Other materials

Classical biological control of the African citrus psyllid Trioza erytreae, a major threat to the European citrus industry

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.

Insect-borne prokaryote-associated diseases in tropical and subtropical perennial crops (TROPICSAFE): improving the production of citrus, grapevine and coconut palms.

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.

Potential insect vectors and alternative host plants of insect-transmitted liberibacter and phytoplasmas in the fynbos biome in the Western Cape

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).

Host range testing of Tamarixia dryi (Hymenoptera: Eulophidae) sourced from South Africa for classical biological control of Trioza erytreae (Hemiptera: Psyllidae) in Europe

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.

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

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.

Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean

“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.

Farmers’ knowledge and farm-level management practices of coconut pests in Ghana: Assessment based on gender differences

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.

Classical biological control of the African citrus psyllid Trioza erytreae, a major threat to the European citrus industry

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 Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease

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.

Genome-informed design of a LAMP assay for the specific detection of the strain of ‘Candidatus Phytoplasma P. asteris’ phytoplasma occurring in grapevines in South Africa.

Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific LAMP assay for detection of a distinct strain of grapevine ‘Candidatus Phytoplasma asteri’ that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South African, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain.