New viral sequences and endogenous viral elements (EVE) in world-wide populations of Trioza erytreae, the African citrus psyllid

The African citrus psyllid (AfCP), Trioza erytreae (Del Guercio) (Hemiptera, Triozidae), recently has been found in northwestern Iberian Peninsula (Spain and Portugal). The insect is an important citrus pest because it transmits the phloem-limited bacteria Candidatus liberibacter spp., the causal agents of huanglongbing (HLB), the most devastating disease affecting citrus. The bacteria can be acquired by both AfCP nymphs and adults, but only adults can spread the pathogen. HLB has been detected neither in the Iberian Peninsula nor in Madeira and the Canary Islands, where the psyllid was recorded more than a decade ago. In the latter European islands, the eradication programmes of AfCP failed. Chemical-based control strategies are costly, could trigger increases of other pests and may have negative impact on the environment. The adoption of rigorous quarantine measures is extremely important for areas free of the psyllid. These measures likely represent the most effective prevention strategies for psyllid containment, because the geographic expansion of psyllids in citrus is mainly due to human activities, especially by the transport of plants and freshly harvested fruits from infested to uninfested areas. Many natural enemies may contribute to the reduction of its populations and consequent spread; hence, conservation biological control should be emphasised, especially in areas where the psyllid is not abundant. Classical biological control programmes should also be implemented in HLB-free areas by introducing effective AfCP parasitoids. In areas where HLB is detected, biological control is difficult to apply, and a rigorous chemical control program targeting the psyllid could complement this strategy. This report is an updated review of AfCP and strategies for its control in anticipation of its possible further spread in Europe and the Mediterranean Basin.

Simple SummaryThe African citrus psyllid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) is an invasive species for citrus crops. In its native range is the main vector of Candidatus Liberibacter africanus (CLaf), a pathogen that causes huanglongbing (HLB). For Mexico, T. erytreae could threat the citrus industry in a potential invasion but until now, the best chances to prevent its damage is analyzing if the country has the ecological conditions suitable for this psyllid. In this study we used the ecological niche modeling approach to explore which areas in Mexico has the environmental suitability for the T. erytreae establishment. Additionally, the potential role of an alternate host, Casimiroa edulis La Llave (Rutaceae), and five points of entry into the country, in the potential T. erytreae dispersion were analyzed. Mexico citrus areas has a wide environmental suitability for T. erytreae, including the main federal entity (Veracruz). The natural distribution of C. edulis matches with the T. erytreae environmental suitability and citrus areas, and could expand its distribution across the country. For preventive monitoring strategies, the port of Veracruz is a vital point for phytosanitary agencies, because of its proximity to citrus areas.The African citrus psyllid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), is a vector of Candidatus Liberibacter africanus (CLaf), a pathogen that causes huanglongbing (HLB) in Africa. Trioza erytreae has invaded areas of Asia and Europe and has threatened citrus production due to its biological habits and the transmission of CLaf. Mexico is a country where citrus production has a vital role from the economic and social point of view. Therefore, ecological niche modeling (ENM) was used to determine if Mexico has the environmental availability that will allow T. erytreae invasion. We analyzed whether or not the distribution of Casimiroa edulis La Llave (Rutaceae) in the country could be a factor that enables the dispersal of T. eytreae. The environmental connectivity between five points of entry into the country (two ports and three airports) was explored to determine possible routes of dispersal of T. erytrae. The results showed that Mexico has wide availability for the invasion of the African citrus psyllid, which coincides with essential citrus areas of the country and with the distribution of C. edulis. Of the entry points studied, the Port of Veracruz showed nearby areas with environmental connectivity. Preventive monitoring measures for T. erytreae in Mexico should focus on Veracruz state because it has an entry point, ideal environmental availability, citrus areas, and specimens of C. edulis.

Simple SummaryThe potential transmission of the bacterium ‘Candidatus Liberibacter solanacearum’ from infected carrot plants to citrus plants by the African citrus psyllid (Trioza erytreae) should be considered and therefore studied, because this psyllid is an efficient vector of citrus huanglongbing disease (associated to bacteria from the same genus). The aim of this study was to assess the bacterium transmission by three different ways: dodder, grafting and the African citrus psyllid. Additionally, the feeding behavior and oviposition of this psyllid were also evaluated. The bacterium was only transmitted from carrot plants to citrus plants through dodder, although the infection was not established. The African psyllid could settle and oviposit in carrot plants, but it was not able to complete its life cycle on them. This psyllid acquired and transmitted the bacterium from carrots to carrots but was not able to transmit it to citrus plants. In conclusion, after having assessed all relevant possibilities by experimental transmissions from infected carrot plants to citrus plants, the bacterium was transmitted but not established. Our data suggest that the bacterium transmission to citrus plants by the African citrus psyllid is unlikely.Bacteria belonging to ‘Candidatus Liberibacter spp.’ are associated with various severe diseases in the five continents. The African citrus psyllid Trioza erytreae (Hemiptera: Triozidae) is an efficient vector of citrus huanglongbing-HLB disease, absent in the Mediterranean basin. This psyllid is currently present in the islands and mainland Portugal and Spain, where the prevalence of ‘Ca. Liberibacter solanacearum’ (CaLsol) associated to a carrot disease is high. Trioza erytreae normally feeds on citrus plants but has also been observed on other crops. It would be a great concern to the Mediterranean citrus industry if T. erytreae could transmit this bacterium from carrots to citrus and cause disease; therefore, the transmission of CaLsol from carrot plants to citrus plants was experimentally assessed. Although CaLsol was initially detected on receptor citrus plants in transmission assays by dodder and budding, the infection was not established. The feeding behavior by electrical penetration graphs and oviposition of T. erytreae on carrot plants versus citrus plants was evaluated. Trioza erytreae only reached the phloem in citrus plants. However, it was able to acquire CaLsol from infected carrots but unable to transmit it to citrus plants. CaLsol was detected in some carrot plants immediately after 7 and 14 days (inoculation access period), but it was not detected after one month. Trioza erytreae was unable to complete its life cycle on carrot plants. In conclusion, the efficient vector of bacteria associated to huanglongbing was unable to transmit CaLsol from carrot to citrus plants, but it acquired and transmitted the bacterium from carrot to carrot plants with low efficiency.

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and is vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the rutaceous tree Calodendrum capensis (Cape chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in citrus, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. When these nucleotide positions are invstigated by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be downgraded as a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as Laf as a species separate into two haplotype groups. The three haplotypes of Laf are therefore: (i) LafA, designated as the first accession sequenced (L22533); (ii) LafC, i.e the former subspecies “capensis”, to recognise the priority in the use of this term; (iii) LafB, the third previously unrecognised haplotype. Thus the cryptic presence of three haplotypes is revealed by this review of the Laf 16S rDNA sequences.

Species distribution models predicting climate suitability for the psyllid Trioza erytreae, vector of citrus greening disease

The center of diversity for citrus was originally found on the northeastern India, eastward through the Malay Archipelago and south to Australia. Today citrus is produced in 140 countries, mainly between the north and south 40o latitudes. Citrus ranked first among fruit crops in the international trade based on value. Citrus production of the world is around 105 million tons per year. Orange ( Citrus sinensis ) accounts for almost two thirds of the total citrus production (65%), followed by tangerine ( Citrus reticulata ) (21%), lemon ( C. limon ) (6%) and grapefruit ( C. paradisi ) (5.5%). Other significant commercially grown species are lime ( C. aurantifolia ), pummelo ( C. grandis ) and citron ( C. medica ). The largest citrus producers are Brazil (20%), United States (14%), China (12%), Mexico (6%) and the countries of the mediterranean basin (15%). Humidity and day-to-night temperature fluctuations influence which varieties are best adapted to an area. Most citrus fruits are produced for fresh market consumption and only around 30% is processed. Fresh fruits are rich in vitamin C which plays a vital role in prevention of scurvy. After extraction of the juice, the skin and fruit pulp can be used as livestock feed or making compost. The rind acid (oil) of the citrus is considered an expensive commodity in international trade (F.A.O 2003). Unfortunately, the citrus industry is threatened by 2 destructive diseases namely Witches’ Broom disease of lime (WBDL) and Huanglongbing (HLB) disease. WBDL has been known to be caused by Candidatus phytoplasma aurantifolia . It is one of the destructive diseases on citrus industry in the Middle East, India and Pakistan. It was reported in 1970 for the first time from lime orchard of Oman, united Arabian emirate (UAE) (1988), Iran (1998) and India and Pakistan (1999). WBDL is a phloem limited phytoplasma disease of lime. HLB disease causing citrus greening ( Candidatus liberibacter spp.) is the second most severe disease on citrus industry all over the world. HLB has destroyed an estimated 60 million trees in Africa and Asia. More than 40 countries were infected by HLB in Africa, Asia and USA (Chau et al., 1996; Bove, 2006; Roux et al., 2006; Batool et al., 2007). The HLB pathogens are highly fastidious phloem-inhabiting bacteria in the genus Candidatus liberibacter . The bacteria have not been cultured yet in laboratory media and do not survive outside the host cells. Three types of phloem limited bacteria causing HLB disease have been described and identified (Bove, 2006). The isolate from South Africa ( Candidatus liberibacter africanus ) is considered heat-sensitive and found in Africa. It is vectored by African citrus psyllid ( Trioza erytreae) and was described by Guercio in 1918. The isolate from Asia ( Candidatus liberibacter asiaticus ) is more severe and widespread; it is vectored by Asian psyllid (Diaphorina citri Kuwayama) (Garnier et al., 2000). This type of HLB is heat-tolerant (Garnier et al., 2000). It can show the symptom on humid, cool and hot temperature, up to 35oC (Garnier et al., 2000; Bove 2006; Le Roux et al., 2006). The isolate from America has been named Candidatus liberibacter americanus ; it was detected in Brazil and Florida (Coletta-Filho et al., 2005; Texeira et al., 2005).

A detailed account is given of the history, aetiology, biology, epidemiology, detection, geographical distribution, and control of huanglongbing (HLB), a destructive disease of citrus that represents a major threat to the world citrus industry, and is slowly invading new citrusgrowing areas. HLB, whose name in Chinese means “yellow dragon disease”, was first reported from southern China in 1919 and is now known to occur in next to 40 different Asian, African, Oceanian, South and North American countries. The agent is a phloem-restricted, non cultured, Gram-negative bacterium causing crippling diseases denoted “greening “ in South Africa, “mottle leaf” in the Philippines, “dieback” in India, “vein phloem degeneration” in Indonesia. The HLB bacterium belongs to the genus Candidatus Liberibacter, three species of which are currently known, Candidatus Liberibacter asiaticus, occurring in Asian countries and, to a lesser extent, in Brazil and the USA (Florida), Candidatus Liberibacter africanus with its subspecies “capensis”, recorded from African countries, and Candidatus Liberibacter americanus present in Brazil. The suggestion is that each liberibacter species has evolved in the continent after which it is named. HLB symptoms are virtually the same wherever the disease occurs. Infected trees show a blotchy mottle condition of the leaves that results in the development of yellow shoots, the early and very characteristic symptom of the disease. Trees are stunted, declining and bear a few, small-sized, and deformed (lop-sided) fruits, that are poorly coloured (greening) and with coloration starting at the peduncular end (colour inversion). HLB can be transmitted by grafting from citrus to citrus and by dodder to periwinkle. The psyllids Trioza erytreae and Diaphorina citri are natural vectors. Two different types of HLB are known: the heat-sensitive African form transmitted by T. erytreae, which develops at temperatures of 22-25°C, and the heat-tolerant Asian form, transmitted by D. citri, which stands temperatures well above 30°C. Although the HLB pathogen can be identified by electron microscopy, other laboratory methods are used for routine detection. ELISA with monoclonal antibodies is not recommended. Better systems are dot blot hybridization with a DNA probe, and various PCR formats (one-step, nested, multiplex) using species-specific primers based on 16S rRNA or rplKAJL-rpoBC operon sequences. Because no curative methods of HLB are available, control is preventive and largely based on inoculum elimination by removal of infected trees and chemical treatments against vectors. Strict quarantine measures must be implemented to impair further international spread of HLB agents and their vectors.

Large-scale metagenomic and -transcriptomic studies have revolutionized our understanding of viral diversity and abundance. In contrast, endogenous viral elements (EVEs), remnants of viral sequences integrated into host genomes, have received limited attention in the context of virus discovery, especially in RNA-Seq data. EVEs resemble their original viruses, a challenge that makes distinguishing between active infections and integrated remnants difficult, affecting virus classification and biases downstream analyses. Here, we systematically assess the effects of EVEs on a prototypical virus discovery pipeline, evaluate their impact on data integrity and classification accuracy, and provide some recommendations for better practices. We examined EVEs and exogenous viral sequences linked to Orthomyxoviridae, a diverse family of negative-sense segmented RNA viruses, in 13 genomic and 538 transcriptomic datasets of Culicinae mosquitoes. Our analysis revealed a substantial number of viral sequences in transcriptomic datasets. However, a significant portion appeared not to be exogenous viruses but transcripts derived from EVEs. Distinguishing between transcribed EVEs and exogenous virus sequences was especially difficult in samples with low viral abundance. For example, three transcribed EVEs showed full-length segments, devoid of frameshift and nonsense mutations, exhibiting sufficient mean read depths that qualify them as exogenous virus hits. Mapping reads on a host genome containing EVEs before assembly somewhat alleviated the EVE burden, but it led to a drastic reduction of viral hits and reduced quality of assemblies, especially in regions of the viral genome relatively similar to EVEs.Our study highlights that our knowledge of the genetic diversity of viruses can be altered by the underestimated presence of EVEs in transcriptomic datasets, leading to false positives and altered or missing sequence information. Thus, recognizing and addressing the influence of EVEs in virus discovery pipelines will be key in enhancing our ability to capture the full spectrum of viral diversity.

Huanglongbing (HLB) is one of the most catastrophic citrus diseases. HLB pathogens Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf), and Ca. L. americanus (Lam) and their insect vectors Asian citrus psyllids (ACP, Diaphorina citri) and African citrus psyllids (AfCP, Trioza erytreae) are invading citrus producing regions where HLB was absent previously, including the Mediterranean basin. Importantly, the Mediterranean region is one of the two major citrus producing areas without HLB. Here, I provide a short perspective regarding a) information related to the distribution of the HLB pathogens and psyllid vectors in this region and neighboring countries, b) predicted distribution of the HLB for this region, c) the possible evolution of Liberibacters and how they could have established their relationship with different hosts, and d) approaches to fend off HLB. Specifically, I emphasized the following measurements: quarantine measures against AfCP, ACP, Las, Laf, and Lam, early detection and diagnosis of HLB and removal of inoculum, surveillance of AfCP and ACP and eradication of citrus psyllids once identified.

The African citrus psyllid, Trioza erytreae (Hemiptera: Triozidae), is a natural vector of Candidatus Liberibacter spp., the causal agents of the citrus greening disease or Huanglongbing (HLB). Despite the global losses of the Citrus industry due to HLB, the vector is rapidly spread to new areas threatening citrus crops and raising concern for stakeholders. Until now, there was a single old register of T. erytreae from the São Tomé island (Bulletin of the British Museum of Natural History, Entomology, 49, 1–102). In this work, we provide up‐to‐date occurrence data on the presence of T. erytreae throughout the isle of São Tomé. The identity of the captured adults was assessed and confirmed through molecular identification by sequencing a portion of the mitochondrial COI. The psyllid was found in lemon, orange and tangerine orchards. Further monitoring is advisable to manage potential outbreaks.

Societal Impact Statement Huánglóngbìng (HLB) is a bacterial disease of citrus that has significantly impacted Brazil and the United States, although citrus production in the Mediterranean Basin remains unaffected. By developing a mathematical model of spread in Spain, we tested surveillance and control strategies before any future HLB entry in the EU. We found while some citrus production might be maintained by roguing, this requires extensive surveillance and significant chemical control, perhaps also including testing of psyllids (which spread the pathogen) for bacterial DNA. Our work highlights the key importance of early detection (including asymptomatic infection) and vector control for HLB management. Summary Huánglóngbìng (HLB; citrus greening) is the most damaging disease of citrus worldwide. While citrus production in the United States and Brazil have been affected for decades, HLB has not been reported in the European Union (EU). However, a HLB vector, the African citrus psyllid, is already in Portugal and Spain. In 2023, the major vector, the Asian citrus psyllid, was first reported in Cyprus. We develop a landscape‐scale, epidemiological model, accounting for heterogeneous citrus cultivation and vector dispersal, as well as climate and disease management. We use our model to predict HLB dynamics for an epidemic vectored by the African citrus psyllid in high‐density citrus areas in Spain, assessing detection and control strategies. Without disease management, we predict large areas infected within 10–20 years. Even with significant visual surveillance, any epidemic will be widespread on first detection, making eradication unlikely. Nevertheless, increased inspection and roguing following first detection, particularly if coupled with intensive insecticide use, could sustain some citriculture for a decade or more, albeit with reduced production. However, effective control may require chemical application rates and/or active substances no longer authorised in the EU. Strategies targeting asymptomatic infection will be more successful. Detection of bacteriliferous vectors—sometimes possible long before plants show symptoms—could reduce lags before disease management commences. If detection of HLB‐positive vectors were followed by intensive insecticide sprays, this may greatly improve outcomes. Our work highlights modelling as a key component of developing epidemiological preparedness for a pathogen invasion that is, at least somewhat, predictable in advance.

Citrus Huanglongbing (HLB), commonly referred to as citrus greening, is one of the major disease challenges of citrus. In Uganda, there was limited information on Liberibacter spp., the causal organisms of HLB disease, and its psyllid vectors despite the presence of HLB symptoms on citrus. The objective of this study was to identify the Liberibacter spp. and psylla vector species responsible for HLB in Uganda. Other native plant species that could be hosts to pathogens and the psyllid vectors, but are not in the citrus genus, were also investigated. A survey was conducted in 15 citrus growing districts, and symptomatic citrus leaf samples collected, as well as citrus psyllid nymphs and adults for isothermal detection of pathogens in the laboratory. Two types of bacterial pathogens responsible for HLB were detected, namely Candidatus Liberibacter africanus (CLaf), known as the African type; and Candidatus Liberibacter asiaticus (CLas), known as the Asian type. CLaf was found in Mukono and Wakiso districts in Central Uganda and in Mbarara in Western Uganda; while CLas was found in Budaka and Tororo districts in Eastern Uganda. Citrus psyllids, which are the major known disease vectors were present in seven out of the fifteen districts. Psyllid vector identification by morphological means indicated Trioza erytreae, the African psyllid to be the insect vector. Psyllids were common on tangerines (66.7%), Sour Orange (13.3%) and Rough Lemon (13.3%); and least on Washington Navel (6.7%). Three non-citrus plants, Stephania abyssinica (Dill. & A. Rich) walp var. tomentella (Oliv.) Deils (Menispermaceae), Diospyros mespiliformis (Ebenaceae) and Ficus spp (Moraceae) were found to be alternative host plants for the psyllid.Key words: Candidatus, Diospyros mespiliformis, Psyllids, Trioza erytreae

Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-β-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

Citrus is considered one of the most important fruit crops globally due to its contribution to food and nutritional security. However, the production of citrus has recently been in decline due to many biological, environmental, and socio-economic constraints. Amongst the biological ones, pests and diseases play a major role in threatening citrus quantity and quality. The most damaging disease in Kenya, is the African citrus greening disease (ACGD) or Huanglongbing (HLB) which is transmitted by the African citrus triozid (ACT), Trioza erytreae. HLB in Kenya is reported to have had the greatest impact on citrus production in the highlands, causing yield losses of 25% to 100%. This study aimed at predicting the occurrence of ACT using an ecological habitat suitability modeling approach. Specifically, we tested the contribution of vegetation phenological variables derived from remotely-sensed (RS) data combined with bio-climatic and topographical variables (BCL) to accurately predict the distribution of ACT in citrus-growing areas in Kenya. A MaxEnt (maximum entropy) suitability modeling approach was used on ACT presence-only data. Forty-seven (47) ACT observations were collected while 23 BCL and 12 RS covariates were used as predictor variables in the MaxEnt modeling. The BCL variables were extracted from the WorldClim data set, while the RS variables were predicted from vegetation phenological time-series data (spanning the years 2014–2016) and annually-summed land surface temperature (LST) metrics (2014–2016). We developed two MaxEnt models; one including both the BCL and the RS variables (BCL-RS) and another with only the BCL variables. Further, we tested the relationship between ACT habitat suitability and the surrounding land use/land cover (LULC) proportions using a random forest regression model. The results showed that the combined BCL-RS model predicted the distribution and habitat suitability for ACT better than the BCL-only model. The overall accuracy for the BCL-RS model result was 92% (true skills statistic: TSS = 0.83), whereas the BCL-only model had an accuracy of 85% (TSS = 0.57). Also, the results revealed that the proportion of shrub cover surrounding citrus orchards positively influenced the suitability probability of the ACT. These results provide a resourceful tool for precise, timely, and site-specific implementation of ACGD control strategies.

African citrus psyllid (Trioza erytreae (Del Guercio)) is a vector insect of the bacterium Candidatus Liberibacter africanus, the putative causal agent of Huanglongbing, the most devastating citrus disease in the world. The insect was found on the island of Madeira in 1994 and in mainland Portugal in 2015. Present in the north and center of the country, it is a threat to Algarve, the main citrus-producing region. Trioza erytreae eggs and first instar nymphs are sensitive to the combination of high temperatures and low relative humidity. Daily maximum air temperature and minimum relative humidity data from 18 weather stations were used to calculate the water vapor pressure deficit (vpd) from 2004 to 2018 at various locations. Based on the mean vpd and the number of unfavorable days (vpd < 34.5 and vpd < 56 mbar) of two time periods (February to May and June to September), less favorable zones for T. erytreae were identified. The zones with thermal and water conditions like those observed in the Castelo Branco and Portalegre (Center), Beja (Alentejo), Alte, and Norinha (Algarve) stations showed climatic restrictions to the development of eggs and first instar nymphs of African citrus psyllid. Effective control measures, such as the introduction and mass release of Tamarixia dryi (Waterson), a specific parasitoid, and chemical control are necessary in favorable periods for T. erytreae development, such as in spring and in areas with limited or no climate restrictions.