Publications

4794

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Candidatus Liberibacter americanus’ (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

In Brazil, citrus huanglongbing (HLB) is associated with ‘Candidatus Liberibacter americanus’ (CLam) and ‘Ca. Liberibacter asiaticus’ (CLas). However, there are few studies about HLB epidemiology when both Liberibacter spp. and its insect vector, the Asian citrus psyllid (ACP, Diaphorina citri), are present. The objective of this work was to compare the transmission of HLB by ACP when both CLam and CLas are present as primary inoculum. Two experiments were performed under screenhouse conditions from April 2008 to January 2012 (experiment 1) and from February 2011 to December 2015 (experiment 2). The experiments were carried out with sweet orange plants infected with CLam or CLas as inoculum source surrounded by sweet orange healthy plants. One hundred Liberibacter-free adult psyllids were monthly confined to the source of inoculum plants for 7 days with subsequent free movement inside the screenhouse. Fortnightly, nymphs and adults of psyllids were monitored. Psyllid and leaf samples were collected periodically for Liberibacter detection by PCR or quantitative PCR. CLas was detected more frequently than CLam in both psyllid and leaf samples. No mixed infections were detected in the psyllids. A clear prevalence of CLas over CLam was observed in both experiments. The final HLB incidences were 16.7 and 14.5% of Liberibacter-positive test plants, and CLas was detected in 92.3 and 93.1% of these infected plants. Mixed infection was observed only in 3.8% of infected test plants in experiment 1. These results endorse the shift in the prevalence of CLam to CLas observed in citrus orchards of São Paulo, Brazil.

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.

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.

‘Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening, is not available in pure culture yet. In addition to trees of citrus and citrus relatives, ‘Ca. L. asiaticus’ can grow in Madagascar periwinkle (Catharanthus roseus). Using gas chromatography-mass spectrometry, we compared the phloem sap composition in sweet orange ‘Valencia’ (Citrus sinensis) and periwinkle plants after the infection with ‘Ca. L. asiaticus’. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared with uninfected phloem sap, the organic acids implicated in the tricarboxylic acid cycle (TCA) cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids, likely benefits bacterial growth and may explain the unequal distribution of ‘Ca. L. asiaticus’ within infected trees. These findings may help in designing media for the pure culturing of ‘Ca. L. asiaticus’.

‘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.

Tomato (Solanum lycopersicum) is one of the most financially important vegetable crops. It is a species belonging to the Solanaceae family and is cultivated in many countries, including Turkey. The natural presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) from the Stolbur group (16SrXII) in tomato plants is extensively characterized based on the 16S rRNA gene worldwide. Tomato plants displaying abnormality and sterility of flower, purpling, and bushy appearance were observed in the Van province, Turkey. DNA extraction from tomato leaves was performed, and the extracted DNA was used to amplify 16S rRNA sequences using universal primer pairs by double PCR assays. After confirming the presence of the probable agent (‘Ca. P. solani’), nested PCR testing was performed using an appropriate primer set amplifying the SecY and Vmp1 genes of the same DNAs. Amplified PCR yields were then cloned into a pGEM T-Easy vector and sequenced by New Generation System (NGS). Sequenced 1438 bp nucleotides for Vmp1 gene (MN104838) and 905 bp nucleotides for SecY gene (MN125054) revealed 97.38% and 100% maximum nucleotide similarity with formerly published Vmp1 and SecY gene sequences of ‘Ca. P. solani’ species belonged to the Stolbur group (16Sr-XII), respectively. According to the phylogenetic tree created using our gene sequences from ‘Ca. P. solani’, Vmp1, and SecY gene sequences showed a highly phylogenetic affinity with the same sequences of the same agent from Serbia and France, respectively. To the best of our knowledge, this study is the first report to reveal the phylogenetic relationships of ‘Ca. P. solani’ in stolbur group (16Sr XII-A) using SecY and Vmp1 genes in the tomato plant in Turkey based on the non-ribosomal genes.

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.