mean rate of parasitism in June (winter) was 99%, but the emergence rate was very low (4%). The number of T. erytreae nymphs was very low during winter in July and August, ranging between zero and two per sampling. No parasitoids were recorded in July and August. The corresponding weather data from the UP experimental orchard from June 6th to September 27th 2018 show that the highest emergence of parasitoids (71%) occurred in spring (September) when the maximum daily temperatures increased (Figure 9).
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
80 70 60 50 40
Parasitism (%) Parasitism 30 20 10 0 Jun Jul Aug Sep Oct
Figure 8: Percentage of parasitism of T. erytreae at the University of Pretoria Experimental Farm between June and September 2018.
40 Precipitation 14 35 Tmax 13 Tmin 30 11
25 9
20 7
15 5 Temperature (°C) Temperature Precipitation (mm) 10 4
5 2
0 0 01 Jun 16 Jun 01 Jul 16 Jul 31 Jul 15 Aug 30 Aug 14 Sep 29 Sep -5 -2 Date Figure 9: Daily minimum and maximum temperatures and rainfall at the University of Pretoria Experimental Farm between June and September 2018.
Pretoria in enclosed into the summer rainfall region with most of the rain falling from October to April. During the study period, no rainfall was received in June, 1 mm in July, 0.8 mm in August and 12 mm in September at the research orchard. Although parasitism level was high in June and September, the number of parasitoids that were able to complete their development was relatively low. From the 138 parasitized individual nymphs collected at the citrus orchard, 52 (38%) parasitoids emerged. From the 187 parasitized nymphs collected in the private garden, 58 (31%) emerged in spring in September 2018. The lack of rainfall and subsequently low humidity during the study period may have negatively impacted on larvae and pupae survival of the parasitoids.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
The percentage of the parasitism in spring was initially higher at the experimental orchard than in the private garden, but the parasitism increased rapidly at the garden before a decline in parasitoid emergence at both sites at the end of September (Figure 10).
80 Experimental Farm 70 Garden
60
50
40
Parsitism (%) Parsitism 30
20
10
0 03-05 Sep 10 Sep 17-20 Sep 25-27 Sep Figure 10: Percentage of parasitism of weekly sampling at the University of Pretoria Experimental Farm and at a garden in Groenkloof (Pretoria) in September 2018.
Conclusions The abundance of T. erytreae and subsequently their parasitism was very low during the winter, possibly due to a combination of factors, i.e. lack of flush on citrus trees, low temperatures and lack of rainfall. Adult psyllids can live for two to three months and overwinter feeding on mature leaves (van den Berg ,1990). The abundance of T. erytreae on citrus is linked to the flushing rhythm of the host plant and if conditions are suitable, such as availability of young flush, T. erytreae may reproduce throughout the year (van Den Berg, 1990). In the spring, the percentage of the parasitism was initially higher at the experimental farm on trees not treated with insecticides, than in the private garden in an urban area, possibly because more citrus trees were available in the orchard than in the private garden to support T. erytreae during the winter. The findings of the TROPICSAFE surveys carried out during the cooler month of the year together with the previous survey during spring and summer (Pérez-Rodríguez et al., 2019) suggest that conservation biological control can be increased with pesticide-free citrus plants grown in private gardens, public areas or orchards.
1.2. America: Diaphorina citri as vector of “huanglongbing”
1.2.1. Cuba: eradication and chemical control for “huanglongbing” management
Introduction Diaphorina citri Kuwayama (Hemiptera: Liviidae), “huanglongbing” vector, has been present in Cuba since 1999 (González-Hernández, 2007). Later, the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) was detected (Luis et al., 2009; Martinez et al.,
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2009). The management of HLB requires an integrated strategy that includes the use of certified propagation material, the elimination of inoculum sources and vector control programs (Bové, 2006; Gottwald et al., 2012). This integrated strategy has been the management proposed by Cuba since the beginning of the epidemic. However, there are no scientific studies supporting that the elimination of infected plants can contribute to manage HLB in Cuba. Therefore, studies are performed to demonstrate the effectiveness of elimination of infected plants in the control of the disease under Cuban conditions. On the other hand, the management of the insect vector, requires the use of insecticides that are toxic for beneficial insects, as well as for animals (Majeed et al., 2018). For this reason, it is necessary to explore the use of alternative insecticides, more environmentally friendly, to control the insect vector. These alternative insecticides should also be available in Cuba. Kaolin is more environmentally friendly than the insecticides used against pests (Karagounis et al., 2006; Hall et al., 2007) and, moreover, it is available in Cuba as it is present in Isla de la Juventud. Therefore, two different and complementary approaches were evaluated to manage HLB in Cuba: i) the eradication of infected trees and ii) the efficacy of kaolin against the psyllid vector D. citri.
Material and Methods Eradication strategy. The effect of eradication was evaluated by comparing the temporal increasing of HLB in orchards in areas with and without eradication programs. Six citrus commercial orchards were selected, three within an area without eradication program (Matanzas province) and three within an area with eradication program (Ciego de Ávila province). Details of the citrus orchards are shown in Table 8. In each orchard, a block of 900 trees was selected to monitor the increase of HLB. In the orchards under eradication programs disease-free trees were used for replanting; chemical control of the insect vector D. citri, and removal of symptomatic trees (eradication) were also performed. In the orchards without the eradication program, the strategy to manage HLB was the same, but without eradication. For both treatments insecticides were sprayed when the D. citri was present during the flush periods. From May 2017 until November 2019 (30 months), the infection levels of HLB were determined in the six orchards with systematic surveys carried every two months. All the 900 citrus trees in the selected plots were inspected to verify the presence of the typical symptoms. A tree was considered infected when its leaves presented the characteristic HLB blotchy mottle (Figure 11).
Figure 11: Typical blotchy mottle symptoms associated with HLB: 1, grapefruit and 2, sweet orange.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Table 8: Citrus orchards selected to evaluate the temporal increasing of HLB with and without eradication of infected trees. Location Planting Area Orchard Variety Rootstock X Y year Valencia 22°38’03.05” 81°14’38.05” C35 1 Criolla sweet 2016 N W Citrange Without orange eradication of Valencia symptomatic 22°37’19.55” 81°14’32.18” Sour 2 Criolla sweet 2016 plants N W orange (Matanzas orange province) 22°37’15.42” 81°15’08.80” Marsh Sour 3 2016 N W grapefruit orange Valencia 21°55’47.11” 78°40’44.33” Citrumelo 4 Criolla sweet 2017 N O Swingle Eradication of orange symptomatic Valencia 21°55’56.15” 78°40’38.70” Citrumelo plants (Ciego 5 Criolla sweet 2017 N O Swingle de Ávila orange province) 21°58’01.19” 78°41’08.90” Marsh Citrumelo 6 2017 N O Grapefruit Swingle
Kaolin strategy. The effect of kaolin on D. citri was evaluated during 2018 and 2019 in a commercial citrus orchard (five years old cultivar Valencia sweet orange / Citrus macrophylla) located in latitude: 21°73’ N and longitude: 82°76’ W. The treatments were kaolin 5% and water as control. Twenty trees were randomly selected, ten per treatment. The applications were done to the whole tree using an agitated knapsack sprayer in March, May and September (Figure 12).
Figure 12: Citrus plants after the foliar application of kaolin.
D. citri density was recorded from March to November in kaolin-treated and untreated trees during both years. In each tree, four shoots (one per each cardinal point) were observed and the number of adult psyllids per shoot was counted. The mean number of adult psyllids per shoot and per tree was calculated. The total number of mean psyllids throughout the year was calculated for each treatment and year. A U Mann-Whitney test was carried to statistically evaluate the results.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Results Eradication strategy. The three citrus orchards under the eradication program showed a very low incidence of symptomatic plants (generally null) (Figure 13A). A few symptomatic trees were detected in January of 2019 and eliminated. On the other hand, the incidence of the disease was higher in the three orchards located in the area without eradication (Figure 13B). In these orchards the incidence of HLB started in March 2018 and ranged between 13% and 18% at the end of 2019.
Incidence of HLB orchards with eradication
orchard 4 orchard 5 orchard 6 0,15
0,10
0,05 (%) 0,00 Incidence of HLB symptomatic plants A
Incidence of HLB orchards without eradication
0,20 orchard 1 orchard 2 orchard 3
0,00 Incidence of HLB symptomatic plants (%)
B
Figure 13: Incidence of HLB in citrus orchards with (A) and without (B) eradication programs in Cuba from May 2017 to November 2019.
Kaolin strategy. Table 9 shows the infestation levels of D. citri in untreated trees and trees treated with kaolin in 2018 and 2019. In general the infestation levels were higher in 2018 than in 2019 in both treatments. The infestation level remained very low in trees treated with kaolin throughout these two years. Only a few individual infested trees were recorded in June, October and November 2018. On the other hand, infestation levels varied throughout the survey period in the untreated trees. The infestation level was medium-high (1 to 9 insects) during the two years (Table 9).
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Table 9: Mean number of individuals of D. citri / shoot / tree observed during the period 2018-2019 on trees treated with kaolin and untreated. Kaolin was sprayed in March, May and September. 2018 2019 Evaluation kaolin water kaolin water March 0102 April 0402 May 0501 June 1803 July 0101 August 0402 September 0702 October 2901 November 2305 TOTAL 542019
Conclusions The results demonstrate that the elimination of symptomatic trees at a regional scale should be implemented to manage HLB in the commercial citrus orchards in Cuba, as it had been previously recommended in other areas (Gottwald, 2010). After two years, the percentage of trees with symptoms was 20% lower in the area where the symptomatic trees had been eliminated. Kaolin can be used to control the psyllid vector D. citri. The efficacy observed in this two-year study can be due to the reduced attraction of citrus trees treated with kaolin for adults of D. citri as it was suggested by Glenn et al. (1999) and Miranda et al. (2018). Overall, the applications of kaolin and the eradication of the symptomatic trees can reduce the use of toxic insecticides to manage HLB. This is important because the intensive use of insecticides such as neonicotinoids and organophosphates to control the vector can have negative impacts as metabolic imbalance of the plant making it more susceptible to attack by pests (Wedding et al., 1953), negative impact on potential biological regulatory organisms (Prabhaker et al., 2006) and resistance against the active ingredients used (Tiwari et al., 2011).
1.2.2. Guadeloupe: organic management of “huanglongbing”
Introduction The Asian citrus psyllid D. citri was first observed in Guadeloupe in 1998. Its main biological control agent, the parasitoid Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) was introduced one year later. The first results of its application were promising and the parasitoid established and reached high parasitism levels (Etienne et al., 2001). However, ‘Ca. L. asiaticus’ was detected in 2012, the disease spread rapidly, and the productions
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dropped dramatically. Nowadays, the infection rates in commercial citrus orchards are very high because the infected trees are not removed. Under these circumstances, conventional production programs based on the use of insecticides to control the insect vector are ineffective, even when D. citri levels are low. In this task, ASSO has followed the dynamic levels of D. citri, the infection levels of HLB, as well as, the mortality of citrus trees in four experimental citrus orchards: two orchards followed an organic program (BIO) and two orchards followed an IPM program (IPM). Moreover, oranges, mandarins, grapefruits, tangelos produced in Guadeloupe are almost no longer available mainly because of their high susceptibility to HLB. The citrus crops still produced are Persian limes (Tahiti lime, Citrus latifolia) because they have more vigor and some tolerance to HLB, likely because they are triploid citrus. Polyploidy confers certain anatomical traits favoring the tolerance to the disease. However, there is still a strong demand for other citrus varieties such as oranges or mandarins. In this context an evaluation of different orange varieties, tangors and mandarins within the same citrus orchards mentioned above is carried out.
Material and Methods Orchards and plant material. The four experimental orchards to evaluate the crop systems and plant materials were located in in Vieux Habitants (two orchards), Capesterre Belle-Eau and Trois Rivières (Figure 14). Two orchards received the same plant material but different management strategy. Thus, the orchards were organized and categorized as “Assay 1 BIO”, “Assay 1 IPM”, “Assay 2 BIO”, “Assay 2 IPM”. Orchards of Assay 1 received the same plant material, but were managed with different strategies. Orchards of assay 2 received the same plant material but were managed with different strategies. The orchards of assay 1 and 2 received different plant material.
Assay 1 BIO, Vieux -Habitants Assay 1 IPM, Trois Rivières Assay 2 BIO, Vieux Habitants Assay 2 IPM, Capesterre Belle Eau
Figure 14: Location of the four experimental citrus orchards in Guadeloupe.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
In the assay 1 the trees were planted between July 2015 and May 2016. In each treatment, there were 162 trees with a mix of six orange varieties: Cara cara Navel, Fisher Navel, Navelate, Navelina, Valencia Rode Red (C. sinensis) and the tangor variety Tangor Ellendale (C. reticulata × C. sinensis). Three rootstocks were used: diploid citrumelo 4475 (C. paradisi × P. trifoliata); Volkamer lemon (C. volkameriana) and Flhorag1 (allotetraploid somatic hybrid Poncirus trifoliata + C. deliciosa Ten.). In the assay 2, all the trees were planted in August 2015. In each treatment, there were 90 trees with a mix of 5 varieties [Jackson & Triumph orangelos (C. sinensis x C. paradisi), Nova tangelo (C. reticulata x C. paradisi), Temple tangor (C. reticulata × C. sinensis), Falglo mandarin (Citrus reticulata)] potentially tolerant to HLB. An orange genotype, Rhode red sweet orange (C. sinensis), was selected a susceptible genotype. All the scion varieties were evaluated in association with three rootstocks (diploid and tetraploid citrumelo 4475 (C. paradisi × P. trifoliata) as well as Flhorag 1 (allotetraploid somatic hybrid P. trifoliata + C. deliciosa Ten.). The characteristics of each orchard are summarized in Table 10.
Table 10: Summary of the main characteristics of the experimental plots in Guadeloupe. ASSAY 1 BIO ASSAY 1 IPM ASSAY 2 BIO ASSAY 2 IPM Density 4x5 4x4 4x5 4x5 Percentage of 33% 33% 66% 66% tetraploids trees Replicates per 3 3 3 3 citrus combination Type of soil Alluvial sol Andosol Alluvial sol Ferraltic sol Type of climate Dry Wet Dry Wet Altitude 50 m 400 m 50 m 306 m Flat - mechanizable Sloping - non Flat - mechanizable Flat - mechanizable Topography dish mechanizable slope dish dish HLB constraint strong lowstrong medium
Management of D. citri and HLB. The management of the citrus orchards was different between those that followed the organic management (BIO) and Integrated Pest Management (IPM). In the organic management (BIO), mineral oils were used to control phloem-feeding insects (psyllid larvae, mealy bugs, aphids). In 2018 and 2019, several species of predators were released to control pests. Finally, herbicides were not used in the alleyways and the cover crop was cut six times per year in order to increase the number of biological control agents. In the IPM management (IPM), Vertimec and Karate were used from 2015 to December 2018 to manage the different pests (about 6 times / year) in association with mineral oil. The orchards were sprayed before pest populations reached high level of development. In January 2019, all pesticide treatments were stopped and only mineral oil and soap were used thereafter. Glyphosate was used from 2015 to 2018 (6 times / year) in order to manage the weeds. Since the beginning of 2019 orchard weeds
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were managed only mechanically. Other details of the management carried out are described in Table 11.
Table 11: Treatments applied in BIO and IPM management systems in Guadeloupe.
Weed Fertilization Irrigation Pest management management
Type Organic + Foliar Aspersion Mechanical Biological control
“Italpolina Guanito”, Black soap+ “Phénix”, Brushcutter on the Vermicompost juice Micro BIO Products “Sheep” manure row / gyro-shredder + effective aspersion + Myr Micro, in the inter-row microorganisms + “Auxym”, PNPPs “Trainer”
Frequency Monthly Daily Monthly Occasionally
Aspersion Mineral (NPK)+ (Assay 1) Mechanical + Integrated pest Type Foliar No (Assay Chemical management 2)
Assay 1: Brushcutter on the row (or glyphosate)/ IPM Urea, “DAP”, Cash crops in the Vertimec + Karate + 11-11-33, 12-6- Micro inter-row areas Oviphyt/black soap Products 20 + Hortal®, aspersion Assay 2: with foliar fertilizers Mérol® Brushcutter on the + PNPPs row (or glyphosate)/ gyro-shredder in the inter-row areas
Frequency Monthly Daily Monthly Bimonthly
Measurements. D. citri abundance was measured monthly from July 2018 to September 2019. In each date, half of the trees were sampled, every two rows alternatively. Psyllid abundance was recorded according to the following scale: 0: no psyllid 1: low abundance of psyllids (1 or 2 / shoot) 2: intermediate abundance of psyllids (3 to 5 / shoot) 3: high abundance of psyllids (more than 4 / shoot) HLB infection was evaluated in 2019. In each orchard, the 30% of the trees was analyzed using a lamp kit (Lamp Amplifire kit, Envirologix). The number of trees dead was counted yearly from 2017 to 2019. The trees killed by the “Maria” hurricane in September 2017 were not considered. Rootstock and variety (scion) diameters were measured once a year between April and July, in all the trees of the plots.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Results D. citri abundance. D. citri abundance was higher in the two organic citrus orchards than in those that followed the IPM program (Figures 15 and 16), almost no psyllids were observed in the IPM managed orchards.
Assay 1 2
1,5
1 abundance
0,5 D. citri 0 dic‐19 dic‐18 ott‐19 ott‐18 giu‐19 lug‐19 set‐19 lug‐18 set‐18 feb‐19 apr‐19 ago‐19 ago‐18 gen‐20 gen‐19 nov‐19 nov‐18 mar‐19 mag‐19
ASSAY 1 IPM ASSAY 1 BIO
Figure 15: Relative abundance of Diaphorina citri in citrus orchards of assay 1. Months without data were not sampled.
Assay 2 1,8 1,6 1,4 1,2 1
abundance 0,8 0,6 0,4
D. citri 0,2 0
ASSAY 2 IPM ASSAY 2 BIO
Figure 16: Relative abundance of Diaphorina citri in citrus orchards of assay 2. Months without data were not sampled.
HLB infection. HLB infection varied among the four orchards. It was lower in the orchard “Assay 1 IPM” than in the rest (Figure 17), likely, because this orchard was planted in 2016 and the other orchards were planted in 2015. The data also show that all the orchards were highly infected, even in the “Assay 1 IPM”, where almost no psyllids were observed (Figure 17). In the orchard “Assay 2 IPM”, all the trees were HLB infected already in 2018. Overall,
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these results show that the rate of HLB infection increased very quickly and the presence of a few psyllids was sufficient to infect very quickly all the orchards.
100,0%
80,0%
60,0%
40,0%
HLB infection rate 20,0%
0,0% Assay 1 IPM Assay 1 BIO Assay 2 IPM Assay 2 BIO
2017 2018 2019
Figure 17: HLB infection rates in the four experimental citrus orchards in Guadeloupe in 2017, 2018 and 2019.
Mortality of trees. Trees mortality could not be directly correlated to HLB presence. Trees’ death was rather due to a combination of biotic and abiotic factors. Indeed, irrigation issues are responsible for the loss of many trees on the “Assay 1 BIO”, since flooding caused the emergence of several diseases (i.e. root rot by Phytophthora spp.). In orchard “Assay IPM 1”, death occurrences were concentrated in one specific block located at the bottom of the field. On the contrary, less than 5% of the threes died in the orchard “Assay IPM 2”. Overall, the data show that the mortality was variable and very high in three orchards, however this mortality cannot be correlated with the high levels of HLB presence (Figure 18).
40%
35%
30%
25%
20%
15%
% of tree mortality 10%
5%
0% Assay 1 IPM Assay 1 BIO Assay 2 IPM Assay 2 BIO
déc‐17 déc‐18 déc‐19
Figure 18: Percentage of trees dead during the assay in the four experimental citrus orchards in Guadeloupe in 2017, 2018 and 2019.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Conclusions The results indicate the complex situation of Guadeloupe to grow citrus under the high levels of HLB. Despite the relatively low abundance of the vector D. citri in some orchards, especially those under an IPM program, HLB levels and the mortality of the trees were very high. This is likely due to the presence of infected trees in private gardens. Based on the experience of previous years and during the TROPICSAFE project, standard strategies to follow in IPM and organic systems to produce citrus in Guadeloupe were prepared. These strategies are presented in Figures 19 and 20 and will be followed by the farmers associated to ASSO.
Figure 19: Scheme of the IPM program developed by ASSO to be followed in Guadeloupe to produce citrus.
Figure 20: Scheme of the organic program developed by ASSO to be followed in Guadeloupe to produce citrus.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
2. Strategies for managing lethal yellowing in palms
2.1. Biological control agents of Haplaxius crudus, lethal yellowing vector in America
2.1.1. Biological control agents of Haplaxius crudus in Mexico
Introduction Haplaxius crudus (van Duzee) (Hemiptera: Cixiidae) has been identified as the vector of lethal yellowing (LY) of coconut crops in America and the Caribbean (Howard and McCoy, 1980). This insect has a hemimetabolous life cycle (Nault and Rodríguez, 1985; Tsai and Kirsch, 1978), going through the stages of egg (11 days), nymph (41 days) and adult (50 days). During the nymph stage, the insect is underground, remaining in the grass roots (Ohler, 1999) and therefore it is hard to detect and sample it. However, within the soil, there are areas of greater reproductive capacity. For example, Howard and Gallo (2007) pointed out that wet sites are more favourable for the development of nymphs. The use of entomopathogenic fungi found in these sites might favour the management of LY. The sustainable management of LY includes the integration of resistant/tolerant varieties of coconuts and cultural control strategies such as monitoring the appearance of symptoms, cutting and destruction of symptomatic plants, vector habitat management, weed control (hosting phytoplasmas and insect vectors) and use of cover crops (Howard, 2015; Ramos- Hernández et al., 2018). The addition of biological control agents would contribute to the sustainable management of the coconut palm pathosystem. Entomopathogenic fungi used in biological control of insects belong mostly to the genera Beauveria and Metarhizium (Nari et al., 2003; Torres de la Cruz et al., 2013). The biocontrol potential of 15 isolates of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (13 isolates) and Metarhizium anisopliae (Metschn.) Sorokin (2 isolates) was tested on H. crudus. In addition, predators of H. crudus present in the field were photographed and identified.
Materials and Methods Entomopathogenic fungi. As it was not possible to collect the cixiids observed as infected with fungi (Figure 21) in the leaves of coconut (Cocos nucifera) or kerpi (Adonydia meriilli) plants at the beginning of 2019, the work began with 15 isolates of entomopathogenic fungi collected from insects captured in the tropical region: 13 isolates of entomopathogenic corresponding to B. bassiana and two to M. anisopliae. Isolates were cultured in 15 x 100 mm Petri dishes with 15 ml of Sabouraud dextrose agar (BD Bioxon®) + yeast extract culture medium using a Socorex® media dosing syringe. After five days, the cultures of entomopathogenic fungi were transferred to Petri dishes with the culture medium (López and Carbonell, 1999). The growth of each isolate was evaluated at three temperatures 20°C, 25°C and 30°C ± 1°C, in complete darkness using three bioclimatic chambers (Incubator NOVATECH®, Incubator BOD 16 Prendo® and Incubators
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
IC103C Yamato®, respectively) and a data logger. The mycelium development was evaluated every two days, and the test finished when the isolate completely covered the box; measures of total radius growth were used for statistical analysis (Dombi et al., 2004). All tests were replicated four times. The measurements were made with a digital Vernier TRUPER®. The number of spores and the germination time were also evaluated for the isolates with faster mycelial development at 25ºC. Predators. During the field inspections of 2019, spiders that were observed searching on the foliage of coconut or kerpi plants were captured and photographed to be identified at the genus or family level (Figure 22). The identifications were confirmed by Dr. Manuel Perez de la Cruz from the Universidad Juarez Autonoma de Tabasco, Mexico.
Figure 21: Derbide insect (Persis foveastis) naturally infected by fungi found on a coconut leaf.
Results Table 12 shows the results of mycelial development of 15 isolates of entomopathogen fungi at the three temperatures 20°C, 25°C and 30°C. In general, the largest mycelial development (measured in mm of mycelial development) of all the isolates studied was achieved at 25°C, followed by development at 30°C and finally at 20°C. At 25°C, the two strains of M. anisopliae (MetaTNK and MetaColpo) and three B. bassiana isolates (BbPACpb, Bb 11 and Bb 12) had the highest growth. All the B. bassiana isolates germinated in 20 hours while M. anisopliae only required a maximum of 10 hours. Spore production also varied among the isolates at 25ºC. For B. bassiana isolates with higher mycelial growth radius, spore production were BppAcpb: 5.6 x 107 ± 1.33 spores/ml; Bp 11: 5.5 x 107 ± 0.46 spores/ml; and Bp 12: 5.4 x 107 ± 6.10 spores/mL. M. anisopliae isolates presented 1.4 x 107 ± 0.61 spores/ml and 2.5 x 107 ± 0.83 spores/ml at 25°C. Based on the previous results, the B. bassiana isolates BbPACpb, Bb 11 and Bb 12 and the two isolates of M. anisopliae were selected for future work and were multiplied to perform the pathogenicity test with H. crudus adults and nymphs captured in the field. However, the low population levels of H. crudus in the field in the last five months, including January 2020, has limited the activity on pathogenicity tests.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Table 12: Mean mycelial growth radius (mm ± standard deviation) of 13 Beauveria bassiana and two Metarhizium anisolpliae isolates cultured in vitro at three temperatures. Mycelial development (mm)
Isolate 20°C 25°C 30°C Mean ± Dst Mean ± Dst Mean ± Dst
Beauveria bassiana
Bb3 19.67 ± 0.44 28.98 ± 1.92 27.29 ± 0.47
BbPACpb 14.93 ± 0.55 31.13 ± 0.41 25.14 ± 0.55
Bb12 18.43 ± 0.97 29.28 ± 1.58 15.82 ± 0.63
Bb11 16.47 ± 0.52 30.52 ± 1.41 25.20 ± 0.65
Bb10 13.91 ± 0.18 28.74 ± 0.46 20.79 ± 0.60
BbMzC01 14.94 ± 0.27 28.73 ± 0.43 22.97 ± 0.56
BbMzAB 15.64 ± 0.70 22.67 ± 1.16 17.98 ± 0.66
Bb8 14.81 ± 0.27 27.49 ± 0.62 22.26 ± 0.45
Bb9 14.14 ± 0.38 18.54 ± 0.55 20.58 ± 0.73
Bbbrc01 9.23 ± 0.90 22.06 ± 0.43 12.61 ± 0.36
Bb7 14.15 ± 0.98 24.10 ± 0.88 17.27 ± 0.45
BbMz02 11.83 ± 0.99 14.55 ± 0.46 19.54 ± 0.48
Bb1 18.79 ± 0.39 23.15 ± 0.41 20.99 ± 0.51
Metarhizium anisopliae
MetaTNK 26.15 ± 2.08 37.40 ± 0.38 29.79 ± 0.69
MetaCOLPOS 33.67 ± 0.17 36.70 ± 0.58 29.36 ± 0.50
Figure 22: Thiodina sp. jumping spider with adult insect in its mouth on a coconut palm leaf.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Figure 23: A Theridion sp. spider and a Haplaxius crudus insect trapped in 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).
Predators. Two species of spiders were identified as potential predators of H. crudus. The jumping spider Thiodina sp. (Saltizidae) (Figure 22) colonizes day and night and throughout the year the coconut and kerpi palm leaves. The spider is distributed along the gulf of Mexico (Tabasco) and on the Pacific coast (Guerrero). Thiodina sp. was observed directly attacking adults of Derbidae and Cixiidae on coconut leaves. The second species is the Theridion sp. (Figure 23.) It was reported as an inhabitant of coastal dunes, where the coconut palm grows. It was observed as active during the day and the night, and in more than one occasion, capturing H. crudus adults on coconut foliage. This species was observed in coconut foliage on the coast of the gulf of Mexico (Tabasco) and in the Yucatan peninsula (Yucatán). Two Theridion species were reported by Howard and Edwards (1985) with great activity on H. crudus in coconut palms in Florida. The results show that the natural control of H. crudus by spiders should be explored in more detail.
Conclusion The identification of the main breeding sites of H. crudus, vector of LY, and the early release of Metarhizium or Beauveria spores in coconut plantations could be used to improve the management of H. crudus. The work started represents the first step to develop this strategy that might slow down the spreading of LY and reduce its impact after further research to test their effect on H. crudus.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
2.1.2. Biological control agents of Haplaxius crudus in Cuba
Introduction The coconut palm is not an economically important crop in Cuba. It is scattered throughout the country as an ornamental plant in gardens and in a few small plantations. The largest areas with coconut palms are located in Baracoa (Guantánamo province), Niquero and Pilón (Granma province) and in several municipalities in Holguín, Pinar del Río and Sancti Spirítus provinces (Cueto, 1986). The coconut plantations located in Baracoa were recently affected by a cyclone and are currently being replanted. The presence of H. crudus, vector of the LY, has been confirmed in insect captures made in the different localities of Cuba; here, the few biological control agents for this insect vector collected during the field samplings using yellow sticky traps are reported.
Materials and Methods Two sites with coconuts (of 16 plants each) present in the experimental station of IIFT in Alquizar Mayabeque province were sampled from February 2019 to February 2020. Six yellow sticky traps were placed in the lower leaves of the plants and were checked and changed every 15 days. The insects captured in the traps were observed with the help of a magnifying glass to identify and count natural enemies. When nymphs were found in the field, these were transported to the laboratory and observed under a binocular microscope to determine symptoms of parasitism or presence of entomopathogenic fungi.
Results Table 13 shows the generalist predators captured in the yellow sticky traps from February 2019 to February 2020. Different species of coccinelids as Cycloneda sanguinea L., Chilocorus cacti L., Brachiacantha sp. and Scymnus sp. were the most abundant generalist predators followed by a Chrysoperla sp. (Neuroptera: Chrysopidae). No specific predators of Cixiidae were identified. Moreover, neither entomopathogenic fungi nor paraditoids were identified in the few H. crudus nymphs collected and transported to the laboratory.
Table 13: Inventory of generalist predators in the yellow sticky traps in Cuba from February 2019 to 2020. 2019 2020 Predator species Feb Mar Apr May June July Aug Sep Oct Nov Dec Jan Feb Cycloneda sanguinea 1 0 2 0 0 0 3 1 0 0 1 2 1
Chilocorus cacti 1 1 1 0 0 0 1 2 0 0 0 3 1
Brachiacantha sp. 1 1 3 0 0 0 0 0 1 1 2 0 1
Scymnus sp. 1 0 0 0 0 0 0 0 0 0 0 1 2
Chrysoperla sp. 0 0 0 1 1 1 0 0 0 0 0 1 0
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2.2. Resistant varieties of coconut
Introduction LY has caused great damage to coconut farmers and industry, killing millions of coconut palms in several countries in America (Figure 24) and it is spreading threatening other countries (Myrie et al., 2019). The disease can be dealt with an integrated pest management approach that includes: i) monitoring to detect palms with early LY symptoms, ii) elimination of these palms as soon as they are detected, iii) immediate replanting, and iv) weeding. Such an approach has been used in Jamaica successfully, keeping low levels of incidence wherever it is applied (Coconut Industry Board, 2013). However, managing LY is more easily achieved if there is LY-resistant germplasm. Both, in Jamaica and Mexico, screening coconut germplasm has been carried out finding materials that can stand LY (Harries, 1995; Zizumbo-Villarreal et al., 2008). In Mexico, management of LY is based on the use of LY-resistant coconut germplasm as the main approach. Replanting with such germplasm in different regions of Mexico that have been affected by LY is working, since no outbreaks have been detected in the areas replanted. Table 14 includes some of these sites and their current status, and this is representative of the situation in Mexico. The resistant germplasm used was identified in field trials, therefore to increase the genetic diversity, new resistance field trials have been established within the TROPICSAFE project. In addition, micropropagation protocols have been developed (Sáenz-Carbonell et al., 2020) to propagate in large scale LY-resistant germplasm. The in vitro plants produced can be transported to other parts of Mexico, and also to partners of the TROPICSAFE project.
Figure 24: Occurrence of lethal yellowing in Latin America and the Caribbean (highlighted in yellow). Red arrows show the path, direction and current limits of spread.
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Table 14: Sites in Mexico with lethal yellowing resistant or presumably resistant coconut genotypes and current status. Location Genotypes Status and observations
Tecpan de Galeana Pacific Tall (8 Ha)
Guerrero Tecpan de Galeana Green Dwarf (7 Ha) No losses due to lethal yellowing
Las Tunas Green Dwarf (12 Ha)
Tecomán Pacific Tall Ecotypes Colima No losses due to lethal yellowing Cuyutlán Pacific Tall Ecotypes
Pacific Tall Ecotype 2 No losses due to lethal yellowing, Mérida (32 Ha) in nearby areas of both sites there were other plants of coconut and Pacific Tall Ecotypes other palm species with lethal San Crisanto yellowing symptoms and LY- Local Green Dwarf positive after PCR detection Yucatán Three LGD coconuts died due to Ticul, Chum Copte I* Local Green Dwarf lethal yellowing (0.5% of the palms in the site)
Brazil Green Dwarf Ticul, Chum Copte II* No losses recently established Alto Saladita
Brazil Green Dwarf Ojoshal* Local Green Dwarf No losses due to lethal yellowing in Tabasco Malayan Yellow Dwarf coconut palms in the vicinity Pailebot Dwarf x Tall hybrids
No losses due to lethal yellowing, Adonidia merilli plants with lethal Veracruz Medellín Malayan Green Dwarf yellowing symptoms and LY- positive after PCR detection *, Sites with evaluations carried out in the TROPICSAFE project
To develop advanced IPM and new pest management strategies for LY in coconut the use of LY-resistant germplasm and LY-resistance screening is evaluated. Towards this aims the transfer of LY-resistant coconut in vitro plants to project partner countries is also carried out. These practices will help to reduce the environmental impact by reducing the use of chemicals (for insect vector control), to prevent the associated pathogen spreading and also to generate knowledge on germplasm susceptibility / resistance to LY. Most importantly is that these LY-resistance screening and germplasm exchange activities can be the basis for the implementation of a permanent system for screening, production and exchange of LY-resistant germplasm in coconut-producing countries. Trial shipments to Cuba and Jamaica where in vitro plants produced in Mexico can be grown and tested under the local conditions were carried out as a basis for replanting programs.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Material and Methods LY-resistant coconut in vitro plants. In vitro plant production and shipment. Plants (Figure 25A) were produced from embryogenic callus according to the methods reported by Oropeza et al. (2018). The calluses were originally obtained from plumule explants from embryos of seed produced by controlled pollination crossing the LY resistant parents MxPT2 x MxPT1. The parent plants were identified as LY-resistant in a field trial reported by Zizumbo-Villarreal et al. (2008). For shipment in vitro plants were transferred from glass containers (Figure 25A) to plastic containers with semi-solid medium without organic components (Figure 25B and C). For this operation, the plastic containers were disinfected chemically and left overnight in flow chamber under UV light before been used. Then within a laminar flow cabinet under sterile conditions, 100 ml of sterile semi-solid Y3 culture medium (Eeuwens, 1976) were added to each container before the medium solidifies. Once the medium is solidified, in vitro plants were transferred. The container is covered with a lid and sealed under sterile conditions (Figure 25D). Finally, the containers with the in vitro plants are placed in cardboard boxes (Figure 25E) and sent to their destination. The acclimatization of in vitro plants is detailed in Figure 26 and Annex B.
Figure 25: Preparation of coconut in vitro plants for shipment. Transfer from glass containers (A) to plastic containers (B, C). When in vitro plants are ready (D) they are boxed (E) before shipment.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Figure 26: LY-resistant coconut plants produced in vitro at CICY. After 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).
Results Coconut in vitro plants of LY-resistant Pacific Tall x Pacific Tall hybrid were produced by micropropagation in CICY and sent to Jamaica and Cuba. A batch of 60 plants was sent to CIB in Jamaica. They were sent through a courier service and they suffered because of
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delays during the journey, probably due to this half of the plantlets have not survived. Also, a batch of 200 was sent later to IIFT Cuba, but this time the batch was taken as luggage by a colleague traveling to Cuba, whom once at IIFT, trained the personnel for acclimatization of the plantlets (Figure 26). Survival is being observed.
Conclusions The germplasm exchange is very valuable to make LY-resistant coconut available to other countries. The exchanged germplasm can be tested under the recipient country conditions and generate more knowledge about the genotypes performance. Shipment of germplasm to other countries is difficult, particularly when it involves long time that may affect the plantlets condition. This might be avoided finding ways to ship the plants more directly and faster, as in the case of the batch taken to Cuba as accompanying luggage, or by finding shipping companies that can fly directly to the destination countries. LY-resistant coconut plants shipping is important to share this coconut germplasm with other countries, and in Mexico it can used as the main approach for managing LY, new shipments will be sent to Cuba and Jamaica within the next three months.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
3. Strategies for managing grapevine yellows
3.1. South Africa: biological control agents of Mgenia fuscovaria, a leafhopper vector of aster yellows phytoplasma
Introduction Mgenia fuscovaria (Stål) (Hemiptera: Cicadellidae) has been identified as a vector of aster yellows (AY) phytoplasma (‘Candidatus Phytoplasma asteris’; 16SrI-B) in South Africa (Engelbrecht et al., 2010; Krüger et al., 2011). Little is known about insect parasitoids and predators of M. fuscovaria or leafhoppers in vineyards in South Africa. In general, leafhoppers are attacked by a variety of natural enemies. These include parasitoids of the Mymaridae (Hymenoptera), which parasitize the egg stage of leafhoppers, several general insect predators, e.g. predatory bugs (Hemiptera), beetles (Coleoptera), and spiders (Costello and Daane, 1999; Hanna et al., 2003; Walton et al., 2012). The control with natural enemies can assist in lowering leafhopper populations and consequently aid in reducing the AY transmission. The current study, therefore, aimed at identifying natural enemies of leafhoppers present in Vredendal, Western Cape, South Africa, one of the three regions where aster yellows phytoplasma has been reported.
Material and Methods Natural enemies were identified from yellow sticky traps placed in a vineyard (cv. Colombard) infected with aster yellows phytoplasma in Vredendal, Western Cape, South Africa before 2016 (Figure 27). Due to a prolonged drought that commenced in 2016 and that led to extremely low numbers of M. fuscovaria trapped from 2017 to 2019 (Figure 31A), natural enemies caught on yellow sticky traps placed into the vineyard prior to the drought were identified.
Results Generalist predators included ants (74%; Hymenoptera: Formicidae), spiders (15%), true bugs (7%; Hemiptera: Miridae, Anthocoridae) and beetles (3%; Coleoptera: Coccinellidae). These were not identified further because their contribution as generalist predators towards regulation of leafhoppers in vineyards is not clear. A total of 487 mymarid parasitoids were recorded during February 2010 (37 specimens), and April (257 specimens), May (181 specimens) and July (12 specimens) in 2013. The majority belonged the genus Polynema (372 specimens, 76%), followed by Gonatocerus (68 specimens, 13%), Stethynium (39 specimens, 8%), Alaptus (4 specimens, 0.8%) and four were unidentified mymarid parasitoids (0.8%).
Conclusions Natural enemies were present in the vineyard infected with aster yellows phytoplasma indicating that natural control occurs. However, the role of parasitoids and generalist
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predators in suppressing M. fuscovaria populations is not clear. Although ants are generalist predators, they have been reported to hinder biological control efforts in vineyards. In South Africa, they have been associated with mealybug outbreaks and disrupting biological control because they tend to honeydew-excreting mealybugs and defend them against natural enemies (Mgocheki and Addison, 2009). Members of the Mymaridae parasitize eggs of other insects. Species of this family are not considered to be very host specific, i.e. they may parasitize eggs of insects belonging to different genera or families, although they have been recorded to most commonly parasitize eggs of Hemiptera, including leafhoppers (Pitkin, 2004). Although the generalist natural enemies recorded may contribute to the population regulation of leafhoppers in vineyards, their specific role has not been established. Therefore, in addition to natural control, several management options in an integrated approach need to be considered to reduce M. fuscovaria populations in South African vineyards.
3.2. South Africa: ecology and management of Mgenia fuscovaria
Introduction Aster yellows (AY) phytoplasma, ‘Ca. P. asteris’, subgroup 16SrI-B, in grapevine has been recorded in South Africa in 2006 (Engelbrecht et al., 2010). It has a limited distribution in the Western Cape (Figure 27). Symptoms of AY include delayed budding on parts of the grapevine, crisp and wrinkled leaves (Figure 28), shoots not lignified, dieback of shoots, abortion of young bunches, partial dieback of fully developed bunches, and infected grapevines may decline and die. The discovery of AY in South Africa led to the identification of the leafhopper vector M. fuscovaria (Krüger et al., 2011). It is important to understand the biology of insect vectors, e.g. life cycle, host plants, population dynamics, overwintering sites and disease epidemiology, for devising sustainable pest management plans. These should incorporate multiple strategies to ensure a successful disease management.
Limpopo
Mpumalanga North West Gauteng
Free State KwaZulu-Natal
Northern Cape
Vredendal Eastern Cape
Wamboomsrevier Western Cape
Cape Town Robertson
Figure 27: Map of South African provinces and distribution of the aster yellows phytoplasma in the Western Cape province in South Africa (red circles).
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Figure 28: An adult Mgenia fuscovaria feeding on a young grapevine shoot in spring (A), symptoms of aster yellows phytoplasma in grapevine (B) and a close up of the symptoms in the leaves (C).
Phytoplasmas rely on both their host plant and insect vectors for survival. They can spread in vineyards through propagation of infected plant material and insect vectors (leafhoppers, planthoppers and psyllids). The management of grapevine yellows includes planting phytoplasma-free material, early detection of infected grapevine plants, planting resistant host plants and managing the insect vectors. A management plan for aster yellows phytoplasma in South Africa has been developed for use by managers and growers. It includes recommendations for leafhopper monitoring, weed control, and chemical control based on research carried out during TROPICSAFE project and previous research findings.
Life stages and life history of M. fuscovaria Origin and distribution. The leafhopper M. fuscovaria, is indigenous to South Africa. Little is known about its distribution in South Africa. It has been recorded in the winter rainfall region in Western Cape province and in the summer rainfall region in KwaZulu-Natal and Limpopo (M. Stiller; personal communication). Egg, nymph, adult. Leafhoppers are hemimetabolous. They have three developmental stages (egg, nymph, adult). They lack a pupa stage and nymphs undergo a gradual metamorphosis. Nymphs of M. fuscovaria resemble the adults, are green/yellowish in color and wingless. Adults of the genus Mgenia Theron are distinct leafhoppers characterized by the shape of their head and face, and arrangement of the large compound eyes (Figure 29). Adults are approximately 5 mm long. Young adults are greenish, turning brown to dark brown as they age. Leafhoppers lay their eggs into the tissue of their host plants. Feeding. Leafhoppers have piercing-sucking mouthparts. M. fuscovaria feeds on the phloem sap of plants. The leafhopper does not cause a direct damage. However, the phloem-feeding habit of M. fuscovaria enables it to transmit phloem-limited pathogens, such as the aster yellows phytoplasma. Host plants. M. fuscovaria has been observed on plant species from different families. However, its host plants for breeding, apart from grapevine, are unknown. The leafhopper has been observed feeding on weeds growing in vineyards mostly during late autumn, winter and early spring (Figure 30). During the growing season, M. fuscovaria is found mostly on grapevine (Kerstin Krüger, personal observation).
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Figure 29: Adult Mgenia fuscovaria, dorsal view (A) and female lateral view (B). (M. Stiller, Agricultural Research Country-Plant Protection Research).
Figure 30: Examples of food plants of Mgenia fuscovaria in vineyards. Urtica sp. (Urticacea) (A), Lamium amplexicaule (Lamiaceae) (B), and Oxalis pes-caprae (Oxalidaceae) (C).
Ecology Seasonal activity. In some vineyards in the Western Cape M. fuscovaria is the most abundant leafhopper on grapevine. Adults and nymphs are active throughout the year (Krüger et al., 2015a). The abundance of the leafhopper is dependent on temperature and rainfall. Monitoring, carried out during the TROPICSAFE project, using 10 yellow sticky traps in a vineyard per week, showed that numbers of M. fuscovaria declined dramatically from 2017 following a prolonged drought in the Western Cape (Figure 31). Most of the rainfall in Vredendal is received during the austral autumn and winter from April to August. In general, M. fuscovaria numbers are highest during June, when the rainfall is highest. The populations tend to decline in July and August, the coldest months in Vredendal and increase in spring in September. The hottest months of the year are from November in spring and December to February in summer. Leafhopper numbers are lowest during November and December. They increase in numbers again in January with a peak in February. M. fuscovaria has three peaks per year suggesting that this species has at least three generations in a year.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
A 70 2009-2016
60 2017-2019
50 (mean ± (mean SE)
40
30 Mgenia fuscovaria 20
Number of 10
0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
B
35 35 Precipitation Mean Tem perature 30 Min. Temperature 30 Max. Temperature
25 25
20 20
15 15 Precipitatiom (mm) Precipitatiom Temperature (°C)
10 10
5 5
0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Figure 31: Seasonal abundance of Mgenia fuscovaria in a vineyard infected 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).
Natural enemies. Generalist predators that feed on leafhoppers include predatory beetles such as lady beetles (Coleoptera, Coccinellidae), true bugs (Hemiptera), lacewings (Neuroptera, Chrysopidae) and spiders (Araneae). No parasitoids (parasitic wasps) have been reared from M. fuscovaria so far. However, species belonging to the Mymaridae (Hymenoptera), a family that includes egg parasitoids of leafhoppers, have been recorded in Vredendal and may contribute to the natural population regulation of M. fuscovaria.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Aster yellows phytoplasma transmission M. fuscovaria experimentally transmitted ‘Ca. P. asteris’ to grapevine and species in the grass (Poaceae) family (Krüger et al., 2011; 2015a). These include wheat (Triticum aestivum L), triticale (Triticosecale sp.), and maize (Zea mays L.). Triticale is a cover crop in vineyards. Maize is sometimes interplanted with grapevine in newly established vineyards (Figure 32). Both nymphs and adults can transmit the pathogen. It is not clear how long the time is between the acquisition of the pathogen by M. fuscovaria and the vector becoming infectious (latent period).
Figure 32: Examples of alternative plant hosts of aster yellows 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.
Management Sampling methods for M. fuscovaria. Methods used for sampling leafhoppers include sweep netting, vacuum sampling, and trapping with yellow sticky traps (Krüger and Fiore, 2019). The choice of methods depends on the purpose. Yellow sticky traps are recommended for monitoring (Figure 33). Sweep netting and vacuum sampling are recommended for surveys and can be combined with yellow sticky traps. Vacuum sampling is easier to standardize than sweep netting for quantitative surveys. Monitoring of M. fuscovaria. In order to support disease management decisions, regular monitoring is used to obtain information on the presence and variation in population size of insects. When comparing different colors for monitoring M. fuscovaria, individuals were more attracted to yellow, which had the highest reflectance of the colors tested, followed by lime green, green, blue and red in a field trial (Krüger et al., 2015b). Furthermore, the leafhopper was more attracted to AY-infected than uninfected leaves. AY-infected leaves had a higher reflectance compared to uninfected leaves (Krüger et al., 2015b). When evaluating whether the leafhopper responds to volatiles emitted from infected and uninfected leaves, the responses to volatiles from grapevines cv. Colombard and cv. Chenin blanc, were very weak (La Grange et al., 2017). Therefore, yellow traps without an olfactory attractant is recommended for monitoring this leafhopper species.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Figure 33: Monitoring of Mgenia fuscovaria with yellow sticky traps (A, B).
Weed management. Weeds may serve as a source of inoculum and a food source for M. fuscovaria. In vineyards, eleven plant species, belonging to seven families and 11 genera tested positive for AY presence with polymerase chain reaction (PCR): Apocynaceae: periwinkle [Catharanthus roseus (L.) G. Don]; Asteraceae: blackjack (Bidens bipinnata L.), white goosefoot (Erigeron bonariensis L.), sowthistle (Sonchus oleraceus L.); Brassicaceae: wild radish (Raphanus raphanistrum L.); Cucurbitaceae: Cucurbita sp.; Poaceae: sticky bristle grass [Setaria verticillata (L.) P. Beauv.], triticale (Triticosecale sp.), maize; Urticaceae: small stinging nettle (Urtica urens L.) (Krüger et al., 2015a); and Solanaceae: tomato (Solanym lycopersicum). A TROPCSAFE survey in the Fynbos and Succulent Karoo biomes in the natural vegetation in the three regions where AY has been reported resulted in the detection of a further plant species, Mesembryanthemum crystallinum (Aizoaceae), as host of AY (Krüger et al., 2019). The species is native to South Africa. Weed control is important for the removal of reservoirs of the pathogen, reducing overwintering sources for M. fuscovaria and thus, indirectly reducing the abundance of the insect vector. Chemical control. Management of AY in vineyards relies largely on the use of insecticides to control the insect vector. Several products with different modes of action have been registered in South Africa, including foliar sprays and soil drenches (Annex C). Chemical control should be combined with other management options to contain the spread of AY and at the same to avoid built up resistance and to reduce the negative impact of pesticides on the environment. Sanitation. An essential component of reducing the disease spread is the planting disease- free material when establishing new blocks. Sanitation measures to reduce inoculum source, potentially leading to fewer infected insect vectors, include the removal of infected grapevines with their roots after harvest, removal of symptomatic branches during the season, and removal of weeds as they may serve as alternative host plants of the aster yellows phytoplasma.
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Recommended management plan Management strategies, frequency and duration. M. fuscovaria is active all year round. Nymphs and adults have been recorded on grapevine during the growing season and on weeds that might host AY, during the remainder of the year. The recommended strategy is therefore to monitor the leafhopper not only through the growing season but throughout the year with yellow sticky traps. Management decisions and timing should be based on the presence and population size of M. fuscovaria, grapevine phenology, and climate. Annex C provides a seasonal management plan for the timing of the different control measures. Sanitation measures to prevent the disease spread include the planting of only aster yellows-free plant material, marking and removal infected plant material, e.g. shoots, and infected plants. A further important measure is the removal of alternative host plants of AY or of the insect vector. Weed management is essential as weeds from different plant families are hosts of aster yellows phytoplasma or, when grapevines are dormant, M. fuscovaria. Care should be taken to plant cover crops that are not hosts of the pathogen. Insecticides for foliar application or soil drenches that have been registered for the management of the insect vector should be used based on the presence and population size of the insect vector according to the manufacturer’s specifications (Krüger et al., 2015a).
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4. Antimicrobial peptides (AMPs) against grapevine yellows
Introduction Towards the end of the previous century an outbreak of grapevine yellows (GY) disease symptoms was observed in vineyards of several wine grape cultivars in South Africa. The disease was limited to the Olifants river production region and the associate agent was identified as aster yellows (AY) phytoplasma at the end of 2006 (Engelbrecht et al., 2010). Since the initial observations, the pathogen was characterized in terms of its host range and epidemiology (Carstens et al., 2011), insect vector (Krüger et al., 2011), and genetics (Zambon et al., 2015). Recently, the draft genome sequence of the South African AY stain was published (Coetzee et al., 2019). Phytoplasmas are intracellular obligate pathogens, and conventional control measures such as antibiotics are not allowed or too expensive and not always effective. In South Africa producers manage the disease by controlling the insect vectors and by rogueing symptomatic plants. Since no known resistance to these diseases is reported, very few breeding programs are in place. Similarly, very few transgenic resistance approaches have been pursued. One such approach that was attempted, was the introduction of an antimicrobial peptide (AMP) gene into the host plant. Rufo et al. (2017) reported the efficacy of transforming periwinkle with the synthetic AMP, BP100, against ‘Ca. P. rubi’ and ‘Ca. P. solani’. The AMP was highly effective when used preventively, and when administered as a treatment the disease symptomatology disappeared completely. However, ‘Ca. P. rubi’ and ‘Ca. P. solani’ were still detected in 75% and 50% of the treated plants, respectively. In earlier research a number of plant-derived AMPs, also from grapevine were identified and isolated (de Beer and Vivier, 2008). These AMPs were therefore tested for their suitability to potentially be used as a means to introduce resistance in grapevine to the AY phytoplasma.
Material and Methods Vector construction. pRSF and pJET GFP vectors were digested with BamHI/HindIII and BamHI/PstI restriction enzymes, respectively. The peptide-encoding fragments were digested with PstI/HindIII. The digested fragments were ligated with a ratio 1:1:1 and then transformed in Escherichia coli JM109. After being confirmed with Sanger sequencing, the clones were transformed into E. coli BL21 cells for expression. For modification of the vectors to include the accessory protein PLAC, both pRSF GFPwThrombin-peptides and the vector carrying PLAC were digested with HindIII/XhoI and then ligated together. The clones were verified again with Sanger sequencing and then transformed in E. coli BL21. Protein expression. The overnight E. coli BL21 cultures were inoculated in 2 liters baffled flasks containing 400 ml of Terrific Broth (Sigma-Aldrich) supplemented with 50 µg/ml kanamycin and incubated at 37°C shaking for 2-5 hours to the logarithmic growth phase. Protein expression was induced by the addition of IPTG at a final concentration of 0.1 mM at 26°C for 24 hours. Cultures were pelleted by centrifugation at 10°C for 15 minutes at 7,500 rpm. Bacterial cell pellets were stored at -20°C.
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His-tag purification by Ni-NTA affinity chromatography. Once thawed on ice for 15 minutes, the cell pellets were resuspended in a 10 ml/g SB containing 1: 300 EDTA-free protease inhibitor cocktail, 0.2 µl/ml DNase, 1 µl/ml RNase and 1 mg/ml lysozyme for cell lysis. The cells were resuspended and then were sonicated, using an Omni-Sonic Ruptor 4000 Sonicator. Lysates were then centrifuged at 12,000 rpm for 2 hours at 10°C. The supernatants (cleared lysates) were collected and transferred to sterile tubes and stored at -20°C. Nickel-nitrilotriacetic agarose (Ni-NTA) superflow columns (5 ml, Qiagen) were equilibrated with 10 column volumes of SB20 [50 mM Tris, 500 mM NaCl, 20 mM imidazole and 10% (v/v) glycerol] before loading the samples. The columns were then equilibrated with SB20 using the ÄKTA Purifier (GE Healthcare), followed by elution of the fusion proteins in SB500 [50 mM Tris, 500 mM NaCl, 500 mM imidazole and 10% (v/v) glycerol]. Eluted samples were stored at -20°C. Tricine-SDS-PAGE analysis. The fractions of eluted proteins were analyzed by 12-15% Tricine-SDS-PAGE electrophoresis. Protein samples were denatured by addition of an equal volume of 2 X sample buffer (200 mM Tris-HCl, pH 6.8, 40% glycerol, 2% SDS, 0.04% Coomassie blue G-250 and 310 mM DTT), followed by incubation at 37°C for 30 minutes. Electrophoresis was initially performed at 40 V for 30 minutes or until the dye front reached the end of the stacking gel, followed by electrophoresis at 200 V until complete. Gels were then immersed in a fixing solution and separated proteins were visualized by staining with the Coomassie blue. Protease cleavage. Peptides were cut with WelQut Protease at a concentration of 0.1 µl/ml. Uncut peptides (no protease treatment) were used as negative control. The digestion was performed at 30°C overnight. For thrombin digestions, 1 X Thrombin Cleavage Buffer, about 50 µg fusion protein and 0.1 U/µl, 0.04 U/µl, 0.02 U/µl of thrombin were added to a final reaction volume of 50 µl. Reactions were incubated at 37°C overnight. Activity test. Quantitative antifungal activity of the peptides against Fusarium oxysporum was measured as reported by de Beer and Vivier (2008). All the readings were corrected by subtracting the time zero readings from the 24- and 48-hour readings.
Results Four antimicrobial peptides (AMPs) VvAMP1, SN1pot, SN1vitis, VvScorpio were studied. The first two (VvAMP1 and SN1pot) were previously isolated and partially characterized in the SUN laboratory. They are active primarily against fungi such as F. oxysporum and Botrytis cinerea, but were been tested against phytoplasmas. SN1vitis was identified by BLAST analysis against the grapevine genome using the sequence of snakin1 potato (SN1pot). VvScorpio was found by differential gene expression analysis between grapevine plants infected with phytoplasmas and non-infected plants. The peptide is significantly overexpressed in the AY-infected plants, and therefore may be involved in the response to the phytoplasma infection. The four peptides were expressed using a bacterial expression system, i.e. the modified pRSF-Duet1 vector in E. coli. The system is based on His-tag purification. The peptide is also fused with GFP, providing easy detection of the expression and translation activities, and a reduction in toxicity of the peptide to the bacteria. All four
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peptides were successfully cloned into the expression vector (Figure 34a), expressed in bacteria and purified using His-tag purification (Figure 34b) and size exclusion chromatography. Subsequently the cleavage of the peptides from the GFP with WelQut protease (protease site between GFP and peptide) was attempted. SDS-PAGE analysis did not show any band corresponding to the peptide size, except for SN1 pot (Figure 35), suggesting either not successful cleavage or low concentration of the peptides.
F Primer HisStart GFP a b lacI PstI wPeptide HindIII pRSF GFPwPeptide
RSF ori KnR
Figure 34: a) Vector map for the expression vector pRSF with GFP, WelQut site (w) and the peptide b) example of His-tag purification of the lysate.
Figure 35: SDS-PAGE gel showing the comparison between peptides 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.
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Different other proteases were therefore considered and three different combinations were selected: thrombin, modified thrombin (missing the last two amino acids) and Nisp. To use these proteases it was necessary to modify the expression vector with the addition of the new protease cleavage sites. The initial strategy used a restriction-free cloning strategy. An overlapping mega-primers containing the new protease cleavage sites was designed and a PCR reaction using a fusion polymerase was performed. However, sequencing of the clones obtained showed that this strategy was not successful. A conventional cloning strategy using restriction/ligation to insert the new protease sites was then adopted. Positive clones were obtained using this strategy that were confirmed by Sanger sequencing (Figure 36). Expression of the peptide fused with GFP was performed and, after purification with His-tag columns, the peptide was digested with the proteases to test which of the three cleavage sites are functional. SDS-PAGE analysis after the digestion showed that only VvAMP1- Thrombin was digested, indeed a band the correct size for the peptide (about 6 kDa) was observed (Figure 37). To confirm the efficacy of the digestion with thrombin, and that the VvAMP1 is active, an assay with F. oxysporum was performed. It was found that VvAMP1 reduces F. oxysporum growth (between 50% and 70% growth inhibition) (Figure 38) as previously demonstrated in literature (de Beer and Vivier, 2008). Therefore, the peptide fused with GFP produced using this expression system and cleaved with thrombin is active and functional. Additionally, the coding sequences of the other peptides (Potato SN1, Vitis SN1 and Vitis Skorpio) were cloned into the same expression vector used for VvAMP1 production, also using the thrombin protease cleavage site. All four the peptides have been successfully expressed and digested with thrombin. The subsequent purification steps have proved to be challenging, indeed most of the peptide was lost during the buffer exchange, making the activity test not feasible at this stage due to the low concentration. A preliminary analysis of the Vitis Skorpio peptide showed a very low activity against F. oxysporum, even before purification. It was hypothesized that the low activity was caused by the incomplete or incorrect formation of disulphide bonds. From previous reports, it is known that some peptides may require accessory proteins to reach the correct structure required for their activity (Bédard et al., 2018). In order to obtain a better activity, the accessory protein PLaC was then expressed in the same vector as the peptides (Figure 39). Analysis of the activities from these protein purifications is in progress. VvAMP1 (with and without PLaC) will be the reference to evaluate the potential advantage of having this accessory protein, since its activity against F. oxysporum is well documented. In the meantime, bigger quantities of the peptides have been produced and the crude extracts will used to test against phytoplasmas.
Conclusions In spite of several technical difficulties and the situation that is are still under improvement, antimicrobial activity by co-expressing the accessory protein for proper folding, it is possible to produce adequate quantities of crude extract of the peptides to be tested for efficacy against the aster yellows phytoplasma.
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Figure 36: Schematic representation of recombinant expression vectors. (A) pRSF GFPwThrombin(long)-Vv-AMP1; (B) pRSF GFPwThrombin(short)-Vv-AMP1; and (C) pRSF GFPwNisP-Vv-AMP1.
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Figure 37: Tricine-SDS-PAGE (15% Tris-Tricine gel) analysis by 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).
Fusarium growth 24 hours Fusarium growth 48 hours
0,25 1,4 0,2 1,2 1 0,15 0,8 0,1 0,6 0,4 0,05 0,2 0 0 Dig Vvamp1 Dig Vvamp1 Dig Vvamp1 non dig Dig Vvamp1 Dig Vvamp1 Dig Vvamp1 non dig 60 30 15 VVamp1 60 30 15 VVamp1
Figure 38: VvAMP1 activity against F. oxysporum. Micro-spectrophotometric readings were recorded at 24 and 48 hours. Different concentrations of thrombin were tested 600 U (60), 300 U (30) and 150 U (15). The non-digested peptide was used as control (non-dig VVamp1).
GFP
WELQut Thrombin primer R primer F VvAMP1 pRSF-GFPVvA MP1 Thrombin Plac HindIII (1008) 5087 bp prsfMCSR MCS2F Forward primer region PlaC XhoI (1613)
Figure 39: Schematic representation of recombinant expression vector with accessory protein PLaC.
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ANNEX A: Sites sampled to study the parasitoid complex of Trioza erytreae in South Africa
Sampling dates and sites, number and variety of sampled trees per site, symptoms and presence of Trioza erytreae. Asterisks indicate parasitized T. erytreae.
Number of Sampling Type of Rutacea T. erytreae Presence of Locality Province Coordinates sampled date orchard Host symptoms T. erytreae trees Western 32°36'27.3"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 18°56'37.8"E sinensis Western 32°36'31.6"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 18°56'39.9"E reticulata Western 32°36'23.3"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 18°56'51.7"E sinensis Western 32°36'37.2"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 18°56'32.1"E limon Western 32°35'43.2"S Private Citrus 21/09/2017 Citrusdal 2 No No Cape 19°00'50.2"E garden sinensis Western 32°51'18.0"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 19°05'55.8"E sinensis Western 32°51'19.0"S Citrus 21/09/2017 Citrusdal Commercial 50 No No Cape 19°05'40.4"E sinensis Western 32°36'37.3"S Organic Citrus 21/09/2017 Citrusdal 50 No No Cape 19°00'01.2"E commercial limon Western 32°36'27.2"S Organic Citrus 21/09/2017 Citrusdal 50 No No Cape 18°59'55.5"E commercial limon Western 32°36'42.4"S Organic Citrus 21/09/2017 Citrusdal 50 No No Cape 19°00'05.5"E commercial reticulata Western 32°37'03.5"S Organic Citrus 22/09/2017 Citrusdal 10 No No Cape 18°57'20.6"E commercial reticulata Western 32°37'03.5"S Private Citrus 22/09/2017 Citrusdal 4 No No Cape 18°57'20.6"E garden sinensis Western 32°37'03.5"S Private Citrus 22/09/2017 Citrusdal 2 No No Cape 18°57'20.6"E garden limon Western 32°21'23.4"S Organic Citrus 22/09/2017 Citrusdal 50 No No Cape 18°55'52.5"E commercial sinensis Western 32°21'28.7"S Organic Citrus 22/09/2017 Citrusdal 50 No No Cape 18°55'56.2"E commercial sinensis Western 33°49'21.8"S Private Citrus 22/09/2017 Stellenbosch 100 No No Cape 18°55'48.1"E garden sinensis Western 33°56'11.0"S Private Citrus 22/09/2017 Stellenbosch 5 No No Cape 18°51'56.3"E garden sinensis Mpumalan 25°27'09.0"S Citrus 26/09/2017 Nelspruit Assay 50 No No ga 30°58'06.4"E sinensis Mpumalan 25°27'07.9"S Citrus 26/09/2017 Nelspruit Assay 50 No No ga 30°58'16.1"E limon Mpumalan 25°23'04.3"S Citrus 27/09/2017 Nelspruit Nursery 20 Yes No ga 30°32'33.8"E sinensis Mpumalan 25°23'06.7"S Citrus 27/09/2017 Nelspruit Commercial 20 No No ga 30°32'34.1"E sinensis Mpumalan 25°22'44.2"S Organic Citrus 27/09/2017 Nelspruit 50 No No ga 30°31'50.7"E commercial limon Mpumalan 25°22'44.2"S Abandoned Citrus 27/09/2017 Nelspruit 10 No No ga 30°31'50.7"E orchard reticulata
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Number of Sampling Type of Rutacea T. erytreae Presence of Locality Province Coordinates sampled date orchard Host symptoms T. erytreae trees Mpumalan 25°22'35.5"S Private Citrus 27/09/2017 Nelspruit 1 No No ga 30°32'30.8"E garden limon Mpumalan 25°22'49.1"S Private Citrus 27/09/2017 Nelspruit 1 No No ga 30°34'03.0"E garden limon Mpumalan 25°22'35.7"S Citrus 27/09/2017 Nelspruit Commercial 1 No No ga 30°32'33.0"E limon Mpumalan 25°26'42.8"S Public Citrus 27/09/2017 Nelspruit 0 No No ga 30°57'57.4"E garden aurantium Mpumalan 25°27'31.5"S Citrus 28/09/2017 Nelspruit Commercial 0 Yes No ga 31°02'46.5"E sinensis Mpumalan 25°27'12.2"S Abandoned Citrus 28/09/2017 Nelspruit 20 No No ga 31°01'58.2"E orchard sinensis Mpumalan 25°27'30.2"S Abandoned Citrus 28/09/2017 Nelspruit 20 No No ga 31°02'35.0"E orchard limon Mpumalan 25°27'58.2"S Citrus 28/09/2017 Nelspruit Commercial 50 No No ga 31°02'32.4"E limon Mpumalan 25°28'48.4"S Citrus 28/09/2017 Nelspruit Assay 30 Yes Yes* ga 30°59'38.2"E limon Mpumalan 25°27'07.7"S Citrus 29/09/2017 Nelspruit Nursery 50 No No ga 30°58'09.5"E sinensis Mpumalan 25°27'07.4"S Citrus 29/09/2017 Nelspruit Assay 50 Yes Yes* ga 30°58'15.9"E limon Mpumalan 25°28'21.9"S Public Citrus 29/09/2017 Nelspruit 10 Yes No ga 30°59'29.8"E garden aurantium Mpumalan 25°28'45.8"S Citrus 29/09/2017 Nelspruit colony 20 No No ga 30°59'38.0"E sinensis 23°52'01.0"S Privat Murraya 03/10/2017 Letsitele Limpopo 1 No No 30°23'20.0"E garden exotica 23°52'07.0"S Privat Murraya 03/10/2017 Letsitele Limpopo 3 No No 30°23'29.6"E garden exotica 23°51'44.7"S Citrus 03/10/2017 Letsitele Limpopo Commercial 50 No No 30°23'01.9"E sinensis 23°55'11.8"S Private Citrus 03/10/2017 Tzaneen Limpopo 3 No No 30°13'58.4"E garden limon 23°50'24.0"S Citrus 03/10/2017 Tzaneen Limpopo Commercial 150 No No 30°18'07.2"E sinensis 23°55'11.8"S Organic Citrus 04/10/2017 Tzaneen Limpopo 150 Yes No 30°13'58.4"E commercial sinensis 23°54'42.1"S Organic Citrus 04/10/2017 Tzaneen Limpopo 150 Yes No 30°13'28.0"E commercial sinensis 23°53'14.7"S Organic Citrus 04/10/2017 Tzaneen Limpopo 150 Yes No 30°19'44.8"E commercial sinensis 23°55'06.3"S Organic Citrus 04/10/2017 Tzaneen Limpopo 150 Yes No 30°13'59.5"E commercial sinensis 23°55'07.9"S Organic Citrus 04/10/2017 Tzaneen Limpopo 150 Yes No 30°13'55.4"E commercial sinensis 23°53'24.4"S Private Citrus 04/10/2017 Nkowankowa-C Limpopo 2 No No 30°19'40.7"E garden limon 23°53'27.9"S Private Citrus 04/10/2017 Nkowankowa-C Limpopo 3 Yes No 30°19'39.6"E garden limon 23°53'25.6"S Private Citrus 04/10/2017 Nkowankowa-C Limpopo 1 No No 30°19'42.1"E garden limon
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
Number of Sampling Type of Rutacea T. erytreae Presence of Locality Province Coordinates sampled date orchard Host symptoms T. erytreae trees 23°53'19.3"S Private Citrus 04/10/2017 Nkowankowa-C Limpopo 2 No No 30°19'43.8"E garden limon 23°53'31.0"S Private Citrus 04/10/2017 Nkowankowa-C Limpopo 1 Yes No 30°19'45.3"E garden limon Citrus 23°50'13.7"S Private 05/10/2017 Tzaneen Limpopo limon/reticu 5 Yes Yes* 30°09'37.8"E garden lata 23°47'56.8"S Private Citrus 05/10/2017 Tzaneen Limpopo 5 No No 30°26'11.0"E garden limon 23°47'54.1"S Private Citrus 05/10/2017 Tzaneen Limpopo 50 No No 30°26'07.9"E garden limon 25°44'52.8"S Experimenta Citrus 05/10/2017 Pretoria Gauteng 10 No No 28°15'32.1"E l farm limon 25°44'52.1"S Experimenta Citrus 05/10/2017 Pretoria Gauteng 25 Yes Yes* 28°15'33.6"E l farm limon 25°45'40.6"S Private Citrus 09/10/2017 Pretoria Gauteng 4 No No 28°14'12.5"E garden sinensis Private Citrus 09/10/2017 Pretoria Gauteng - 3 Yes Yes garden limon Private Murraya 09/10/2017 Pretoria Gauteng - garden exotica 25°44'21.4"S Public Citrus 09/10/2017 Pretoria Gauteng 10 No No 28°16'24.7"E garden aurantium Private Citrus 10/10/2017 Pretoria Gauteng - 5 Yes No garden limon 25°45'30.5"S Public Citrus 20/10/2017 Pretoria Gauteng 3 No No 28°14'31.4"E garden sinensis Private Citrus 01/11/2017 Pretoria Gauteng - 1 Yes No garden limon 25°49'55.6"S Public Citrus 17/11/2017 Pretoria Gauteng 5 No No 27°53'20.3"E garden sinensis
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
ANNEX B: Protocol for coconut palm micropropagated plants acclimatization
Materials and facilities. Soil, sand, peat moss, black polyethylene bags (10 x 26 cm), clear polyethylene bags (24.5 x 33 cm), 3-hole paper punch, duct tapes in several colors, permanent marker, 500-ml wash bottles, scissors, 20 liter plastic buckets, rubber bands, plastic containers to transport plants. For the acclimatization process of coconut in vitro plants, it is required a 15 x 15 m greenhouse (or larger depending on the size of the operation). The greenhouse must have: shade cloth in roof and the surrounding area 70% to 80%; water nebulizers or micro-sprinklers for plant irrigation with temperature and humidity regulation; in the area around the greenhouse, sinks with jetted tap water must be available for washing the plants; work tables, minimum three 1.5 m long; a clean and dry area for the preparation of the substrate mixture and filling of bags with it. Acclimatization procedure. Prepare the substrate by mixing soil, sand, and peat moss in a 1: 1: 1 ratio; fill the black polyethylene bags with the substrate, these bags must already be perforated at the bottom; wet the substrate contained in the bags with enough water. Then remove the plants from their containers (Figure 26A); wash the plants with tap water to eliminate culture medium residues (Figure 26B); with the sterile scissors remove the necrotic leaves, taking care not to damage the plants. Then introduce each plant within a black polythene bag with substrate and wet it, taking care not to break the root (Figure 26C); cover the plants with a clear polyethylene bag, this bag should have three cuts on each side approximately 2 cm long (Figures 26D and 26E); the clear bag should overlap the black bag, seal them with rubber bands (Figures 26F and 26G). Then, one week after planting, the number of cuts in the clear bag should increase to six on each side. After two weeks from planting, the clear bag is removed and the plant is taken to the greenhouse (Figure 26H).
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727459
ANNEX C: Seasonal plan for aster yellows management in the Western Cape
Seasonal plan for aster yellows phytoplasma management in grapevines in the Western Cape. The season commences in the austral winter in July.
Aster yellows management matrix for grapevines
Month *(peak vector activity) July Aug * Sep Oct Nov Dec Jan * Feb March April May * June
Pruning/ Shoot Post Post Vineyard stage Pruning Bud burst Flowering Veraison Crop ripening Harvest Harvest Winter rest bud burst growth harvest harvest
Weed control Weed control Weed control (herbicide, (herbicide, (herbicide, Alternative plant hosts control mechanically mechanically mechanically or manually) or manually) or manually) Removal of Removal or marking infected grapevine Planting only healthy marked Sanitation measures material (shoots, cordons, whole vine) as grapevines infected soon as symptoms appear grapevines Imidacloprid Soil drench (Kohinor 350 SC) Vector Chlorpyrifos control (Dursban 480 EC; Foliar spay Use Dursban 750 WG) registered Indoxacarb (InCide products 300 WG; Margin 300 Foliar spay only; mind WDG; Steward; withholding Steward 150 EC) periods Natural pyrethrins Foliar spay (Xterminator)
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