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.

Evaluating the mitochondrial genomic diversity, global distribution and niche overlap of two invasive Phthorimaea species.

This study sought to evaluate the genetic diversity of two invasive Phthorimaea species (Phthorimaea operculella Zeller and Phthorimaea absoluta Meyrick), and identify potential niche overlap of both species. The complete mitogenomes of P. operculella and P. absoluta were sequenced and compared. Furthermore, the diversity within the family Gelechiidae was assessed. Subsequently, two species distribution models (MaxEnt and BIOCLIM) were used to identify niche overlaps of both species globally. The complete mitogenomes of both species were similar in size and structure, with a pairwise identity of 92.3%. The models showed a niche overlap of both species and revealed areas of marginal to high suitability for both pests in countries where they have not been reported. Therefore, these results present a compelling case for a deeper genetic and ecological investigation of the Gelechiidae family for taxonomic harmonization, an early warning for surveillance, stricter phytosanitary considerations and preventive management against the spread of the pests.

Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta.

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

Biochemistry and transcriptomic analyses of Phthorimaea absoluta (Lepidoptera: Gelechiidae) response to insecticides.

Phthorimaea absoluta is an invasive solanaceous plant pest with highly devastating effects on tomato plant. Heavy reliance on insecticide use to tackle the pest has been linked to insecticide resistance selection in P. absoluta populations. To underline insights on P. absoluta insecticide resistance mechanisms to diamides and avermectins, we evaluated the transcriptomic profile of parental (field-collected) and F8 (lab-reared) populations. Furthermore, to screen for the presence of organophosphate and pyrethroid resistance, we assessed the gene expression levels of acetylcholinesterase (ace1) and para-type voltage-gated sodium channel (VGSG) genes in the F1 to F8 lab-reared progeny of diamide and avermectin exposed P. absoluta field-collected populations. The VGSG gene showed up-regulation in 12.5% and down-regulation in 87.5% of the screened populations, while ace1 gene showed up-regulation in 37.5% and down-regulation in 62.5% of the screened populations. Gene ontology of the differentially expressed genes from both parental and eighth generations of diamide-sprayed P. absoluta populations revealed three genes involved in the metabolic detoxification of diamides in P. absoluta. Therefore, our study showed that the detoxification enzymes found could be responsible for P. absoluta diamide-based resistance, while behavioural resistance, which is stimulus-dependent, could be attributed to P. absoluta avermectin resistance.

Population dynamics and insecticide resistance in Tuta absoluta (Lepidoptera: Gelechiidae), an invasive pest on tomato in Kenya.

Tuta absoluta feeds on solanaceous plants with preference on tomato. Management of the pest is mostly with chemical insecticides. This study identified insecticide resistant populations and predicted resistance to insecticides. Insecticide resistance development was modelled using system thinking, and system dynamics approaches. The model showed the pest resistance development is alarming with an exponential increase of the resistance strength mostly in recent years. Furthermore, we used seven insecticide-resistance gene markers to resolve the population structure and genetic differentiation of insecticide-resistant populations in Kenya. The genes for resistance (knockdown resistance (kdr) mutations, acetylcholinesterase (AChE) and voltage gated sodium channel (para)) were detected in all populations. Population structure analyses separated T. absoluta populations into three genetic clusters with resistant genes that are interconnected. A better insight on the population dynamics and the genetic structure T. absoluta resistant genes in Kenya will help estimate resistance strength and determine the most effective pest control strategies.

Immunological responses and gut microbial shifts in Phthorimaea absoluta exposed to Metarhizium anisopliae isolates under different temperature regimes.

The invasive tomato leaf miner, Phthorimaea absoluta, is conventionally controlled through chemical insecticides. However, the rise of insecticide resistance has necessitated sustainable and eco-friendly alternatives. Entomopathogenic fungi (EPF) have shown potential due to their ability to overcome resistance and have minimal impact on non-target organisms. Despite this potential, the precise physiological mechanisms by which EPF acts on insect pests remain poorly understood. To attain a comprehensive understanding of the complex physiological processes that drive the successful control of P. absoluta adults through EPF, we investigated the impacts of different Metarhizium anisopliae isolates (ICIPE 665, ICIPE 20, ICIPE 18) on the pest's survival, cellular immune responses, and gut microbiota under varying temperatures. The study unveiled that ICIPE 18 caused the highest mortality rate among P. absoluta moths, while ICIPE 20 exhibited the highest significant reduction in total hemocyte counts after 10 days at 25°C. Moreover, both isolates elicited notable shifts in P. absoluta's gut microbiota. Our findings revealed that ICIPE 18 and ICIPE 20 compromised the pest's defense and physiological functions, demonstrating their potential as biocontrol agents against P. absoluta in tomato production systems.

Genetic diversity of Diaphorina citri (Hemiptera: Liviidae) unravels phylogeographic structure and invasion history of eastern African populations.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is a key pest of Citrus sp. worldwide, as it acts as a vector for Candidatus Liberibacter asiaticus, the bacterial pathogen that causes citrus Huanglongbing. Diaphorina citri has been reported in Kenya, Tanzania, and more recently in Ethiopia. This study assessed the genetic diversity and phylogeographic structure of the pest to gain insights into the potential sources of its introduction into Africa. Population structure and differentiation of D. citri populations from China, Ethiopia, Kenya, Tanzania, and the USA were assessed using 10 microsatellite loci. Additionally, five new complete mitogenomes of D. citri collected in China, Ethiopia, Kenya, Tanzania, and the USA were analyzed in the context of publicly available sequences. Genotype data grouped the D. citri populations from Kenya and Tanzania in one cluster, and those from Ethiopia formed a separate cluster. The two genetic clusters inferred from genotype data were congruent with mitochondrial sequence data. The mitogenomes from Kenya/Tanzania/China had 99.0% similarity, and the Ethiopia/USA had 99.9% similarity. In conclusion, D. citri populations in eastern Africa have different sources, as the Kenyan and Tanzanian populations probably originated from southeastern Asia, while the Ethiopian population most probably originated from the Americas.

Mitogenomic analysis of diversity of key whitefly pests in Kenya and its implication to their sustainable management.

Whiteflies (Hemiptera: Aleyrodidae) are devastating agricultural pests of economic importance vectoring pathogenic plant viruses. Knowledge on their diversity and distribution in Kenya is scanty, limiting development of effective sustainable management strategies. The present study is aimed at identifying whitefly pest species present in Kenya across different agroecological zones and establish predictive models for the most abundant species in Africa. Whiteflies were sampled in Kenya from key crops known to be severely infested and identified using 16S rRNA markers and complete mitochondrial genomes. Four whitefly species were identified: Aleyrodes proletella, Aleurodicus dispersus, Bemisia afer and Trialeurodes vaporariorum, the latter being the most dominant species across all the agroecology. The assembly of complete mitogenomes and comparative analysis of all 13 protein coding genes confirmed the identities of the four species. Furthermore, prediction spatial models indicated high climatic suitability of T. vaporariorum in Africa, Europe, Central America, parts of Southern America, parts of Australia, New Zealand and Asia. Consequently, our findings provide information to guide biosecurity agencies on protocols to be adopted for precise identification of pest whitefly species in Kenya to serve as an early warning tool against T. vaporariorum invasion into unaffected areas and guide appropriate decision-making on their management.

Mitochondrial genetic variation reveals phylogeographic structure and cryptic diversity in Trioza erytreae.

Trioza erytreae is the main vector for 'Candidatus Liberibacter africanus', the causative agent of African Citrus Greening disease. The insect is widespread in Africa, and has recently disseminated to Southwestern Europe. This study aimed at generating reference mitogenome sequences for T. erytreae, as a background for future genetic diversity surveys. Complete mitochondrial sequences of three specimens collected in Ethiopia, Uganda and South Africa were recovered using Ion Torrent technology. The mitogenomes of T. erytreae from Uganda and Ethiopia were highly similar, and distinct from that found in South Africa. The phylogeographic structure of T. erytreae was assessed using genetic clustering and pairwise distances, based on a dataset of public COI sequences recorded as T. erytreae. The dataset revealed ten haplotypes with strong phylogeographic structure in Africa and Europe. Three haplotypes found in Kenya on Clausena anisata belonged to pairs separated by distances as high as 11.2%, and were basal to all other sequences. These results indicate that not all sequences identified as T. erytreae belong to the same species, and that some degree of specificity with different plant hosts is likely to exist. This study provides new baseline information on the diversity of T. erytreae, with potential implications for the epidemiology of African Citrus Greening disease.

Author Correction: Mitochondrial genetic variation reveals phylogeographic structure and cryptic diversity in Trioza erytreae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Microbiome diversity in Diaphorina citri populations from Kenya and Tanzania shows links to China.

The Asian citrus psyllid (Diaphorina citri) is a key pest of Citrus spp. worldwide, as it acts as a vector for "Candidatus Liberibacter asiaticus (Las)", the bacterial pathogen associated with the destructive Huanglongbing (HLB) disease. Recent detection of D. citri in Africa and reports of Las-associated HLB in Ethiopia suggest that the citrus industry on the continent is under imminent threat. Endosymbionts and gut bacteria play key roles in the biology of arthropods, especially with regards to vector-pathogen interactions and resistance to antibiotics. Thus, we aim to profile the bacterial genera and to identify antibiotic resistance genes within the microbiome of different populations worldwide of D. citri. The metagenome of D. citri was sequenced using the Oxford Nanopore full-length 16S metagenomics protocol, and the "What's in my pot" (WIMP) analysis pipeline. Microbial diversity within and between D. citri populations was assessed, and antibiotic resistance genes were identified using the WIMP-ARMA workflow. The most abundant genera were key endosymbionts of D. citri ("Candidatus Carsonella", "Candidatus Profftella", and Wolbachia). The Shannon diversity index showed that D. citri from Tanzania had the highest diversity of bacterial genera (1.92), and D. citri from China had the lowest (1.34). The Bray-Curtis dissimilarity showed that China and Kenya represented the most diverged populations, while the populations from Kenya and Tanzania were the least diverged. The WIMP-ARMA analyses generated 48 CARD genes from 13 bacterial species in each of the populations. Spectinomycin resistance genes were the most frequently found, with an average of 65.98% in all the populations. These findings add to the knowledge on the diversity of the African D. citri populations and the probable introduction source of the psyllid in these African countries.