Publications

3918

AbstractThe Asian citrus psyllid, Diaphorina citri is an invasive insect 1 and a vector of ’Candidatus Liberibacter asiaticus’ (CLas), a bacterium whose growth in Citrus species results in citrus greening disease 2,3. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification 4 and nevertheless contain significant quantities of host DNA 5,6. To overcome these hurdles, we developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to directly sequence CLas and the complete, primarily uncultivable, microbiome from D. citri adults. The PDF protocol yielded CLas abundances upwards of 60% and enabled detection of 156 genetic variants in these strains compared to progenitor strains in Florida, which included prophage encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can inform agricultural management strategies related to pathogen evolution 7, insect microbiome surveillance 8, antibiotic resistance screening 9, and gut content analysis 10.

A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds in boreal and subarctic areas. However, it has probably escaped detection in many previous studies that used 16S rRNA gene amplicon sequencing due to insufficient database coverage, as previously analysed metagenome-assembled genomes (MAGs) affiliated with Ca. Methylumidiphilus do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG data set. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we found 5 MAGs, each containing one 1532 bp sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in 2 unique 16S rRNA gene sequences. These sequences represented 2 different putative species: Ca. Methylumidiphilus alinenensis (GenBank accession OK236221) and another unnamed species of Ca. Methylumidiphilus (GenBank accession OK236220). We suggest that including these 2 sequences in reference databases will enhance 16S rRNA gene-based detection of members of this genus from environmental samples.

Huanglongbing (HLB) has turned into a devastating botanical pandemic of citrus crops, caused by Candidatus Liberibacter asiaticus (CLas). However, until now the disease has remained incurable with very limited control strategies available. Restoration of the affected microbiomes in the diseased host through the introduction of an indigenous endophyte Bacillus subtilis L1-21 isolated from healthy citrus may provide an innovative approach for disease management. A novel half-leaf method was developed in vitro to test the efficacy of the endophyte L1-21 against CLas. Application of B. subtilis L1-21 at 104 colony forming unit (cfu ml−1) resulted in a 1,000-fold reduction in the CLas copies per gram of leaf midrib (107 to 104) in 4 days. In HLB-affected citrus orchards over a period of 2 years, the CLas incidence was reduced to < 3%, and CLas copies declined from 109 to 104 g−1 of diseased leaf midribs in the endophyte L1-21 treated trees. Reduction in disease incidence may corroborate a direct or an indirect biocontrol effect of the endophytes as red fluorescent protein-labeled B. subtilis L1-21 colonized and shared niche (phloem) with CLas. This is the first large-scale study for establishing a sustainable HLB control strategy through citrus endophytic microbiome restructuring using an indigenous endophyte.