Transcriptome Profiling of ‘Candidatus Liberibacter asiaticus’ in Citrus and Psyllids

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Graft Compatibility Classification within Aurantioideae Based on Biometric Traits and the Anatomy of Graft Union

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respectively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by ‘Candidatus Liberibacter asiaticus’. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their graft compatibility, as follows: fully compatible with high graft-take and plant growth; potentially compatible with high graft-take but lower plant growth; partially incompatible with lower graft-take and poor plant growth; and fully incompatible with a complete absence of graft-take.

Different Sweet Orange–Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Scion

The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.

Different Sweet Orange‒Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Roots

Grafting a scion onto a rootstock results in physical and physiological changes in plant growth and development, which can affect tree vigor, productivity, and tolerance to stress and disease. Huanglongbing (HLB) is one of the most destructive citrus diseases and has become endemic in Florida since its introduction in 2005. It is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), which cause severe metabolic disruptions in affected plants. Although most scion cultivars are highly susceptible, some rootstock cultivars are tolerant and allow the grafted tree to cope better with the disease. The objectives of this study were to identify rootstock traits that can be used to assess cultivars under controlled greenhouse conditions in advance of longer-term field trials. We used 10 commercially important rootstocks with different genetic backgrounds and known field performance in graft combination with ‘Valencia’ sweet orange scion. Trees were graft-inoculated with CLas and compared against mock-inoculated trees. Tree health and CLas populations were assessed regularly, and root growth was monitored using a minirhizotron imaging system. Plants were excavated and destructively sampled 21 months after inoculation to assess biomass distributions and other CLas-induced effects. We found significant differences between healthy and infected trees for most variables measured, regardless of the rootstock. In contrast to leaf CLas titers, root titers were significantly influenced by the rootstock, and highest levels were measured for ‘Ridge’ sweet orange and sour orange. Root growth and root biomasses were reduced upon infection but differences among rootstocks did not always agree with reported field performances. Despite severe biomass reductions plants maintained their relative distribution of biomass among different components of the root system, and no dead roots were observed. Root respiration was reduced by CLas infection and was overall higher in tolerant cultivars suggesting its potential as a physiological marker. This study improves our knowledge about the strengths and weaknesses of assessing rootstock traits of grafted trees in a controlled greenhouse setting. Results from the study suggest that in addition to HLB tolerance, other rootstock traits will ultimately have major contributions to field survival and productivity of the grafted trees in an HLB endemic production environment.

Survey for ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ in Citrus in Chile

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

Population diversity of “Candidatus Liberibacter asiaticus” and Diaphorina citri in Sichuan: A case study for Huanglongbing monitoring and interception

Citrus Huanglongbing (HLB) is present in 10 provinces in China and is associated with “Candidatus Liberibacter asiaticus” (CLas), which is transmitted by the Asian citrus psyllid (Diaphorina citri, ACP). To date, HLB and ACP have expanded to Yibin city of Sichuan Province, posing an imminent threat to the citrus belt of upper and middle reach of Yangtze River, an important late maturing citrus-producing area in China. To understand the epidemiological route of CLas and ACP in newly invaded regions of Sichuan and thereby better establish an HLB-interception zone ranging from Leibo to Yibin, we evaluated the molecular variability of 19 CLas draft genomes from citrus or dodder (Cuscuta campestris). They include three type-specific prophage loci, three variable numbers of tandem repeat (VNTR) loci, a miniature inverted-repeat transposable element (MITE) types, and population diversity of 44 ACP mitochondrial genomes. The results indicated that CLas isolates in the newly invaded area (Pingshan) were more diverse than those in the HLB endemic areas (Leibo and Ningnan). Phylogenetic analysis based on mitochondrial genomes demonstrated that ACPs in Leibo, Pingshan and Xuzhou (rural areas) represent a new group (MG4), distinguished by the three unique SNPs in cox1, nad4 and cytb. However, the ACPs sampled from the urban areas of Cuiping and Xuzhou belonged to the southeastern China group (MG2-1). Altogether, our study revealed multiple sources of ACP and CLas in the HLB-interception zone and proposed their transmission route. This study contributes to the formulation of precise HLB prevention and control strategies in the HLB-interception zone in Sichuan and could be useful for HLB management efforts in other regions.

‘Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress in Planta

The lipopolysaccharides (LPS) of Gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid (VLCFA) modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on non-host tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host citrus (Citrus sinensis, confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione (GSH) pool, callose deposition and activation of the salicylic acid (SA) and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ BCP peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (a) attenuates NO-mediated SAR signaling and (b) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.