Candidatus Liberibacter Africanus Research Articles

Molecular identification and characterization of “Candidatus Liberibacter spp.” from major citrus growing region of Malwa, Madhya Pradesh, India

One of the most damaging diseases impacting yearly yield and productivity is huanglongbing, often known as greening of citrus fruits and plants. Huanglongbing is caused by the Candidatus Liberibacter species, of which three primary species are known worldwide: Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter americanus (CLam), and Candidatus Liberibacter asiaticus (CLas). In the current study, Candidatus Liberibacter infection and the occurrence of huanglongbing disease were examined in the Malwa region of Madhya Pradesh, which is home to the Nagpur Mandarin cultivar. A total of 100 samples were gathered for the current study from 14 villages in the chosen area, primarily Mandsaur, Neemuch, Ratlam, Ujjain, Dewas, Indore, Shajapur, Rajgarh, Dhar, and Jhabua. Here, out of 100 samples, 22 were determined to be positive, while the remaining 100 were negative despite having modest physiological and morphological characteristics that resembled symptoms of HLB. Sequencing results from 08 samples in the current investigation indicate that Candidatus Liberibacter asiaticus (05), Candidatus Liberibacter americanus (02), and Candidatus Liberibacter africanus (01) are all positive. In the study, eight 16S rRNA genes sequences targeting OI1/OI2c were submitted to GenBank with for Candidatus Liberibacter africanus (1) accession no: OQ725636.1 for Candidatus Liberibacter americanus (2) Accession: OQ725635.1 and OQ725634.1 and Candidatus Liberibacter asiaticus (5) for Accession: OQ725633.1, OQ725632.1, OQ725631.1, OQ725630.1 and OQ725629.1. Candidatus Liberibacter species isolated and sequenced in the current study had a greater degree of mutation (41 point mutations), according to single nucleotide polymorphism analysis. Present study provides occurrence and prevalence of Candidatus Liberibacter asiaticus as key causative agent for huanglongbing in the Nagpur Mandarin cultivar. Higher prevalence and occurrence of Candidatus Liberibacter species also affect other agriculture crops and hence a trend and pattern in genomic profile was investigated via SNP analysis that provides details of mutation. Genomic profile of Candidatus Liberibacter species is highly recommended for the development of antibacterial to tackle Candidatus Liberibacter species driven agriculture economical losses.

Trapping and Repellent Techniques for Huanglongbing Management in Citrus Orchards: Innovative Strategies to Combat Vector-Mediated Disease Transmission

The global increase in human population has heightened the demand for citrus fruits and their derivatives, necessitating effective management strategies to mitigate the impact of huanglongbing (HLB) disease. Caused by gram-negative bacteria such as ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’, and ‘Candidatus Liberibacter americanus’, HLB is primarily transmitted by the insect vector Diaphorina citri. While traditional vector suppression methods using insecticides have proven ineffective, innovative approaches are required to control disease spread. This study aims to address the persistent nature of the vector and its ability to transmit HLB throughout its life cycle by emphasizing trapping and repellent techniques. By implementing early detection methods, utilizing disease-resistant citrus varieties, employing disease-free seeds, enhancing nutritional regimes, and reducing vector populations, promising results have been achieved in reducing vector populations and limiting HLB inoculum. The implications of this research underscore the importance of adopting trapping and repellent strategies, alongside the use of disease-free citrus plants, to effectively manage HLB and safeguard citrus plantations worldwide.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Predicting the potential global distribution of an invasive alien pest Trioza erytreae (Del Guercio) (Hemiptera: Triozidae)

The impact of invasive alien pests on agriculture, food security, and biodiversity conservation has been worsened by climate change caused by the rising earth’s atmospheric greenhouse gases. The African citrus triozid, Trioza erytreae (Del Guercio; Hemiptera: Triozidae), is an invasive pest of all citrus species. It vectors the phloem-limited bacterium “Candidatus Liberibacter africanus”, a causal agent of citrus greening disease or African Huanglongbing (HLB). Understanding the global distribution of T. erytreae is critical for surveillance, monitoring, and eradication programs. Therefore, we combined geospatial and physiological data of T. erytreae to predict its global distribution using the CLIMEX model. The model’s prediction matches T. erytreae present-day distribution and shows that parts of the Mediterranean region have moderate (0 < EI < 30) to high (EI > 30) suitability for the pest. The model predicts habitat suitability in the major citrus-producing countries, such as Mexico, Brazil, China, India, and the USA. In the Special Report on Emissions Scenarios (SRES) A1B and A2 scenarios, the model predicts a reduction in habitat suitability from the current time to 2070. The findings show that global citrus production will continue to be threatened by T. erytreae. However, our study provides relevant information for biosecurity and risk assessment.

Differentiation of ‘Candidatus Liberibacter asiaticus’ in Saudi Arabia based on tandem repeat variability in genomic locus

Citrus greening, or huanglongbing, is a destructive disease threatening many citrus worldwide, and drastically altering the global dynamics of the citrus industry. The disease is caused by one of several unculturable bacterial species belonging to ‘Candidatus Liberibacter’. The recent availability of complete genome sequences of ‘Candidatus Liberibacter asiaticus’ (CLas) has facilitated comprehensive assessments of genomic variability using a range of approaches, including short tandem repeat analysis. The objective of this study was to evaluate the genetic diversity of CLas populations in Saudi Arabia based on tandem repeat number (TRN) within the CLIBASIA_01645 locus, predicted to encode the bacteriophage C1 repressor protein. Results indicated that the genotype richness of the Saudi Arabian CLas isolates was conserved by 27% based on the TRN locus. Four different genotypes TRN2, TRN3, TRN4, and TRN5 were identified. However, the TRN2 and TRN5 were the most dominant genotypes. All four of the TRN genotypes were associated with CLas-positive mandarin (Citrus reticulata) or sweet orange (C. sinensis) citrus trees. The diversity (H = 0.69) and evenness (H'=0.914) were overall relatively high, with the northern region of Saudi Arabia harboring the highest diversity (0.7) and evenness score (0.9–1.0). Phylogenetic analysis of the CLas-bacteriophage C1 repressor protein of the Saudi Arabian isolates indicated CLas was more closely related to ‘Candidatus Liberibacter africanus’ than to ‘Candidatus Liberibacter americanus’.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

A Review of the 'Candidatus Liberibacter africanus' Citrus Pathosystem in Africa.

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as 'Candidatus Liberibacter africanus'. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five 'Ca. L. africanus' subspecies have been identified in various rutaceous species, with 'Ca. L. africanus subsp. clausenae' the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.

Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

Temperature‐based phenology model of African citrus triozid (Trioza erytreae Del Guercio): Vector of citrus greening disease

AbstractThe African citrus triozid (ACT), Trioza erytreae Del Guercio (Hemiptera: Triozidae), is the major vector of the ‘Candidatus Liberibacter africanus’, associated with the African citrus greening disease [huanglongbing (HLB)]. Although T. erytreae is a very destructive pest in citrus production with yield losses up to 100%, there is limited information on its thermal biology, which is a prerequisite for developing an environmentally friendly management strategy. This study investigated the effect of temperature on the development and survival of T. erytreae under eight constant temperatures (10, 15, 18, 20, 22, 24, 27 and 30°C), relative humidity of 65 ± 5% and a photoperiod of 12D:12L. Using Insect Life Cycle Modeling software, we fitted linear and non‐linear models to T. erytreae development and mortality data. The best‐fitted functions were compiled for each life stage to yield a phenology model. Our findings revealed that temperature had a significant effect on the development of all the immature stages. No complete development was observed at 10, 27 and 30°C. The developmental time of eggs ranged from 5.27 days at 24°C to 13.5 days at 15°C. The lowest developmental temperature threshold ranged between 3 and 15°C, with thermal constants ranging between 16.7 and 100 degree‐days. The 1st nymphal instars recorded the highest mortality at 15°C. The highest survival rates of the 3rd instar nymphs were recorded at 20°C. The model predicted that temperatures between 20 and 25°C were optimal for the survival of all the immature life stages, except for the 1st nymphal instars. Our results provide relevant information on the impact of temperature on the development and survival of T. erytreae, which is essential for the development of more efficient management strategies.

PM 7/121 (2) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

Introduction to Diagnostics Standards

Introduction to Diagnostics Standards

Does the African Citrus psyllid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), Represent a Phytosanitary Threat to the Citrus Industry in Mexico?

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.

Exploring non‐host plant‐based management strategy with lemongrass, garlic and guava volatiles for the African citrus triozid

AbstractThe African citrus triozid (ACT), Trioza erytreae, is an important pest of citrus. Both nymphs and adults damage the plant by feeding on the sap causing young shoots to die. Trioza erytreae also vectors Candidatus Liberibacter africanus, the bacteria that cause citrus greening disease. Since certain non‐host plants are known to repel insect pests, it is important to investigate how such plants can be exploited to manage T. erytreae. Here, we screened effects of odours of three non‐host plants namely guava (Psidium guajava), garlic (Allium sativum) and lemongrass (Cymbopogon citratus) against T. erytreae's location of a common host plant, rough lemon (Citrus jambhiri) and showed that repellence varied interspecifically with the plants. Using cage assays, we found that guava and garlic decreased the attraction of females but not males of T. erytreae to rough lemon volatiles. Chemical analysis by coupled gas chromatography/mass spectrometry (GC/MS) showed that volatiles of three of the plants were dominated by terpenoids; guava (69% comprised of limonene, 34%, (E)‐β‐ocimene, 29% and (Z)‐β‐Ocimene, 6%), lemongrass (56% comprised of geranial, 26%, neral, 19% and myrcene, 11%) and rough lemon (74% comprised of limonene, 53%, sabinene, 11% and (E)‐β‐ocimene, 10%). On the other hand, the volatile profile of garlic was dominated by benzenoids and saturated compounds (85% comprised of benzaldehyde, 12%, benzyl alcohol, 17%, nonanal, 31%, decanal, 13% and hexadecane, 12%). Our results suggest that non‐host plant volatile composition and richness in specific compounds may contribute to influencing T. erytreae response to its host, with garlic and guava as potential non‐host plants that can be exploited in the management of the pest.

First report of 'Candidatus Liberibacter africanus' associated with African Greening of Citrus in Angola.

Huanglongbing (HLB, Asian Citrus Greening), the most devastating disease of citrus has not been detected in southern Africa (Gottwald, 2010). HLB is associated with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium vectored by Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian Citrus Psyllid (ACP). African Citrus Greening, associated with 'Candidatus Liberibacter africanus' (CLaf) and its vector the African Citrus Triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), are endemic to Africa, although not previously reported from Angola. African Greening is less severe than HLB, largely due to heat sensitivity of CLaf and its vector. Introduction of HLB into southern Africa would be devastating to citrus production in commercial and informal sectors. Concern was raised that CLas or ACP might hae inadvertently been introduced into Angola. In July 2019, a survey was conducted in two citrus nurseries in Luanda and Caxito and in different orchards on 7 farms surrounding Calulo and Quibala. Yellow sticky traps for insects were placed at the various localities and collected after c. 3 weeks. Breeding signs of T. erytreae (pit galls) were observed on citrus in some locations, but no insect vectors were detected on traps. Trees were inspected for signs and symptoms of citrus pests and diseases, particularly those that resemble HLB (foliar blotchy mottle, shoot chlorosis, vein yellowing and corking, lopsided fruit with aborted seeds and colour inversion) and its vectors (pit galls on leaves or waxy exudates). Leaves and shoots with suspect symptoms were sampled for laboratory analysis (43 samples). DNA was extracted from petiole and midrib tissue of leaves using a modified CTAB extraction protocol of Doyle and Doyle (1990). Real-time PCR was done using universal Liberibacter primers of Roberts et al. (2015), CLaf specific primers of Li et al. (2006) and CLas specific primers of Bao et al. (2019). All real-time PCR protocols indicated the presence of CLaf in 6 samples (Tab. S1). CLas or other citrus Liberibacter species were not detected. The presence of CLaf in sample 37 was confirmed by constructing a library (NEXTFLEX® DNA Sequencing Kit, PerkinElmer) with extracted DNA and performing high-throughput sequencing on an Ion Torrent™ S5™ platform (Central Analytical Facility, Stellenbosch University). To improve the quality of the reads, all 233,617,700 obtained reads were trimmed from the 3' end to a maximum length of 240 nt using Trimmomatic (Bolger et al. 2014). The high quality reads were mapped to the Citrus sinensis reference genome (NC_023046.1) using Bowtie 2.3.4 (Langmead and Salzberg 2012) to subtract all the reads that had high identity to the host plant (number of mismatches allowed in the seed was set to 1). The 14,691,369 unmapped reads (6.2% of original data) were mapped to the CLaf reference genome NZ_CP004021.1 using CLC Genomics Workbench 10.1.1 (Qiagen) (Length fraction = 0.8; Similarity fraction = 0.9). A CLaf consensus genome was generated that spanned 99.7% of the reference genome and the 163001 mapped reads had a 22.9 mean read coverage. The consensus sequence was 99.7% identical to NZ_CP004021.1 and was submitted to Genbank as accession: CP054879. The positive CLaf detections were from trees with typical HLB or African Citrus Greening symptoms, viz. lopsided fruit with green stylar ends, aborted seed and stained columella at base of fruit button; yellow shoots with leaves showing symptoms of blotchy mottle and vein yellowing and corking (Fig. S1) in a commercial citrus farm outside Calulo and included 2 'Ponkan' mandarin (C. reticulata), 2 Valencia and 1 'Navelina' tree (C. sinensis), and a citrus nursery in Luanda (1 lime tree; C. aurantifolia) (Tab. S1). This first report of CLaf in Angola highlights the need to prevent spread by removing infected trees and managing the insect vector, as well as the need for further surveys to determine the occurrence of African Greening and its vectors in other provinces and to confirm the absence of exotic citrus pests and diseases. References Bao, M. et al. 2020. Plant Dis. 104:527 Bolger, A. M. et al. 2014. Bioinformatics. 30:2114-2120. Doyle, J.J. and Doyle, J.L. 1990. Focus 12:13 Gottwald, T.R. 2010. Annu. Rev. Phytopathol. 48:119 Langmead, B. and Salzberg, S. 2012. Nature Methods. 9:357-359. Li, W. et al. 2006. Jnl. Microbiol. Methods 66:104 Roberts, R. et al. 2015. Int. J. Syst. Evol. Micr. 65:723.

Diversity and phylogenetic analysis of endosymbionts from Trioza erytreae (Del Guercio) and its parasitoids in Kenya

AbstractThe African citrus triozid (ACT), Trioza erytreae (Del Guercio), is the primary vector of Candidatus Liberibacter africanus (CLaf), the causative agent of Africa citrus greening disease (ACGD). This study evaluates the diversity of ACT parasitoids and further characterizes endosymbionts associated with both T. erytreae and its parasitoids that could be used as biological control agents of T. erytreae and management of ACGD. Mitochondrial cytochrome oxidase I gene was used to reconstruct T. erytreae and its parasitoids phylogeny, while 16S rRNA gene was used for the bacterial phylogeny. One well‐supported clade of ACT was detected within the Triozidae phylogeny, while the parasitoid species clustered into four groups within eulophid and encyrtid phylogeny. The phylogenetic result of parasitoids was supported by morphological identification where five different parasitoid species could be identified, that is Tamarixia dryi, Psyllaephagus pulvinatus, Tetrastichus sp., Aphidencyrtus cassatus and Charipine species. Moreover, four eubacterial symbionts (Wolbachia,Rickettsia,Arsenophonus and Candidatus Liberibacter sp.) were detected in T. erytreae and three symbionts (Wolbachia,Rickettsia and Cardinuim) in the parasitoid specimens. Maximum likelihood phylogenetic inferences clustered the identified eubacterial symbionts within α and γ proteobacteria subdivisions. Phylogenetic inferences of 16S rRNA gene sequences indicated that Wolbachia strains from ACT and the parasitoids did not form a single monophyletic clade; however, both clustered within Supergroup B. The impacts of these parasitoid species and endosymbionts on ACT are still unknown, but their occurrence and broad distribution indicate the possibility of future use for control of T. erytreae.

Comparison of the performance of the main real-time and conventional PCR detection tests for ‘Candidatus Liberibacter’ spp., plant pathogenic bacteria causing the Huanglongbing disease in Citrus spp.

Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species: Candidatus Liberibacter asiaticus, africanus and americanus. These species are considered quarantine pathogens in the world, and pose major risks for citrus production and industry. Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.

Detection of Asian Citrus Psyllid (Hemiptera: Psyllidae) in Ethiopia: A New Haplotype and its Implication to the Proliferation of Huanglongbing.

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid, is a pest of citrus known for its transmission of Candidatus Liberibacter asiaticus (Ca. L. asiaticus), the causal bacterium of Huanglongbing. The African citrus triozid Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) has been the putative vector of Candidatus Liberibacter africanus (Ca. L. africanus) which causes the African citrus greening disease, until the recent detection of D. citri on the continent. Following reports of D. citri in Kenya and Tanzania, we surveyed citrus plants to establish the presence/absence of D. citri in Ethiopia in citrus-growing regions ranging from 900 to 2,460 m above sea level (masl). Diaphorina citri adults were detected in five of the surveyed sites in Ethiopia. Adult insects encountered were collected using an aspirator and stored in 97% ethanol. The mitochondrial cytochrome oxidase 1 (mt COI) gene of the collected insects was amplified using LepF1/LepR1 primers, and sequences obtained showed low variation, which fell within the acceptable range of species. BLAST was used to query the sequences obtained, and all the sequences linked to D. citri accessions that are available in GenBank. The analysis of the sequences revealed a new haplotype of the species that differs from haplotypes previously reported. Phylogenetic relationships of our samples and other D. citri reference sequences was inferred using the Maximum-likelihood method. Monophyly was observed between the samples and the publicly available sequences from global accessions. This is the first report of the presence of D. citri in Ethiopia.

Anatomical study of the female reproductive system and bacteriome of Diaphorina citri Kuwayama, (Insecta: Hemiptera, Liviidae) using micro-computed tomography

Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus. It is caused by (1) Candidatus Liberibacter africanus, transmitted by Trioza erytreae and (2) C.L. asiaticus and C.L. americanus, transmitted by Diaphorina citri. As part of a multidisciplinary project on D. citri (www.citrusgreening.org), we made a detailed study, using micro-computed tomography, of the female abdominal terminalia, reproductive system (ovaries, accessory glands, spermatheca, colleterial (= cement) gland, connecting ducts, and ovipositor) and bacteriome, which we present here. New terms and structures are introduced and described, particularly concerning the spermatheca, ovipositor and bacteriome. The quality of images and bacteriome reconstructions are comparable, or clearer, than those previously published using a synchrotron or fluorescence in situ hybridisation (FISH). This study: reviews knowledge of the female reproductive system and bacteriome organ in D. citri; represents the first detailed morphological study of D. citri to use micro-CT; and extensively revises existing morphological information relevant to psylloids, hemipterans and insects in general. High quality images and supplementary videos represent a significant advance in knowledge of psylloid anatomy and are useful tools for future research and as educational aids.

Water Vapor Pressure Deficit in Portugal and Implications for the Development of the Invasive African Citrus Psyllid Trioza erytreae.

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.

Distribution of Candidatus Liberibacter species in Eastern Africa, and the First Report of Candidatus Liberibacter asiaticus in Kenya

Huanglongbing (HLB) is a serious disease of Citrus sp. worldwide. In Africa and the Mascarene Islands, a similar disease is known as African citrus greening (ACG) and is associated with the bacterium Candidatus Liberibacter africanus (Laf). In recent years, Candidatus Liberibacter asiaticus (Las) associated with the severe HLB has been reported in Ethiopia. Thus, we aimed to identify the Liberibacter species affecting citrus, the associated vectors in Eastern Africa and their ecological distribution. We assessed the presence of generic Liberibacter in symptomatic leaf samples by quantitative PCR. Subsequently, we sequenced the 50 S ribosomal protein L10 (rplJ) gene region in samples positive for Liberibacters and identified the species by comparison with public sequence data using phylogenetic reconstruction and genetic distances. We detected generic Liberibacter in 26%, 21% and 66% of plants tested from Uganda, Ethiopia and Kenya, respectively. The rplJ sequences revealed the most prevalent Liberibacters in Uganda and Ethiopia were LafCl (22%) and Las (17%), respectively. We detected Las in Kenya for the first time from three sites in the coastal region. Finally, we modelled the potential habitat suitability of Las in Eastern Africa using MaxEnt. The projection showed large areas of suitability for the pathogen in the three countries surveyed. Moreover, the potential distribution in Eastern Africa covered important citrus-producing parts of Ethiopia, Kenya, Uganda and Tanzania, and included regions where the disease has not been reported. These findings will guide in the development of an integrated pest management strategy to ACG/HLB management in Africa.