Assessment of incidence, severity and distribution patterns of citrus greening in Kenya and Tanzania; the role of African Citrus Triozid endosymbionts in disease epidemiology

Abstract

Infestation by insect pests and infection of disease causing pathogens are among the major biotic constraints hindering production, profitability and contribution of citrus to economic development. The African citrus triozid (ACT), Trioza erytreae (Del Guerico) (Hemiptera: Triozidae) and the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), known vectors of Candidatus Liberibacter spp. pathogens, causal agents of the incurable and deadly citrus greening disease (CGD) are regarded as the most economically important citrus pests across all major citrus producing areas in the world. The objective of this study is to assess the incidence, severity and distribution patterns of CGD in Kenya and Tanzania, and to provide molecular characterization of potential circulating citrus greening pathogens. This study further investigates the interactions between Candidatus Liberibacter africanus (CLaf), causal agent of the African form of CGD and ACT, with special reference to the impact of CLaf infection on various fitness parameters and dispersal ability of the vector. This study also evaluates the diversity of ACT parasitoids and further characterizes endosymbionts associated with both T. erytreae and its parasitoids. Citrus trees were randomly selected in orchards and rated for visual CGD symptoms in various geographic regions across Kenya and Tanzania, representing the low, mid and high altitudes areas. PCR and phylogenetic analysis were used for identification of the CLaf pathogen in collected leaves and insect samples. The results indicated a widespread occurrence of the CLaf subsp. clausena (CLafcl) in citrus trees throughout the surveyed regions. In contrast, only in a very low proportion of the ACT vectors (21%) could CLaf infection be detected, and none of the sampled and analysed ACP showed any signs of a CLaf or Candidatus Liberibacter asiaticus (CLas) infection. The level of disease incidence and severity varied across the different regions, with chronic greening situation observed in the Upper midland (UM) (1,300-1,800 meter above sea level [m.a.s.l.]) and Lower midland (LM) (800-1,300 m.a.s.l.) regions in both countries. Moreover, both T. erytreae and D. citri vectors coexisted at high elevations in the UM and LM regions, illustrating that D. citri was spreading fast in these regions, quickly adapting to new geographical areas and ecologies. The findings on the effects of a CLaf infection on fitness traits of ACT established that CLaf infected (CLaf+) ACT conferred some fitness benefits, which included increased fecundity, fertility, faster egg-to-adult development time, higher female ratio and dispersal capability, all of which could potentially help to promote the spread of infection. However, the CLaf+ ACT vectors also incurred some fitness costs such as reduced nymphal and adult survival. Overall, the results suggested that CLaf infection had fitness effects on ACT, but there was no clear‐cut distinction between benefits and costs of an infection, since the pathogen affected several fitness parameters of the vector in different directions, resulting in simultaneous fitness gains and costs for CLaf and ACT. These complex and subtle interactions between fitness traits and the effects of a pathogen infection could all contribute to strong ecological impacts and evolutionary pressures on the host and the pathogen, and thus ultimately on the epidemiology of disease. Morphological and molecular characterization identified several ACT parasitoid species like Tamarixia dryi, Psyllaephagus pulvinatus, Tetrastichus sp, Aphidencyrtus cassatus and Charipinae sp. In addition, four eubacterial symbionts (Wolbachia, Rickettsia, Arsenophonus and Candidatus Liberibacter sp.) were detected in T. erytreae, while parasitoids harboured three symbionts (Wolbachia, Rickettsia and Cardinuim). Maximum likelihood phylogenetic inferences clustered the identified eubacterial symbionts within the 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. These identified parasitoids and endosymbionts could be explored as part of future biological and integrated pest and disease management strategies against ACT and CGD, respectively