Other materials

This video shows the highlights of the research activities conducted by the consortium from 2017 until 2022.

The partner of Tropicsafe CIRAD has published a research article under teh title "Specific Physiological and Anatomical Traits Associated With Polyploidy and Better Detoxification Processes Contribute to Improved Huanglongbing Tolerance of the Persian Lime Compared With the Mexican Lime".

Cécile Morillon and TROPICSAFE Guadelaoupe partners, CIRAD, designed this entertaining comic about Huanglongbing (HLB), the Citrus Disease. HLB is one of bacterial diseases threatening selected tropical and subtropical areas and is one of the study object in the project. The comic tells the story of the "Dragon Yellow Disease", the insects vectors and the interesting process followed in the TROPICSAFE project to give solution to this problem.

Citrus production has been widely affected by pathogens which causes economic losses to the growers resulting in severe social effects in many of the areas where the economy is based on agriculture. In the past decade citrusassociated phytoplasmas have originated widespread epidemics in some citrus-growing regions of the world. They are associated to either specific symptoms such as witches’ broom or non-specific symptoms like leaf yellowing, leaf mottling and lopsided fruit. So far about 21 citrus cultivars have been reported to be infected by phytoplasmas enclosed in 11 ribosomal groups; among these 16SrI (aster yellows) and 16SrII (peanut witches’ broom) are the most widespread. Here symptomatology, economic importance, transmission and strategies used for disease management are reported together with the molecular classification of the detected phytoplasma strains in Asian, American and African continents.

The confirmation of the presence of 16SrXXII-B phytoplasmas associated with coconut lethal yellowing disease in Ghana and of 16SrIV in the Caribbean (Jamaica, Cuba, and Mexico) were the basis to study alternative plant species and potential insect vectors of this economically relevant disease carried out by the TROPICSAFE project started in 2017. In South Africa grapevine yellows disease associated with ‘Candidatus Phytoplasma asteris’, (subgroup 16SrI-B) and transmitted by the leafhopper Mgenia fuscovaria was studied in its main epidemiological aspects that are relevant to its management. In Chile and in Italy phytoplasmas belonging to diverse ribosomal subgroups and several alternative host plants were detected. Some insect species have been described as phytoplasma vectors or potential vectors in grapevine or in vineyard environments.

Citrus greening or huanglongbing (HLB) is the main threat to the European citrus industry since one of its vectors, the African citrus psyllid, Trioza erytreae, has recently become established in mainland Europe. In this context, classical biological control programmes should be implemented to reduce the spread of the psyllid. The aims of this study were to: i) disentangle the parasitoid complex of T. erytreae combining morphological and molecular characterization; and ii) to study the biology of its main parasitoids in its area of origin in South Africa for their future importation into Europe. The main citrus producing areas of South Africa were surveyed during 2017. In contrast to previous studies, the parasitoid complex of T. erytreae included three species of primary parasitoids: Tamarixia dryi, Psyllaephagus pulvinatus and another parasitoid of the genus Tamarixia. Molecular analysis showed that it is a new species closely related to T. dryi. Tamarixia dryi was the most abundant parasitoid but its relative abundance varied among sampling sites. The sex ratio (males/females) of T. dryi and Tamarixia sp. decreased with T. erytreae size and became female biased when psyllid nymphs were larger than 0.6 and 1.2 mm2, respectively. These parasitoids were attacked by three species of hyperparasitoids, Aphidencyrtus cassatus, Marietta javensis and a species of the genus Aphanogmus. Aphidencyrtus cassatus, the most abundant hyperparasitoid, tended to emerge from large nymphs, and adult females lived as long as those of T. dryi. The implications of these results are discussed within the framework of the introduction of T. dryi into Europe.

A new species of cixiid planthopper (Hemiptera: Fulgoroidea) in the genus Oecleus Stål, Oecleus mackaspringi sp. n., is described from Spring Garden, Jamaica. This new taxon is associated with coconut palm (Cocos nucifera L.) and was found while surveying palm plots with active cases of lethal yellowing (LY). This is the first report of the genus Oecleus from Jamaica. Sequence data for the COI gene and 18S gene are also provided.

Coconut lethal yellowing (LY) is the single most important disease presently affecting the coconut production worldwide. Symptomatic and asymptomatic coconut plants were sampled in some selected areas to verify the identity of phytoplasmas associated with LY in Cuba. Diverse phytoplasma ribosomal groups were detected in the samples from symptomatic palms such as 16SrXII, 16SrVII, and 16SrI. In several other palms 16SrIV-A subgroup phytoplasmas were identified, in the groEL gene the only positive plant from Pilón resulted diverse from the others and identical to the 16SrIV-A strains detected in Jamaica LY infected coconut palms. This is the first record of occurrence of 16SrI, -VII and -XII groups in coconut palms in Cuba.

Two of the most common leafhoppers present in Chile are the Cicadellidae Paratanus exitiosus and Bergallia valdiviana. They commonly occur in vineyards of central Chile, including some vineyards infected by phytoplasmas. The present study demonstrates that P. exitiosus and B. valdiviana can transmit 16SrIII-J phytoplasmas to grapevine and periwinkle plants. This provides improved understanding of the 16SrIII-J phytoplasma epidemiology in Chilean vineyards.

The development of methods for culturing phytoplasmas in artificial media suggested the evaluation of strategies to produce antibodies against a phytoplasma target. To this purpose, an aster yellows phytoplasma isolate obtained from field collected infected grapevine was used for the production of a polyclonal antiserum. It was then tested by ELISA and IFAS methods in order to check and evaluate its suitability for phytoplasma detection in plant samples.

“Huanglongbing” (HLB) is one of the most devastating citrus diseases in the world. Citrus samples with symptoms referable to HLB were collected in different Caribbean Countries to verify the possible association of ‘Candidatus Phytoplasma’ and HLB. In Cuba, 140 samples out of 142, resulted positive for HLB and 32 were positive also for phytoplasmas. In Jamaica, out of 6 samples, 5 were positive to HLB and 1 was positive to phytoplasmas. In Guadeloupe, 17 samples were positive for HLB and 7 for phytoplasmas. These results indicate that the symptomatology is not discriminating between the two pathogens and the mixed infection is not an uncommon event.

Surveys in and nearby vineyards in some Italian provinces were carried out to detect alternative host plants and possible insect vectors of phytoplasmas detected in grapevines. Aster yellows is the prevalent phytoplasma detected in both beside the 16SrXII-A and 16SrV-C phytoplasmas. Moreover other phytoplasmas belonging to diverse ribosomal groups were identified indicating a relevant epidemiological role of spontaneous vegetation and possible new insect vectors in the grapevine yellows.

Potential insect vectors and alternative host plants of the phytoplasmas associated with grapevine yellows (GY) were surveyed in the Fynbos and Succulent Karoo biomes in the Western Cape, South Africa. Aster yellows phytoplasma (16SrI-B), which has been reported infecting grapevine in three regions in the Western Cape, was identified in a plant species belonging to the Aizoaceae. Other phytoplasmas were detected from species belonging to the Brassicaceae, Montiniaceae, Proteaceae and Zygophyllaceae and in a few insect specimens. The information will be used to confirm the insect vector status and the role of the plant species identified as alternative host plants in controlled transmission experiments.

A survey to verify the presence of grapevine yellows was carried out in a restricted area of the Marche region (central east Italy) in a small geographic area. In two locations six grapevine varieties were examined and phytoplasmas belonging to four ribosomal groups were detected by PCR/RFLP analyses. The surveyed varieties were Passerina, Pecorino, Merlot, Sangiovese, Fedit and Montepulciano. The different phytoplasmas detected were present in a scattered manner in all the varieties, but only Passerina, Pecorino and Sangiovese where shown to be infected by “bois noir” (16SrXII-A) phytoplasmas. Considering the landscape shape and the age of the vineyards the source of BN infection and of the other phytoplasmas is very likely in the surrounding environment or vineyards.

Grapevine yellows (GY) are often associated with molecularly differentiable phytoplasmas according to their geographic distribution and sometimes also to different varieties. A review of the most recent findings worldwide is presented.

The project addresses insect-borne prokaryote-associated diseases of palm, citrus and grapevine in tropical and subtropical areas seriously affecting the trade and import of agricultural products and materials worldwide. These diseases are due to the presence of ‘Candidatus Phytoplasma’ associated with “lethal yellowing” in palms and “yellows” in grapevine, and ‘Ca. Liberibacter’ associated with “huanglongbing” in citrus. Their negative impact on agriculture may be worsened as a result of climate change. The management of these diseases is exploited in Africa (Ghana, South Africa and Mozambique), the Americas (Mexico, Chile, Guadalupe, Jamaica, and Cuba), and Europe in selected areas where the prevalence of these diseases is severe giving rise to social and economic threats, affecting local agriculture and export of products. Knowledge and technologies available for detection and identification of these pathogens and new ones implemented by the project will be applied in the studied regions for epidemiological studies aimed at filling knowledge gaps. Integrated pest management strategies based also on innovative diagnostic and on exploited prevention tools such as reduced insecticide treatments, and improved resistant germplasm are applied and pest risk assessment schemes developed. The solutions that are under testing are evaluated to assess the economic and social impact of these diseases, and their distributive effects on the rural communities in the target countries The 22 partners from 12 countries are collaborating with local plant protection organizations, farmers and producers who are actively participating in training and practical application of the developed tools and schemes for the exploitation of the project outcome and results.

The diseases associated with phytoplasmas in grapevine are collectively called yellows and occur in the majority of grapevine growing regions over the world. Among these diseases the “bois noir” is the most widespread and in most of the cases is it endemic in vineyards. Molecular variability was reported in the ‘Candidatus Phytoplasma solani’ (“stolbur”, 16SrXII-A) infecting grapevines, and in some cases a link to epidemiological aspect of the genomic features detected by MLT was hypotized. Statistical modeling to allow the prediction of the disease in specific grapevine growing areas is also developed. Studies about grapevine- “bois noir” phytoplasma interaction were focusing on some of the main genes regulated in the plant response. A study linking wine production and quality from healthy and “bois noir” infected grapevines showed the negative influence of the disease on wine production but only when the weather conditions were favorable to the production. Perspectives for improved management of the “bois noir” disease were also explored.

Potential insect vectors and alternative host plants of pathogens associated with citrus greening in citrus (Citrus spp., Rutaceae) and grapevine yellows (GY) in grapevine (Vitis vinifera, Vitaceae) were surveyed in the fynbos and succulent karoo biomes in the Western Cape in South Africa. The insect-transmitted diseases are associated with the presence of bacterial pathogens (‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’) that threaten the production and international trade of these crops. Despite previous efforts, the knowledge concerning potential insect vectors and the host range in indigenous host plants is limited. Three surveys were carried out in September (spring) in 2017, and January (summer) and August (winter) in 2018 in the natural vegetation at nine sites. Insects were collected with vacuum sampling from 20 randomly selected plant samples per species at the specific sites. Branches with leaves from the same plant species were collected, and both insects and plant samples were tested for the presence of phytoplasmas and liberibacters by quantitative polymerase chain reaction (real time PCR) or nested PCR and sequencing. Alternative host plants from 989 samples representing 42 species belonging to 19 families were analysed. None of the 42 plant species tested positive for ‘Ca. Liberibacter africanus’, the agent of citrus greening in South Africa. Aster yellows phytoplasma (16SrI-B), which has been reported infecting grapevine in three regions in the Western Cape, was identified in a plant species belonging to the Aizoaceae. Other phytoplasmas were detected from species belonging to the Brassicaceae, Montiniaceae and Proteaceae and in a few insect specimens belonging to the Auchenorrhyncha (Hemiptera).

Three 16SrIII-J phytoplasma-positive vineyards were visited each 15 days from September 2017 to August 2018 to capture insects using entomological sweep nets and yellow sticky traps. Additionally weeds showing typical symptoms associated with the presence of phytoplasmas were also collected. The phytoplasma 16SrIII-J was detected for the first time in the leafhoppers Amplicephalus ornatus, A. pallidus and Bergallia sp., and in the weed Brassica campestris. Another phytoplasma classified as 16SrXI, was detected in one leafhopper belonging to the genus Paratanus. The identification of the 16SrXI ribosomal subgroup and the species of Bergallia sp. and Paratanus sp. are in progress, as well as the transmission trials with these two insects plus A. pallidus and A. ornatus.

Several phytoplasmas were associated to grapevine yellows in Chile. Among them, 16SrIII-J is the prevalent in the vineyards of the central zone of the country (Gajardo et al. 2009; Gonzalez et al. 2010). This phytoplasma has been reported infecting various crops and spontaneous plants in Chile, suggesting the necessity of epidemiological studies to identify its insect vectors (Gonzalez et al. 2011; Quiroga et al. 2017). Several leafhoppers (Hemiptera, Auchenorryncha, Cicadellidae) were found positive to phytoplasma presence, including Bergallia valdiviana Berg 1881 among the most common species present in infected vineyards. Transmission trials carried out using periwinkle [(Cantharanthus roseus L. (G. Don.)] as indicator plant, show that B. valdiviana is vector of 16SrIII-J phytoplasmas (Quiroga et al. 2015). The purpose of this work was to demonstrate that B. valdiviana transmits the 16SrIII-J phytoplasma also to the grapevine.

The 16SrIV-A phytoplasmas are associated with the devastating disease lethal yellowing (LY) of palms. In Tabasco (Mexico), the death of Cocos nucifera, Adonidia merrillii, and Attalea butyracea palms have been suspected to be associated with LY based on symptomatology. Samples from the trunk of both symptomatic and nonsymptomatic palms were collected in three different environments: two species of palms within a rural zone and the other within an urban zone. DNA was extracted to perform a nested PCR with phytoplasma primers P1/P7-LY16SF/R16R2. A 1,345-bp fragment was amplified from the DNA extracted from each of the 29 LY-symptomatic palms sampled. Phytoplasma identification was achieved by amplicon sequencing and virtual restriction fragment length polymorphism analyses. Three 16SrIV phytoplasma subgroups were detected: 16SrIV-A in C. nucifera, 16SrIV-B in A. merrillii, and 16SrIV-D in C. nucifera, A. merrillii, and A. butyracea. Phylogenetic analysis showed also that the three phytoplasma strains found in the palm species clustered with phytoplasmas reported in the literature in the three subgroups identified. This is the first report of phytoplasmas associated with these palm species in Tabasco.

The Cape St. Paul wilt disease is a lethal yellowing type disease of coconut in Ghana. It is associate with the presence of a phytoplasma belonging to the 16SrXXII-B subgroup also referred to as ‘Candidatus Phytoplasma palmicola’-related strain. A leucyl tRNA gene based assay was used to discriminate the two strains of the phytoplasma detected in Ghana since sequences from the 16S rRNA gene were unable to reveal these genetic differences in the phytoplasmas.

At present serological assays for the detection of “flavescence dorée” (FD), a quarantine disease of grapevine, are not available. The aim of this study was to produce polyclonal antibodies targeting a surface abundant protein of FD and related phytoplasmas, Imp (immunodominant membrane protein), and to verify its use for the detection of FD phytoplasmas in grapevine, other host plant species, and insect vectors. Two antisera were obtained using recombinant Imp proteins from two FD strains. Preliminary results confirmed the specificity and good ELISA performance in the detection of the target in grapevine, clematis and insect vectors tissues.

The African citrus psyllid, Trioza erytreae, vectors citrus greening or huanglongbing (HLB) disease. The psyllid has been reported from mainland Europe, where it is rapidly spreading from the northwest to the southwest of the Iberian Peninsula. In order to reduce its spread and population levels, a classical biological control program with the parasitoid Tamarixia dryi is under development in Spain. We evaluated the host specificity of T. dryi using 11 non-target psyllid (NTP) species, including five species of the genus Trioza. The psyllids were selected based on phylogenetic and ecological criteria. Tamarixia dryi exhibited a high host specificity. Females did not parasitize any of the 11 NTPs tested, except for one nymph of a gall-forming Trioza species closely related to Trioza montanetana. Tamarixia dryi only laid one egg on a nymph when it was removed from the gall on Convolvulus canariensis and exposed directly to the parasitoid. However, the immature parasitoid died before emerging. We further confirmed that T. dryi did not parasitize a representative triozid species, Trioza laurisilvae, of the endemic Canarian fauna after long time exposure. Our results demonstrate that T. dryi is a highly specific parasitoid and its introduction, release and establishment in Europe within the classical biological control program of T. erytreae should not affect other psyllid species. Therefore, no significant environmental impact is expected.

Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) vectors the most important citrus disease, namely huanglongbing (HLB) or citrus greening. This vector has been recently found on the European mainland in the north-western region of the Iberian Peninsula (Spain and Portugal), although HLB has not yet been detected in Europe. Thus, preventing the spread of the vector is currently an urgent priority. Here, we show two prevention methods to avoid the spread of this psyllid from areas where it is present to psyllid-free areas. Our results showed that two types of anti-insect mesh with a pore size of 0.4 × 0.77 mm2 and 0.27 × 0.77 mm2 were effective in excluding T. erytreae in two field experiments. These results have direct implications in the nursery plant production system, which should adopt meshes with pore sizes small enough to guarantee the exclusion of T. erytreae. Additionally, we evaluated the longevity of T. erytreae on fruit to understand the risk of dispersion of the psyllid with unprocessed fruit movement between infested and non-infested areas. The longevity of T. erytreae on mandarin and lemon fruit under winter conditions (13.8°C and 85.4% of relative humidity) was 11 days for both fruits.

In the field of plant health, and in particular for unculturable pathogens or pathogens difficult to grow, there is a lack of availability of reference materials, here defined as well-characterized material suitable to be used as reference positive test control. Reference materials differ in their commutability and complexity, as well as the effort required for their preparation and characterization. Here the preparation of two types of reference materials based on synthetic DNA and well characterized DNA of naturally contaminated plant material of grapevine on an example of “stolbur” phytoplasma associated with “bois noir” disease of grapevine is described. The materials are suitable as controls for quantitative PCR with the specific target of the 16SrXII group and for a wide variety of molecular tests. They provide comparability among tests, laboratories and over time, and are a useful reference in validation studies, test performance studies and proficiency tests.

Genome sequence data is becoming more available and represents a source of potential target sequences for specific molecular tests to verify the feasibility and efficiency of genome-inform test design the attempt to identify novel specific sequences in the publicly available genomes of aster yellows phytoplasmas and use them to design primers for a spcific LAMP assays. Coupling RUCS automated analysis with PrimerExplorer V5 primer designer and applying criteria for selection of assays at each stage of the process resulted in 13 promising LAMP assays targeting four newly identified regions specific to aster yellows phytoplasmas.

To date, phytoplasmas belonging to six ribosomal subgroups have been detected to infect grapevines in Chile in 36 percent of the sampled plants. A new survey on the presence of grapevine yellows was carried out from 2016 to 2020, and 330 grapevine plants from the most important wine regions of the country were sampled and analyzed by nested PCR/RFLP analyses. Phytoplasmas enclosed in subgroups 16SrIII-J and 16SrVII-A were identified with infection rates of 17% and 2%, respectively. The vineyards in which the phytoplasma-infected plants were detected were further inspected to identify alternative host plants and insects of potential epidemiological relevance. Five previously unreported plant species resulted positive for 16SrIII-J phytoplasma (Rosa spp., Brassica rapa, Erodium spp., Malva spp. and Rubus ulmifolius) and five insect species were fully or partially identified (Amplicephalus ornatus, A. pallidus, A. curtulus, Bergallia sp., Exitianus obscurinervis) as potential vectors of 16SrIII-J phytoplasmas. The 16SrVII-A phytoplasmas were not detected in non-grape plant species nor in insects. This work establishes updated guidelines for the study, management, and prevention of grapevine yellows in Chile, and in other grapevine growing regions of South America.

Huanglongbing (HLB) is presently a major threat to the citrus industry. Because of this disease, millions of trees are currently dying worldwide. The putative causal agent is a motile bacteria belonging to Candidatus Liberibacter spp., which is transmitted by psyllids. The bacteria is responsible for the synthesis of callose at the phloem sieve plate, leading to the obstruction of the pores that provide connections between adjacent sieve elements, thus limiting the symplastic transport of the sugars and starches synthesized in leaves to the other plant organs. The Persian triploid lime (Citrus latifolia) is one of the most HLB-tolerant citrus varieties, but the determinants associated with the tolerance are still unknown. HLB-infected diploid Mexican lime (Citrus aurantiifolia) and Persian lime were investigated. The leaf petiole was analyzed using scanning electron microscopy (SEM) to observe callose deposition at the phloem sieve plate. Leaf starch contents and detoxification enzyme activities were investigated. In the field, Persian lime leaves present more limited symptoms due to HLB than the Mexican lime leaves do. Photosynthesis, stomatal conductance, and transpiration decreased compared with control plants, but values remained greater in the Persian than in the Mexican lime. Analysis of the petiole sieve plate in control petiole samples showed that pores were approximately 1.8-fold larger in the Persian than in the Mexican lime. SEM analyses of petiole samples of symptomatic leaves showed the important deposition of callose into pores of Mexican and Persian limes, whereas biochemical analyses revealed better detoxification in Persian limes than in Mexican limes. Moreover, SEM analyses of infected petiole samples of asymptomatic leaves showed much larger callose depositions into the Mexican lime pores than in the Persian lime pores, whereas biochemical traits revealed much better behavior in Persian limes than in Mexican limes. Our results reveal that polyploids present specific behaviors associated with important physiological and biochemical determinants that may explain the better tolerance of the Persian lime against HLB compared with the Mexican lime.

“Huanglongbing” (HLB) is one of the most devastating diseases of citrus orchards worldwide. Samples from 183 citrus plants of different cultivars and rootstock/cultivar combinations, showing HLB symptoms in three Caribbean countries (Cuba, Jamaica, and Guadeloupe-France), were collected to verify the possible co-infection of ‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’ species. The 64% of the samples resulted positive to the ‘Ca. L. asiaticus’ and the 27% to diverse ‘Ca. Phytoplasma’-related species, moreover about the 14% of the samples infected with ‘Ca. Liberibacter’ were also found positive to phytoplasmas, indicating the presence of mixed infection especially in the orchards located in Cuba. Moreover, in one of the samples from Jamaica mixed phytoplasma infection was detected. Moreover the detection of only phytoplasmas in 11 symptomatic citrus samples collected from Cuba and Guadeloupe without ‘Ca. Liberibacter’ detection, confirmed that the symptomatology cannot be the sole criterium to discriminate between the presence of the two pathogens, and molecular detection is necessary to identify single or mixed infections. Diaphorina citri insects collected from Cuba and Guadeloupe resulted infected with ‘Ca. L. asiaticus’ confirming its active role in the dissemination of the pathogen. Only one insect of the Cicadidae family, collected in Guadeloupe, was found positive for phytoplasma presence. Considering that the phytoplasmas belonging to some ‘Candidatus species’ were detected in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute to citrus orchards.

Citrus Greening disease (CG) in South Africa (SA) is associated with the fastidious bacterium ‘Candidatus Liberibacter africanus’ (Laf). It has been observed that Laf isolates obtained from different geographic localities in SA differed in the rate of transmission during grafting experiments leading to the hypothesis that genetic variation of Laf may exist in this country. To determine this, 167 Laf isolates obtained from Limpopo, North West, Mpumalanga and the Western Cape were subjected to microsatellite analyses, using four polymorphic markers. From UPGMA and STRUCTURE analysis, it was shown that most sources belong to one of two major genetic groups of Laf and these comprise 25 distinct haplotypes. Four samples included within this study did not group with these two major groups, suggesting a potential third and fourth genetic group of Laf being present, which can be validated by further sampling. Results further indicate that Laf populations in SA are formed by geographic locality. The high genetic diversity observed for Laf within this study is consistent with the hypothesis that Laf originated on the African continent, warranting further genetic analysis of Laf populations from Africa. This is the first study to unveil the genetic diversity of Laf.

Coconut production is significantly constrained by agricultural pests. Given that management of these pests is influenced by gender differences, there was a need to assess farmers’ knowledge about coconut pests, farm-level pest management strategies, and institutions offering training to farmers to develop an ecologically sound management strategy. To achieve this, we surveyed six coconut growing districts, three each from the Western and Central Regions of Ghana using face-to-face interviews, discussions, and direct observations. In addition, a multistage sampling technique was used to sample the coconut farmers. The sample population for each town was determined using a proportion to population size approach. The sample population was randomly drawn from each town/village using a sampling frame based on the agricultural sector records. Most of the farmers mentioned Oryctes monoceros as the most important pest of coconut. Significantly, more females than males mentioned weaver birds in their plantations (p = 0.035). Women who did not mention pests in their farms were significantly higher than men (P = 0.007). We observed a significant difference (p = 0.018) between male and female farmers who used indigenous knowledge (i.e., knowledge accumulated by an indigenous (local) population over generations of living in a certain area). However, pest management strategy did not vary in Central Region. Our findings showed that some of the farmers did not use any of the management strategies, suggesting that future studies and training are required to develop sustainable pest management strategies for the coconut pests.

The involvement of the immunodominant membrane protein Imp of 16SrV-D phytoplasma in the interaction with insect proteins was investigated. The His-tagged partial Imp protein and a rabbit polyclonal antibody were produced and used in Far-Western dot Blot (FWdB) assays. Total native proteins were extracted from batches of six insect species (Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Ricania speculum, Metcalfa pruinosa and Zyginidia pullula). FWdB showed interaction of Imp fusion protein with total proteins of S. titanus, E. variegatus (known as efficient vectors) and M. quadripuncltulatus (potential vector), while no interaction signal was detected with the other three species that are reported as non-phytoplasma vectors.

In this study, binding between the immunodominant membrane protein Imp of the 16SrV-D phytoplasma associated with Flavescence dorée disease (FD-Dp) and insect proteins of vectors and non-vectors of FD-Dp was tested.

Citrus greening or huanglongbing (HLB) is the main threat to the European citrus industry since one of its vectors, the African citrus psyllid, Trioza erytreae, has recently become established in mainland Europe. In this context, classical biological control programmes should be implemented to reduce the spread of the psyllid. The aims of this study were to: i) disentangle the parasitoid complex of T. erytreae combining morphological and molecular characterization; and ii) to study the biology of its main parasitoids in its area of origin in South Africa for their future importation into Europe. The main citrus producing areas of South Africa were surveyed during 2017. In contrast to previous studies, the parasitoid complex of T. erytreae included three species of primary parasitoids: Tamarixia dryi, Psyllaephagus pulvinatus and another parasitoid of the genus Tamarixia. Molecular analysis showed that it is a new species closely related to T. dryi. Tamarixia dryi was the most abundant parasitoid but its relative abundance varied among sampling sites. The sex ratio (males/females) of T. dryi and Tamarixia sp. decreased with T. erytreae size and became female biased when psyllid nymphs were larger than 0.6 and 1.2 mm2, respectively. These parasitoids were attacked by three species of hyperparasitoids, Aphidencyrtus cassatus, Marietta javensis and a species of the genus Aphanogmus. Aphidencyrtus cassatus, the most abundant hyperparasitoid, tended to emerge from large nymphs, and adult females lived as long as those of T. dryi. The implications of these results are discussed within the framework of the introduction of T. dryi into Europe.

The African citrus psyllid Trioza erytreae is one of the major threats to citrus industry as the vector of the incurable disease known as huanglongbing (HLB) or citrus greening. The psyllid invaded the northwest of the Iberian Peninsula 6 years ago. The invasion alarmed citrus growers in the Mediterranean basin, the largest citrus producing area in Europe, which is still free of HLB. Before our study, no research had been carried out on the genetic diversity of T. erytreae populations that have invaded the Iberian Peninsula and the archipelagos of the Macaronesia (Madeira and the Canary Islands). In this study, combining microsatellites markers and mtDNA barcoding analysis, we characterize the genetic diversity, structure and maternal relationship of these new invasive populations of T. erytreae and those from Africa. Our results suggest that the outbreaks of T. erytreae in the Iberian Peninsula may have derived from the Canary Islands. The populations of T. erytreae that invaded Macaronesia and the Iberian Peninsula are likely to have originated from southern Africa. We anticipate our results to be a starting point for tracking the spread of this invasive pest outside of Africa and to be important for optimizing contingency and eradication plans in newly invaded and free areas.

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.

In 2015, a disease of unknown origin appeared in Torreon, Coahuila, in the Northeast of Mexico, causing great mortality in Phoenix palms, especially in Phoenix canariensis. Until early 2019, around 1300 palms died from this disease. The aim of this study was to determine its etiology. The symptoms registered in affected palms were similar to those described for Texas Phoenix Palm Decline (TPPD). Phytoplasmas were detected in samples from nine P. canariensis individuals using a TaqMan/real-time PCR assay specific for group 16SrIV detection. DNA of positive samples was amplified by nested PCR using primer pair P1/P7 followed by LY16Sf/LY16-23Sr and R16F2n/R16R2. In silico analysis of the sequences obtained revealed the presence of phytoplasmas associated with TPPD, belonging to subgroup 16SrIV-D. This is the first report of a disease associated with subgroup 16SrIV-D phytoplasmas in the Northeast of Mexico, further extending the known geographical range of this pathogen.

The 16SrIV-A phytoplasmas are associated with the devastating disease lethal yellowing (LY) of palms. In Tabasco (Mexico), the death of Cocos nucifera, Adonidia merrillii, and Attalea butyracea palms have been suspected to be associated with LY based on symptomatology. Samples from the trunk of both symptomatic and nonsymptomatic palms were collected in three different environments: two species of palms within a rural zone and the other within an urban zone. DNA was extracted to perform a nested PCR with phytoplasma primers P1/P7-LY16SF/R16R2. A 1,345-bp fragment was amplified from the DNA extracted from each of the 29 LY-symptomatic palms sampled. Phytoplasma identification was achieved by amplicon sequencing and virtual restriction fragment length polymorphism analyses. Three 16SrIV phytoplasma subgroups were detected: 16SrIV-A in C. nucifera, 16SrIV-B in A. merrillii, and 16SrIV-D in C. nucifera, A. merrillii, and A. butyracea. Phylogenetic analysis showed also that the three phytoplasma strains found in the palm species clustered with phytoplasmas reported in the literature in the three subgroups identified. This is the first report of phytoplasmas associated with these palm species in Tabasco.

Coconut (Cocos nucifera L.) encounters many problems due to the lethal diseases that severely affect its growth and development. Over the last decades, plant breeders have realized that certain genes are directly involved in disease resistance, known as resistance genes (R genes). Most of them encode for proteins (R proteins) with a nucleotide-binding site and a leucine-rich repeat domain (NBS-LRR). However, there are certain sequences of unknown function that code for these motifs and have been linked to known R genes and have been denominated as ‘resistance gene candidates’ (RGC). Currently, 340 RGCs have been identified in the genome of C. nucifera, and 231 sequences have been well characterized phylogenetically indicating the existence of specific clades of genes in C. nucifera. Other clades also comprise a mixture of NBS sequences from C. nucifera, E. guineensis, and P. dactylifera, indicating that similar resistance genes are still shared in different genera of the Arecales. The identification of RGCs in plants is a valuable resource for the discovery of new resistance genes. It has great potential in breeding programs using marker-assisted selection, with special emphasis on the resistance to phytoplasma, a pathogen that causes a high mortality on the plantations of this species.

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

Maritza Luis Pantoja and Camilo Paredes Tomás from the Research Institute of Tropical Fruit Crops (IIFT) explain the evolution of the Cuban citriculture from the beginning of the citrus program in 1968, highlighting the impact of HLB in the production. The authors describe the most frequently observed symptoms, the pathogens detected, and the control measures used to reduce the incidence of the Diaphorina citri vector in the orchards.

Nicola Fiore from the Faculty of Agricultural Sciences of the University of Chile Santiago presents an overview of the GY disease in Chile, where its presence in V. vinifera was first reported in 1971. The symptoms observed, the most widespread phytoplasmas and the insect vectors that can transmit the disease are explained in this article

In Jamaica, coconut palms are an important source of income and a vital part of the subsistence agriculture in rural villages. This article explains how the spread of Lethal Yellowing through the coconut growing areas is impacting in many vulnerable communities, by threatening income as well as food security. The authors, Sonia Marongiu from the Council for Agricultural Research and Economics (CREA) Italy and Wayne Myrie from the Coconut Industry Board (CIB) Jamaica, explain the evolution of the disease in this country.

The text explains the associations between phytoplasmas and the most widespread diseases in coconut palms in the Americas, the Caribbean region and Africa. Assunta Bertaccini from UNIBO, Tropicsafe coordinator and author of the article, introduces the economic impact of LY that is killing millions of palm plants in the main coconut producing areas.

The article describes HLB, a devastating disease affecting citrus. Authors, Alejandro Tena and Jessica Pérez-Rodríguez from IVIA , explain the impact on the production and how the affected countries are managing it. Some symptoms of the diseases are also explained. Insect vectors (Diaphorina citri in Asia and America, Trioza erytreae in Africa and very recently also in Europe) are presented, as well as some guidelines to identify them. Finally, a short preview of Tropicsafe activity to improve the biological control of T. erytreae in Europe is included.

A table reporting phytoplasmas associated with the main GY in the world is presented in this article, which also describes differences among epidemiological cycles of GY in the different geographical areas, due to phytoplasma and insect vector diversity. Assunta Bertaccini from UNIBO and Elisa Angelini from CREA introduce the work planned under Tropicsafe framework to tackle the lack of information about grapevine susceptibility to GY, its vectors, their seasonal trend, and management strategies.

Citrus producers and associations from Guadeloupe are talking about the impact of HLB in their plots and production systems. “Several producers have lost all their plots and are having big financial difficulties” says Uneau Youri, Tropicsafe partner representing ASSOFWI, an association that aims to develop fruit diversification in Guadeloupe.