Citrus is the most widely grown specialty fruit crop in the world, containing a variety of health-promoting compounds, including vitamin C. The crop is highly vulnerable to various fungal, bacterial, and viral diseases, owing to its narrow genetic diversity [1]. Huanglongbing (HLB, aka citrus greening) is one of the most devastating diseases, which has affected the global citrus industry during last few decades ^[2][3][2,3]. The disease was first reported in southern China [4]. The discovery of HLB in India was attributed to a citrus dieback in the 1700s ^[5][6][5,6], resulting in a hypothesis that the disease was established in India before spreading to China ^[3][7][3,7]. A similar malady was observed in South Africa in 1929 and named “citrus greening disease” based on the poor color development of the stylar end of affected fruit [8]. The disease was also confirmed in South America, in the state of Sao Paulo in Brazil in 2004 [9], and in the state of Florida in the USA [10]. It has seriously impacted the US citrus industry, with an approximate loss of USD 3.6 billion per year [11]. In the USA, the disease was also detected in other states, including two significant citrus-producing states, Texas [12] and California [13], as well as in South Carolina, Georgia, and Louisiana [14]. The disease had also become established in several Caribbean countries such as Cuba [15], Jamaica [16], Belize [17], and Mexico [18]. Other major citrus-growing areas of the Mediterranean Basin and Australia are under threat. The disease also has moved west from Pakistan into Iran [19] and is threatening the neighboring areas. Presently, the disease is distributed in over 58 countries of Asia, America, Africa, Oceania, and the Caribbean. Reports are based on symptomatology, DNA-DNA hybridization with specific probe, PCR followed by Xbal restriction digestion of the amplified DNA, electron microscopy, and real-time PCR (Figure 1, Table 1) [20].

. On the other hand, T. erytreae, African citrus psyllid (AfCP), the vector for spread of CLaf in Africa [29], is heat-sensitive. The adult and juvenile forms grow in a cool, moist environment and cannot withstand temperatures above 32 °C [55]. The rapid spread of HLB throughout the globe sparked research interest in understanding the genomics, transcriptomics, and proteomics of the host/vector/pathogen virulence and diversity.

Despite the unculturable nature of CLas, the complete circular genome sequence was generated from CLas-infected psyllid by metagenomics, which became the foundation platform for further research in functional genomics [56]. Presently, 42 complete isolate sequences of CLas are available in GenBank.Ten genomes are fully assembled in a single scaffold: psy62 [56], gxpsy [57], Ishi-1 [58], A4 [59], JXGC [60], AHCA1 [61], JRPAMB1 [62], TaiYZ2 [63], CoFLP1 [64], and ReuSP1 [65] (Table 2). A total of 4.5% and 8% of genes are involved in cell motility and active transport mechanism, respectively, and they might contribute to its virulence activity in the citrus plant phloem system [56]. Bacterial plant pathogens use the secreted proteins (effectors) in their defense mechanism to suppress plant immunity and create a favorable environment for colonization and proliferation ^[66][67][66,67]. The CLas genome consists of all Type I secretion system genes that encode proteins involved in multidrug efflux and toxin effectors: HlyD (membrane fusion protein, CLIBASIA_01355), PrtD (ABC transporter, CLIBASIA_1350), and TolC (outer membrane export protein, CLIBASIA_04145). However, CLas lacks type III, type IV, and type VI secretion systems and typical degradative enzymes, which are required for its free-living state ^[67][68][69][67,68,69].

CLas also possesses the general Sec secretion system/Sectransloconcapable of pathogenicity to the host plants by secreting effectors directly outside bacterial cells. CLas secretory proteins CLIBASIA 05315, CLIBASIA 03875, CLIBASIA 00460, and CLIBASIA 04025 have been reported as Sec-dependent secretory proteins engaged in starch accumulation, cell death, and host plant infections ^{[67][68][69][70][71]}[67,68,69,70,71]. Different peroxidase enzymes, such as SC2_gp095 and CLIBASIA_RS00445, have been identified as non-classical secretory proteins in CLas, which counter the reactive-oxygen-species (ROS)-mediated defense-signaling response, including H₂O₂, used by plants to combat disease progression [69]. This indicates that CLas may have developed a non-classical secretion pathway to release virulence proteins to combat the host. According to secretome analysis, the CLas genome contains a total of 27 non-classically secreted proteins (ncSecPs), the majority of which are involved in suppressing early plant defense mechanisms by diminishing the hypersensitive response [69]. The peroxiredoxin (Prx) superfamily proteins are ubiquitous cysteine-based non-heme peroxidases present in CLas. For example, bacterioferritin comigratory protein (BCP) is involved in the oxidative stress defense system of CLas due to its ROS scavenging activity [72]. Lipopolysaccharides (LPS), the most important outer membrane module of CLas encoded by 21 genes, not only play a critical role in maintaining the robust structural integrity to the bacterial cell, but also play a role in the virulence mechanism. However, there are some differences between CLas, CLaf, and CLam for type I secretion system, and LPS production has been reported ^[67][73][67,73].

Quorum sensing is a cell-to-cell signaling cascade where chemical-based regulatory communications occur among bacterial populations for their motility, biofilm formation, and virulence mechanism [11]. The mechanism of quorum sensing is regulated by two genes: luxI and luxR. The luxI gene encodes different quorum-sensing molecules, acyl-homoserine lactone (AHL), which induce biofilm formation by activation of luxR genes [68]. As CLas has a solo LuxR system but lacks LuxI [56], there is currently no evidence on how the CLas pathogen employs a quorum-sensing-based mechanism to cause the pathogenicity in citrus plants, although it is speculated that the disease is established like other phytopathogens ^[74][75][74,75]. It has been hypothesized that the communication among the CLas, endosymbiont, and psyllid is based on luxR and luxl genes [74]. CLas potentially communicates with the endosymbiont (Wolbachia spp.) and psyllid after adhering in the saliva sheath. Proteins like Mucin-5AC protein (23.46 kDa) were identified in D. citri saliva in a proteome study, which might be involved in the formation of the salivary sheath. Studies have shown that the down-regulation of Mucin 5AC results in reduced bacterial pathogen acquisition by inhibiting bacterial adhesion to the insect gut [76]. It has been reported that some proteins of psyllid (haemocyanin protein and myosin protein) and CLas (phosphopantothenoylcysteine synthetase and pantothenate kinase) interact with each other after the acquisition of CLas [76]. Therefore, a comprehensive understanding of the quorum-sensing system in CLas and the interaction with the citrus and the vector with respect to co-evolved protein interaction networksmay provide a target for combating HLB by hampering acquisition, growth, and biofilm formation of CLas.

Typical symptoms of the disease are yellowing of shoots with mottled blotchy leaves (partly yellow/green, with several shades of yellow blending), corky veins, and green islands as depicted in Figure 2 ^[41][42][41,42]. The localized symptoms of greening eventually spread on the entire canopy and finally cause defoliation and tree dieback [43]. The symptoms of the chlorotic pattern often resemble zinc and iron deficiencies, as well as other diseases such as citrus tristeza, citrus stubborn, and phytophthora infection ^[44][45][46][44,45,46]. It is often seen that fruits from infected trees are small, lopsided, poorly colored, and bitter in taste (Figure 2). The root system is found to be underdeveloped due to starvation that leads to loss of fibrous roots [47]. All the species and hybrids of citrus, irrespective of their rootstock, are susceptible to the greening disease. However, symptoms vary from cultivar to cultivar, with the most severe found on sweet orange (C. sinensis), mandarin (C. reticulata), tangelo (C. tangelo), and grapefruit (C. grandis). Less severe symptoms are observed on lemon, rough lemon, and sour orange [2]. There are no known resistant citrus species for the disease, but some cultivars are more tolerant. For example, grapefruit is more tolerant than sweet orange. The pomelo (Citrus maxima) and kumquat (Fortunella margarita) cultivar were initially considered as tolerant but eventually became infected and started showing mottling symptoms ^[2][48][2,48].

The pathogen associated with HLB was initially thought to be a mycoplasma-like organism. Subsequent electron microscopic studies confirmed that the causative organism is a bacterium. The fastidious nature of the pathogen was an impediment in traditional taxonomical classification like the study of morphology and growth characteristics. The phloem-limited causal agent was classified based on the 16S rRNA gene sequence and grouped under the α-subdivision of proteobacteria, genus Candidatus Liberibacter in the family Rhizobiaceae ^[49][50][49,50]. So far, three species of bacterium are known to be associated with citrus greening disease: ‘Candidatus Liberibacter asiaticus’ (CLas), ‘Candidatus Liberibacter africanus’ (CLaf), and ‘Candidatus Liberibacter americanus’ (CLam). To date, no successful attempts have been made to grow these bacteria in culture.

Among them, CLas is the most destructive, widely prevalent, highly divergent, and has caused significant economic loss in citrus production globally [51]. CLam and CLaf are only present in Brazil and Africa, respectively. CLam, originally identified in Brazil, was the major species, but later CLas became the most prevalent species [52]. This intracellular plant pathogen acts as an insect symbiont and is transmitted by two sap-sucking insect species, Diaphorinacitri and Triozaerytreae. D. citri isalso known as the Asian citrus psyllid (ACP) (Figure 3). The ACP is responsible for the spread of CLas and CLam in Asia as well as in the Americas [53]. The ACP is heat-tolerant and can withstand high temperatures (up to 45 °C) but is sensitive to high humidity (above 90%) [54]

Recent studies have put emphasis on understanding the virulence mechanisms of CLas in the citrus host. The contribution of prophages in CLas pathogenicity towards the suppression of plant defense has been reported [88]. Initially, it was reported that CLas bacterium carries two prophages, Type 1 (SC1) and Type 2 (SC2). Recently, the prophages have been classified into three types, i.e., Type 1 (SC1), Type 2 (SC2), and Type 3 (P-JXGC-3), based on functional and comparative genomic analysis of 15 different CLas genomes [89]. The SC1 prophage is reported tobe lytic as it produces proteins necessary for the lytic cycle and becomes replicative in plants [90]. Phage particles were observed in the phloem of infected periwinkle and sweet orange plants [91]. SC2, on the other hand, is a replicative excision plasmid that lacks lytic genes and may play a role in the lysogenic cycle. SC2 encodes proteins, i.e., peroxidase (SC2_gp095) and glutathione peroxidase (SC2_gp100); it has been observed that transient expression of SC2 gp095 leads to suppression of H₂O₂-mediated defense signaling in plants ^[88][90][88,90]. The CLas may use the peroxidase enzyme as a defense mechanism against the host immune response by suppressing the plant’s H₂O₂-mediated hypersensitive response ^[88][90][88,90]. Zheng et al. (2016) studied the dominating CLas strains in southern China, revealing a single prophage, SC1 (90.4%) or SC2 (82.6%), over other strains [92]. The in silico analyses of CGdP2 have identified the presence of CRISPR/cas systems in SC1 and SC2 prophages. Based on this analysis, it was hypothesized that the presence of a CRISPR/cas system in dominating species allows them to overcome an invading phage/prophage into the CLas genome. CLas also contains other virulence factors, like serralysin (CLIBASIA_01345) and hemolysin (CLIBASIA_01555). Serralysin is a metalloprotease, which inactivates various antimicrobial proteins involved in the plant defense mechanism. This enzyme is believed to be used by the CLas to defend against the citrus immune response [93]. To promote virulence, the endosymbiont-like pathogen ‘Ca. L. psyllaurous’ suppresses the expression of genes involved in the plant defense mechanism, i.e., genes regulated by jasmonic acid (JA) and salicylic acid (SA), by introducing protein effectors [94]. CLas also degrades SA, which plays a critical role in the plant defense mechanism against pathogens using salicylate hydroxylase.Salicylate hydroxylase reduces the defense action of the citrus plant by attenuating the response to exogenous SA [95]. The secretion and transport of the effector proteins in the host plant cells is one of the most important virulence factors of the bacterial pathogen [66]. Thevirulence factor CaLas5315 (Sec-delivered effector 1) hinders the papain-like cysteine protease’s activity to suppress the defense mechanism of citrus. It also induces the callose deposition inside the vascular tissue, starch formation, chlorosis, and plant cell death after localization in the chloroplast of Nicotiana benthamiana ^[69][71][96][69,71,96]. Ying et al. (2019) have assessed 60 total putative virulence factors of CLas and identified four candidates (detrimental virulence factors) which are responsible for growth inhibition (CLIBASIA_00470 and CLIBASIA_04025) and cell death (CLIBASIA_05150 and CLIBASIA_04065C) in N. benthamiana [97].