Candidatus Liberibacter' species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from 'Candidatus Liberibacter asiaticus' (Las). In order to gain greater insight into 'Ca. Liberibacter' biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse 'Ca. Liberibacter' species, including those that can infect citrus. Our phylogenetic analysis differentiates 'Ca. Liberibacter' species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic 'Ca. Liberibacter' species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of 'Ca. Liberibacter' species, the introduction of Las in the United States and identifies promising Las targets for disease management. K E Y W O R D S 'Candidatus Liberibacter' sp., citrus greening disease, HLB, huanglongbing, phylogenomics, SEC effector | 717 THAPA eT Al.

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The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.