Pandey, Sheo Shankar

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

‘ Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant−pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant−pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development.

Citrus Huanglongbing (HLB), also known as citrus greening, is one of the most devastating citrus diseases worldwide. Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain associated with HLB, which is yet to be cultured in vitro. None of the commercial citrus cultivars are resistant to HLB. The pathosystem of Ca. Liberibacter is complex and remains a mystery. In this review, we focus on the recent progress in genomic research on the pathogen, the interaction of host and CLas, and the influence of CLas infection on the transcripts, proteins, and metabolism of the host. We have also focused on the identification of candidate genes for CLas pathogenicity or the improvements of HLB tolerance in citrus. In the end, we propose potentially promising areas for mechanistic studies of CLas pathogenicity, defense regulators, and genetic improvement for HLB tolerance/resistance in the future.

AbstractThe immune system is critical for keeping animals and plants healthy from pathogens. However, immune-mediated diseases are also common for human. Immune-mediated diseases have not been reported for plants. Here, we present evidence that citrus Huanglongbing (HLB), caused by phloem-colonizing Candidatus Liberibacter asiaticus (CLas), is an immune-mediated disease. CLas infection of Citrus sinensis stimulated systemic and chronic immune response in the phloem tissues including reactive oxygen species (ROS) production as indicated by H2O2, callose deposition, and induction of immune related genes. Systemic cell death of companion and sieve element cells, but not surrounding parenchyma cells, was observed following ROS production triggered by CLas. ROS production triggered by CLas localized in phloem tissues. The H2O2 concentration in exudates extracted from phloem enriched bark tissue of CLas infected plants reached a threshold of killing citrus protoplast cells, which was suppressed by uric acid (a ROS scavenger) and gibberellin. Foliar spray of HLB positive citrus with antioxidants (uric acid and rutin) and gibberellin significantly reduced both H2O2 concentrations and cell death in phloem tissues induced by CLas and reduced HLB symptoms. RNA-seq analyses of CLas infected and health C. sinensis support that CLas causes oxidative stress. In sum, HLB is an immune-mediated disease and both mitigating ROS via antioxidants and promoting plant growth can reduce cell death of the phloem tissues caused by CLas, thus controlling HLB.

‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ‘Candidatus Liberibacter asiaticus’ and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ‘Ca. L. asiaticus’ inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ‘Ca. L. asiaticus’. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ‘Ca. L. asiaticus’ transmission by ACP. In this study, we report a new method for targeted early detection of ‘Ca. L. asiaticus’ in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that ‘Ca. L. asiaticus’ remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ‘Ca. L. asiaticus’ in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ‘Ca. L. asiaticus’ inoculum removal and preventing HLB from spreading.