Publications

4545

In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). Thermotherapy for HLB-affected trees was proposed as a short-term management solution to maintain field productivity. It was hypothesized that thermotherapy could eliminate HLB from affected branches; therefore, the study objectives were to show which time–temperature combinations eliminated CLas from woody tissues. Hardening, rounded Valencia twigs collected from HLB-affected field trees were treated in a steam chamber at different time–temperature combinations (50°C for 60 s; 55°C for 0, 30, 60, 90, and 120 s; 60°C for 30 s; and an untreated control). Three independent repetitions of 13 branches per treatment were grafted onto healthy rootstocks and tested to detect CLas after 6, 9, and 12 months. For the RNA-based CLas viability assay, three branches per treatment were treated and bark samples were peeled for RNA extraction and subsequent gene expression analyses. During the grafting study, at 12 months after grafting, a very low frequency of trees grafted with twigs treated at 55°C for 90 s and 55°C for 120 s had detectable CLas DNA. In the few individuals with CLas, titers were significantly lower (P ≤ 0.0001) and could have been remnants of degrading DNA. Additionally, there was a significant decrease (P ≤ 0.0001) in CLas 16S rRNA expression at 55°C for 90 s, 55°C for 120 s, and 60°C for 30 s (3.4-fold change, 3.4-fold change, and 2.3-fold change, respectively) in samples 5 days after treatment. Heat injury, not total CLas kill, could explain the limited changes in transcriptional activity; however, failed recovery and eventual death of CLas resulted in no CLas detection in most of the grafted trees treated with the highest temperatures or longest durations.

‘Candidatus Liberibacter asiaticus’ (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

Abstract Candidatus Liberibacter solanacearum (Lso) is a phloem-limited, Gram-negative, unculturable bacterium that is primarily spread by psyllid insect vectors. It is considered very invasive due to its ability to be transported primarily in infective psyllids (Munyaneza et al., 2007a; 2010a,b; 2012a,b; Munyaneza, 2012; Alfaro-Fernandez et al., 2012a,b). It has been shown that Lso distribution in the Americas, New Zealand and Europe follows the distribution of its known psyllid vectors (Munyaneza, 2010; 2012).In New Zealand, where Lso was introduced along with Bactericera cockerelli, supposedly from Western USA in early 2000s, the bacterium had already spread to both North and South Island by the time it was first documented in 2006 (Gill, 2006). It is clear that introduction of the psyllid vectors of Lso into new regions is likely to result in the rapid spread of this bacterium. Lso and several of its vectors are already on several alert lists, including the EPPO A1 Regulated Quarantine Plant Pests.

AbstractThe Candidate Phyla Radiation (CPR) constitutes a large group of mostly uncultured bacterial lineages with small cell sizes and limited biosynthetic capabilities. They are thought to be symbionts of other organisms, but the nature of this symbiosis has been ascertained only for cultured Saccharibacteria, which are epibiotic parasites of other bacteria. Here, we study the biology and the genome of Vampirococcus lugosii, which becomes the first described species of Vampirococcus, a genus of epibiotic bacteria morphologically identified decades ago. Vampirococcus belongs to the CPR phylum Absconditabacteria. It feeds on anoxygenic photosynthetic gammaproteobacteria, fully absorbing their cytoplasmic content. The cells divide epibiotically, forming multicellular stalks whose apical cells can reach new hosts. The genome is small (1.3 Mbp) and highly reduced in biosynthetic metabolism genes, but is enriched in genes possibly related to a fibrous cell surface likely involved in interactions with the host. Gene loss has been continuous during the evolution of Absconditabacteria, and generally most CPR bacteria, but this has been compensated by gene acquisition by horizontal gene transfer and de novo evolution. Our findings support parasitism as a widespread lifestyle of CPR bacteria, which probably contribute to the control of bacterial populations in diverse ecosystems.

Abstract Zebra chip, is a potato disease associated with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) and vectored by the potato psyllid, Bactericera cockerelli Šulc. Potato psyllids are native to North America, where four haplotypes have been described. They are able to colonize a wide range of solanaceous species, crops, and weeds. The epidemiology of zebra chip disease is still poorly understood and might involve the different haplotypes of psyllids as well as two haplotypes of Lso. As several perennial weeds have been recognized as potential host for potato psyllids and Lso, a yearly monitoring of several patches of bittersweet nightshade (Solanum dulcamara) and field bindweed (Convolvulus arvensis) located in the potato-growing region of southern Idaho was conducted from 2013 to 2017, to gain insight into psyllid dynamics in non-potato hosts and Lso presence in the fields. Potato psyllids caught on each host were individually tested for Lso, and a subset were haplotyped based on the CO1 gene, along with the haplotyping of Lso in positive samples. On bittersweet nightshade, the Northwestern haplotype was numerically dominant, with around 2.7% of psyllids found to be carrying either Lso haplotype A or B, suggesting a limited role in zebra chip persistence, which has infected Idaho fields at a low occurrence since the 2012 outbreak. Field bindweed was found to be a transient, non-overwintering host for potato psyllid of Northwestern, Western and Central haplotypes late in the season, suggesting minor, if any, role in persistence of Lso and field infestation by potato psyllids.