African Citrus Greening Disease in East Africa: Incidence, Severity, and Distribution Patterns.

African citrus greening disease (ACGD) is considered as one of the major diseases of citrus threatening citrus production in East Africa. Our study aimed for the first time to assess the incidence, severity, and distribution patterns of ACGD in Kenya and Tanzania. In total, 105 citrus orchards were assessed in 13 regions representing low, mid, and high altitude areas. In each backyard and orchard, trees were randomly selected and rated for visual ACGD symptoms; then leaves and insect samples collected for analysis of 'Candidatus Liberibacter africanus' (CLaf), the presumptive causal agent of ACGD. Endpoint PCR, sequencing, and molecular phylogenetic tools were employed to confirm the identity of potential circulating pathogens. Incidence and severity of ACGD varied significantly among the different regions. Both Trioza erytreae (Del Guerico) (Hemiptera: Triozidae) and the invasive Asian citrus psyllid vector Diaphorina citri (Kuwayama) (Hemiptera: Liviidae) were found to co-occur in upper and lower midland regions. Molecular characterization identified 'Candidatus Liberibacter africanus spp. Clausenae' (CLafCl) as the main causal agent of ACGD in most of the citrus plants and insect samples. No instances of Candidatus Liberibacter asiaticus infection were found. These findings provide valuable insights into understanding and management of ACGD by employing stringent and early disease detection tools to curb the spread of the disease.

DNA Barcode Reference Library for the African Citrus Triozid, Trioza erytreae (Hemiptera: Triozidae): Vector of African Citrus Greening.

Citrus (Citrus spp.) production continues to decline in East Africa, particularly in Kenya and Tanzania, the two major producers in the region. This decline is attributed to pests and diseases including infestation by the African citrus triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae). Besides direct feeding damage by adults and immature stages, T. erytreae is the main vector of 'Candidatus Liberibacter africanus', the causative agent of Greening disease in Africa, closely related to Huanglongbing. This study aimed to generate a novel barcode reference library for T. erytreae in order to use DNA barcoding as a rapid tool for accurate identification of the pest to aid phytosanitary measures. Triozid samples were collected from citrus orchards in Kenya, Tanzania, and South Africa and from alternative host plants. Sequences generated from populations in the study showed very low variability within acceptable ranges of species. All samples analyzed were linked to T. erytreae of GenBank accession number KU517195. Phylogeny of samples in this study and other Trioza reference species was inferred using the Maximum Likelihood method. The phylogenetic tree was paraphyletic with two distinct branches. The first branch had two clusters: 1) cluster of all populations analyzed with GenBank accession of T. erytreae and 2) cluster of all the other GenBank accession of Trioza species analyzed except T. incrustata Percy, 2016 (KT588307.1), T. eugeniae Froggatt (KY294637.1), and T. grallata Percy, 2016 (KT588308.1) that occupied the second branch as outgroups forming sister clade relationships. These results were further substantiated with genetic distance values and principal component analyses.

Identification of aphid (Hemiptera: Aphididae) species of economic importance in Kenya using DNA barcodes and PCR-RFLP-based approach.

Aphids are among pests of economic importance throughout the world. Together with transmitting plant viruses, aphids are capable of inflicting severe crop production losses. They also excrete honeydew that favours the growth of sooty mold which reduces the quality of vegetables and fruits and hence their market values. Rapid and accurate identification of aphids to the species level is a critical component in effective pest management and plant quarantine systems. Even though morphological taxonomy has made a tremendous impact on species-level identifications, polymorphism, morphological plasticity and immature stages are among the many challenges to accurate identification. In addition, their small size, presence of cryptic species and damaged specimens dictate the need for a strategy that will ensure timely and accurate identification. In this study, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based on mitochondrial cytochrome c oxidase subunit I gene and DNA barcoding were applied to identify different aphid species collected from different agro-ecological zones of Kenya. Three restriction enzymes RsaI, AluI and Hinf1 produced patterns that allowed unambiguous identification of the species except Aphis craccivora and Aphis fabae. Analyses of the barcode region indicated intraspecific and interspecific sequence divergences of 0.08 and 6.63%, respectively. DNA barcoding identified all species, including the morphologically indistinguishable A. craccivora and A. fabae and separated two subspecies of A. fabae. Based on these results, both PCR-RFLPs and DNA barcoding could provide quick and accurate tools for identification of aphid species within Aphididae subsequently aiding in effective pest management programmes and enhance plant quarantine systems.